JP2023506306A - Bifunctional molecules containing IL-7 variants - Google Patents

Abstract

The present invention relates to IL-7 variants, bifunctional molecules containing same, and uses thereof.

Description

The present invention relates to the field of immunotherapy. The present invention provides bifunctional molecules comprising IL-7 variants.

Interleukin-7 is an immunostimulatory cytokine member of the IL-2 superfamily and plays an important role in the adaptive immune system by promoting immune responses. This cytokine activates immune function by stimulating T- and B-cell survival and differentiation, lymphocyte cell survival, and natural killer (NK) cell activity. IL-7 also regulates lymph node development via lymphoid tissue inducer (LTi) cells and promotes survival and division of naive or memory T cells. In addition, IL-7 enhances human immune responses by promoting the secretion of IL-2 and interferon-γ. The receptor for IL-7 is a heterodimer, consisting of IL-7Rα (CD127) and a common γ chain (CD132). Although the γ chain is expressed on all hematopoietic cell types, IL-7Rα is primarily expressed by lymphocytes, including B and T lymphocyte progenitors, naive T cells, and memory T cells. It has been observed that IL-7Rα expression on regulatory T cells is lower compared to effector/naive T cells, which express higher levels. CD127 is therefore used as a surface marker to distinguish between these two populations. IL-7Rα is also expressed on innate lymphoid cells as NK and gut-associated lymphoid tissue (GALT)-derived T cells. IL-7Rα (CD127) chain is shared with TSLP (tumor stromal lymphopoietin) and CD132 (γ chain) is associated with IL-2, IL-4, IL-9, IL-15 and interleukin-21. shared with. Two major signaling pathways: (1) the Janus kinase/STAT pathway (ie, Jak-Stat-3 and 5) and (2) the phosphatidyl-inositol-3 kinase pathway (ie, PI3K-Akt) are linked to CD127/CD132 is induced by IL-7 administration is well tolerated by patients and is associated with expanded proliferation of CD8 and CD4 cells and a relative decrease in CD4+ T regulatory cells. Recombinant naked IL-7 or IL-7 fused to the Fc N-terminal domain of an antibody has been tested clinically. The latter is based on the rationale that fusion with the Fc domain extends the IL-7 half-life and enhances the long-lasting efficacy of therapy.

Recombinant IL-7 cytokines have poor pharmacokinetic properties, limiting their clinical use. After injection, recombinant IL-7 is rapidly distributed and cleared, with half-lives of IL-7 in humans (ranging from 6.8 to 9.5 hours) (Sportes et al., Clin Cancer Res. 2010 Jan 15;16). (2):727-35) or mice (2.5 hours) (Hyo Jung Nam et al., Eur. J. Immunol. 2010.40:351-358). Fusing an IgG Fc domain to IL-7 prolongs the half-life of IL-7. This is because IgG can bind to neonatal Fc receptors (FcRn) and participate in transcytosis and endosomal recycling of the molecule. For IL-7 Fc fusion molecules, prolonged circulation half-life was observed in mice (t1/2=13 hours), with IL-7 Fc fusion molecules at detectable levels (200 pg/mL) up to 8 days after dosing. (Hyo Jung Nam et al., Eur. J. Immunol. 2010.40:351-358). Although IL-7 cytokines fused to the Fc domain have increased half-lives, frequent in vivo injections of this molecule were required for biological effects. In the context of immunocytokine molecules, cytokines are fused to antibodies (e.g. targeting cancer antigens, immune checkpoint blockers, co-stimulatory molecules...) that preferentially concentrate the cytokine to target antigen-expressing cells. It is However, the affinity of IL-7 cytokines for CD127/CD132 receptors (in the nanomolar to picomolar range) can be higher than that of antibodies for their targets. Cytokines would then drive the pharmacokinetics of the product, leading to rapid depletion of available drug in vivo by target-mediated drug disposition (TMDD) mechanisms. This rapid clearance has been described for immune cytokines such as IL-2 or IL-15, indicating that the pharmacokinetic properties of fusion proteins can directly affect drug performance (List et Neri Clin Pharmacol. 2013; 5(Supplement 1):29-45).

International Publication No. 2006061219 WO 19222294 WO 19215510 WO 19178362 International Publication No. 19178364 International Publication No. 19144309 WO 19046313 WO 18215937 WO 18201047 International Publication No. 18064611 International Publication No. 17216223 U.S. Patent Application No. 2018319858 WO 17158436 WO 16200219 International Publication No. 05063820 WO2014/194302 WO2017/040790 WO 2017/19846 WO2017/024465 WO 2017/025016 WO2017/132825 WO2017/133540 WO 2006/121168 International Publication No. 2013006490 WO2016/161270 WO2018/085469 WO2018/129553 WO2011/155607 U.S. Patent No. 8,552,156 European Patent No. 2581113 U.S. Patent Application No. 2014/044728 WO 18025178 WO 19179388 WO 19179391 WO 19174603 International Publication No. 19148444 WO 19120232 WO 19056281 WO 19023482 WO 18209701 WO 18165895 International Publication No. 18160536 WO 18156250 WO 18106862 WO 18106864 WO 18068182 WO 18035710 WO 18025178 WO 17194265 WO 17106372 WO 17084078 WO 17087588 WO 16196237 WO 16130898 WO 16015675 WO 12120125 International Publication No. 09100140 International Publication No. 07008463 International Publication No. 2008132601 EP 2320940 WO 19152574 International Publication No. 08076560 WO 10106051 WO 11014438 WO 17096017 WO 17144668 International Publication No. 19232484 WO 16028656 WO 16106302 International Publication No. 16191643 WO 17030823 WO 17037707 WO 17053748 WO 17152088 WO 18033798 WO 18102536 WO 18102746 WO 18160704 WO 18200430 WO 18204363 WO 19023504 WO 19062832 International Publication No. 19129221 WO 19129261 WO 19137548 WO 19152574 WO 19154415 International Publication No. 19168382 WO 19215728 International Publication No. 06015886 WO 10006071 International Publication No. 10084158 WO 18077926 WO 01/58957 WO 96/34103 WO 94/04678 WO2014/145806 U.S. Patent No. 8,216,805 U.S. Patent Application Publication No. 2012/0149876 WO 2020/127366 WO 2018/053106, pp. 36-43

Sportes et al., Clin Cancer Res. 2010 Jan 15; 16(2): 727-35. Hyo Jung Nam et al., Eur. J. Immunol. 2010.40: 351-358 List et Neri Clin Pharmacol. 2013; 5(Supplement 1): 29-45. Jiang, Y., Li, Y., and Zhu, B. (Cell Death Dis vol. 6, e1792 (2015)) Gao S H, Huang K, Tu H, Adler A S., BMC Biotechnology. 2013:13:55 Soding, J. (2005) "Protein homology detection by HMM-HMM comparison", Bioinformatics 21:951-960. Bitar et al., Front. Immunol., 2019, vol. 10 Scheff et al., Pharm Res. 2011 May;28(5):1081-9. Gebauer and Skerra, 2009, Curr Opin Chem Biol 13(3): 245-255. Nygren, 2008, FEBS J, 275, 2668-2676. Innovations Pharmac. Technol. (2006), pp. 27-30 Meth, Mol. Biol., 352 (2007), 95-109. Drug Discovery Today (2005), Vol. 10, pp. 23-33 Nat. Biotechnol. (2004), vol.22, pp.575-582 Nat. Biotechnol. (2005), vol.23, pp.1556-1561 FEBS J, (2007), vol.274, pp.86-95 Expert. Opin. Biol. Ther. (2005), vol.5, pp.783-797 J. Pharmacol. Exp. Ther. (2006) 318, 803-809. Trends. Biotechnol. (2005), vol.23, pp.514-522 Krehenbrink et al., 2008, J. Mol. Biol. 383(5):1058-68. Desmet, J. et al., 2014, Nature Communications. 5:5237 Grabulovski D et al., 2007, J Biol Chem. 282(5):3196-3204. Avacta Life Sciences, Wetherby, UK Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017) Angal S et al. (1993) Mol. Immunol. 30:105-8. Edelman, G.M. et al., Proc. Natl. Acad. USA, 63:78-85 (1969); www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html#refs Ridgway et al., Protein Engineering 9(7):617 (1996) Atwell et al., J. Mol. Biol. 1997, 270:26. Merchant et al., Nature Biotech. 16:677 (1998). Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010) Coligan et al. (eds), Current protocols in immunology, pp. 10.19.1-10.19.11 (Wiley Interscience, 1992). "Antibody engineering: a practical guide" W. H. Freeman and Company (1992) Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2005) Remington's Pharmaceutical Sciences, 16th ed., Osol, A., ed. (1980) Antonia et al., Immuno-oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res.

Therefore, there is an important interest in the art for new and improved IL-7 variants that can improve distribution and reduce clearance of IL-7 products, especially bifunctional molecules containing IL-7. A need still exists. The inventors have made significant progress with the invention disclosed herein.

We provide mutated and optimized Fc scaffolds of IL-7 to improve the distribution and clearance of bifunctional molecules to enhance their biological effects in vivo. We have observed that IL-7 variants, especially in combination with IgG isotype and linker length, allow better distribution and longer in vivo half-life of the bifunctional molecule.

In particular, the bifunctional molecules provided herein exhibit good pharmacokinetics and pharmacology in vivo, especially compared to bifunctional molecules comprising IL-7 wild-type. In addition, advantageous and unexpected properties are associated with these new molecules, as detailed in the Introduction and Examples in the Detailed Description.

In a first aspect, the invention provides a bifunctional molecule comprising an interleukin-7 (IL-7) variant conjugated to a binding moiety, comprising:
- the binding moiety binds to a target specifically expressed on the immune cell surface;
- the IL-7 variant exhibits at least 75% identity with wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant i) reduce the affinity of IL-7 variants for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R; containing at least one amino acid mutation that improves the pharmacokinetics of the bifunctional molecule, including the IL-7 variant, compared to the functional molecule;
It relates to bifunctional molecules.

In particular, at least one mutation is (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F, or W142Y, (iii) D74E , D74Q, or D74N, iv) Q11E, Y12F, M17L, Q22E, and/or K81R; or any combination thereof (i.e., the amino acid numbering is , as shown in SEQ ID NO: 1).

In particular, the invention provides a bifunctional molecule comprising an interleukin-7 (IL-7) variant conjugated to a binding moiety, comprising:
- the binding moiety binds to a target specifically expressed on the immune cell surface;
- the IL-7 variant exhibits at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant comprises (i ) W142H, W142F, or W142Y, (ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (iii) D74E, D74Q, or D74N, iv) Q11E, Y12F, M17L, Q22E, and/or K81R; or any combination thereof, wherein the amino acid numbering is as shown in SEQ ID NO: 1, and at least A single amino acid mutation i) reduces the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R, ii) wth - improve the pharmacokinetics of bifunctional molecules containing IL-7 variants compared to bifunctional molecules containing IL-7,
It relates to bifunctional molecules.

In one aspect, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, and C47S-C92S and C34S-C129S (i.e. Amino acid numbering is as shown in SEQ ID NO: 1).

In another aspect, the IL-7 variant comprises an amino acid substitution selected from the group consisting of W142H, W142F, and W142Y (ie, amino acid numbering is as shown in SEQ ID NO:1).

In another aspect, the IL-7 variant comprises an amino acid substitution selected from the group consisting of D74E, D74Q, and D74N (ie, amino acid numbering is as shown in SEQ ID NO:1).

In particular, IL-7 variants comprise or consist of the amino acid sequences shown in SEQ ID NOs:2-15.

In one aspect, the binding moiety is optionally T250Q/M428L; M252Y/S254T/T256E+H433K/N434F; /Q311;K326W/E333S;S239D/I332E/G236A;N297A;L234A/L235A;N297A+M252Y/S254T/T256E;K322A; N297A in combination with L234A/L235A, and a heavy chain constant domain of human IgG1, preferably an Fc domain, having a substitution or combination of substitutions selected from the group consisting of L234A/L235A.

In particular, the binding portion optionally has a substitution or combination of substitutions selected from the group consisting of S228P; L234A/L235A, S228P+M252Y/S254T/T256E.17, and K444A. , preferably comprising an Fc domain.

Preferably the immune cells are T cells, preferably exhausted T cells.

In one aspect, the target is expressed by a T cell and the binding moiety is PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, Consisting of HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8 Binds a target selected from the group.

Preferably, the target is expressed by T-exhausted cells and the binding moiety preferably binds to a target selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, LAG3 and TIM3.

In one aspect, the binding moiety is an antibody or antigenic fragment thereof and the N-terminus of the IL-7 variant is C-terminal to the heavy or light chain constant domain of the antibody or antibody fragment thereof, preferably the heavy chain constant It is fused to the C-terminus of the domain, optionally via a peptide linker.

In another aspect, the IL-7 variant is selected from the group consisting of GGGGS (SEQ ID NO: 68), GGGGSGGGS (SEQ ID NO: 67), GGGGSGGGGS (SEQ ID NO: 69), and GGGGSGGGGSGGGGS (SEQ ID NO: 70), preferably (GGGGS ) ₃ (SEQ ID NO: 70) to the binding moiety.

In one aspect, the molecule comprises a first monomer and a second monomer, wherein the first monomer is optionally C-terminal to the N-terminus of the first heterodimeric Fc chain. wherein the first heterodimeric Fc chain is C-terminally to the N-terminus of the IL-7 variant, optionally via a peptide linker Covalently linked, the second monomer comprises a complementary second heterodimeric Fc chain lacking the antigen-binding domain. Preferably, in the second monomer, the complementary second heterodimeric Fc chain is covalently linked to the IL-7 variant, optionally via a peptide linker, preferably C The terminus is covalently linked to the N-terminus of the IL-7 variant, optionally via a peptide linker.

In another aspect, the molecule comprises a first monomer and a second monomer, wherein the first monomer is optionally C-terminal to the N-terminus of the first heterodimeric Fc chain. optionally comprising an antigen-binding domain covalently linked via a peptide linker, wherein the first heterodimeric Fc chain lacks the IL-7 variant and the second monomer comprises the antigen-binding domain wherein the second heterodimeric Fc chain is covalently linked to the IL-7 variant, optionally via a peptide linker, preferably at the C-terminus is linked to the N-terminus of the IL-7 variant, optionally via a peptide linker.

In another aspect, the molecule comprises a first monomer and a second monomer, wherein the first monomer is optionally C-terminal to the N-terminus of the first heterodimeric Fc chain. Optionally comprising an antigen-binding domain covalently linked via a peptide linker, the second monomer is at the N-terminus of a second heterodimeric Fc chain with complementary C-terminus wherein only one of the heterodimeric Fc chains, preferably the first, is C-terminal to the N-terminus of the IL-7 variant at Covalently linked.

In particular, the antigen binding domain of the bifunctional molecule is a Fab domain, Fab', single chain variable fragment (scFV) or single domain antibody (sdAb).

Preferably, the antigen-binding domain comprises (i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:55, 56, 57, 58, 59, 60, 61, or 62; and (ii) comprising or consisting essentially of a light chain comprising CDR1 of SEQ ID NO:64 or SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16.

In particular, the antigen-binding domain is
(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, or 25;
(b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28
comprising or consisting essentially of

Preferably, the antigen binding domain comprises or consists essentially of a heavy chain variable region (VH) of SEQ ID NO:24 and a light chain variable region (VL) of SEQ ID NO:28.

The invention also relates to isolated nucleic acid sequences or groups of isolated nucleic acid molecules that encode the bifunctional molecules according to the invention.

The invention also relates to host cells containing an isolated nucleic acid according to the invention.

The invention also relates to pharmaceutical compositions comprising a bifunctional molecule, nucleic acid or host cell according to the invention, optionally together with a pharmaceutically acceptable carrier.

Finally, the invention relates to a bifunctional molecule, nucleic acid, host cell or pharmaceutical composition according to the invention for use as a medicament, in particular for the treatment of cancer or infectious diseases.

PD-1 binding ELISA assay. Human recombinant PD-1 (rPD1) protein was immobilized and antibody was added at different concentrations. Development was performed with an anti-human Fc antibody conjugated to peroxidase. Colorimetry was determined at 450 nm using TMB substrate. A. PD-1 binding of bifunctional molecules comprising an anti-PD1 antibody and IL-7 mutated at amino acids D74, Q22, M17, Q11, K81. B. PD-1 binding of bifunctional molecules containing IL-7 mutated at amino acid W142C. C. PD-1 binding of bifunctional molecules mutated at IL-7 disulfide bonds (SS1, SS2 and SS3 mutations). All molecules tested in this figure were constructed with the IgG4m isotype and a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. CD127 binding ELISA assay of IgG fused to mutated IL-7. PD-1 recombinant protein was immobilized on a plate, then bifunctional anti-PD-1 IL-7 molecules were pre-incubated with CD127 recombinant protein (histidine tag, Sino reference 10975-H08H) and wells were plated. Added to Development was performed with a mixture of biotin-conjugated anti-histidine antibody plus peroxidase-conjugated streptavidin. Colorimetry was determined at 450 nm using TMB substrate. A. CD127 binding of bifunctional molecules containing IL-7 mutated at amino acids D74, Q22, M17, Q11, K81. B. CD127 binding of bifunctional molecules containing IL-7 mutated at amino acid W142. IL-7R signaling pathways of different bifunctional molecules as measured by STAT5 phosphorylation. Human PBMCs isolated from peripheral blood of healthy volunteers were incubated with bifunctional anti-PD-1 IL-7 molecules for 15 minutes. Cells were then fixed, permeabilized and stained with AF647-labeled anti-pSTAT5 (clone 47/Stat5(pY694)). Data were obtained by calculating MFI pSTAT5 in CD3 T cells. A. pSTAT5 activation of anti-PD-1 IL-7 bifunctional molecules containing IL-7 mutated at amino acids D74, Q22, M17, Q11, K81. B. pSTAT activation of anti-PD-1 IL-7 bifunctional molecules containing IL-7 mutated at amino acid W142C. C. Anti-PD-1 IL-7 doubles containing IL-7 disulfide bonds, IL-7 mutations in SS2 (black) and SS3 (▴) compared to anti-PD-1 IL-7 WT (● gray). pSTAT5 activation of functional molecules. All molecules tested in this figure were constructed with the IgG4m isotype and a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules in mice. Mice were injected intravenously with a single dose of IgG fused to IL-7 wild-type or mutant IL-7. Molecule concentrations in serum were assessed at multiple time points after injection by ELISA. A. Injection of IgG4-G4S3 IL7 WT (■ gray); IgG4-G4S3 IL7 D74E (● black). B. Injection of IgG4-G4S3 IL7 WT (■ gray) or IgG4-G4S3 IL7 W142H (● black). C. Injection of IgG4-G4S3 IL7 WT (■ gray); IgG4-G4S3 IL7 SS2 (●) or IgG4-G4S3 IL7 SS3 (▲). D. Correlation between PK of each molecule versus area under the curve (AUC) calculated from ED50 pSTAT5 (nM). All molecules tested in this figure were constructed with the IgG4m isotype and a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. Addition of a disulfide bond between anti-PD-1 and IL-7 reduces pSTAT5 activation while increasing drug exposure in vivo. A. Human PBMC after treatment with anti-PD-1 IL-7 bifunctional molecule WT (grey ●) or anti-PD-1 IL-7 bifunctional molecule with an additional disulfide bond (black ●). IL7R signaling measured by pSTAT5 activation. B. Pharmacokinetics in mice of anti-PD-1 IL-7 bifunctional molecule WT (grey ●) or anti-PD-1 IL-7 bifunctional molecule with an additional disulfide-linked molecule (black ●). Mice were injected intravenously with a single dose containing the anti-PD-1 IL7 bifunctional molecule. Concentrations of the molecule in serum were assessed by ELISA at multiple time points after injection. All molecules tested in this figure were constructed with the IgG4m isotype and a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. PD-1 binding ELISA assay. Human recombinant PD-1 (rPD1) protein was immobilized and antibody was added at different concentrations. Development was performed with an anti-human Fc antibody conjugated to peroxidase. Colorimetry was determined at 450 nm using TMB substrate. A. Anti-PD-1 IL-7 WT bifunctional molecule containing IgG4m (● gray), anti-PD-1 IL-7WT bifunctional molecule containing IgG1m (▲ black), anti-PD-1 IL containing IgG1m isotype. PD-1 binding of the -7 D74E bifunctional molecule (■) or anti-PD-1 IL-7 W142H bifunctional molecule containing IgG1m (⋄). B. In a separate experiment, PD-1 binding of anti-PD-1 IL-7 SS2 bifunctional molecules containing the IgG4m isotype (■) or anti-PD-1 IL-7 SS2 bifunctional molecules containing IgG1m (▲). was tested. CD127 binding ELISA assay of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1N298A or IgG4 isotypes. The recombinant protein targeted by the antibody scaffold was immobilized and then the antibody fused to IL-7 was pre-incubated with CD127 recombinant protein (histidine tag, Sino reference 10975-H08H). Development was performed with a mixture of anti-histidine antibody conjugated to biotin and streptavidin conjugated to peroxidase. Colorimetry was determined at 450 nm using TMB substrate. A. Anti-PD-1 IL-7 W142H bifunctional molecule with IgG4m isotype (grey), anti-PD-1 IL-7 W142H bifunctional molecule with IgG1m (black), or anti-PD with IgG1m isotype. -1 CD127 binding of IL-7 WT bifunctional molecule (black). B. Anti-PD-1 IL-7 SS2 bifunctional molecule containing IgG4m isotype (● gray), anti-PD-1 IL-7 SS2 bifunctional molecule containing IgG1m (▲ black) or anti-PD-1 containing IgG1m. CD127 binding of IL-7 WT bifunctional molecule (black). C. Anti-PD-1 IL-7 SS3 bifunctional molecule containing IgG4m isotype (● gray), anti-PD-1 IL-7 SS3 bifunctional molecule containing IgG1m (▲ black) or anti-PD-1 IL-7. CD127 binding of the WT bifunctional molecule IgG1m (black). D. CD127 binding of anti-PD-1 W142H bifunctional molecules containing isotype IgG1m (black) or isotype IgG1m+YTE (grey). Anti-PD-1 D74E bifunctional molecules containing isotype IgG1m (black) or isotype IgG1m+YTE (black) were also tested for CD127 binding. All molecules tested in this figure were constructed with a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. IL-7R signaling analysis of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1N298A or IgG4 isotypes. Human PBMC or Jurkat PD1+CD127+ cells were incubated with anti-PD-1 IL7 bifunctional molecules for 15 minutes. Cells were then fixed, permeabilized and stained with AF647-labeled anti-pSTAT5 (clone 47/Stat5(pY694)). Data were obtained by calculating the % of pSTAT5 on CD3 T cells. A. pSTAT5 signaling on human PBMCs after treatment with the bifunctional molecule anti-PD-1 IL-7 with the mutation D74E containing the IgG4m isotype (● gray) or the IgG1m isotype (▲ black). B. pSTAT5 signaling on human PBMC after treatment with anti-PD-1 IL-7 SS2 with IgG4m isotype (● gray) or anti-PD-1 IL-7 SS2 with IgG1m (black)). C. pSTAT5 signaling on human PBMC after treatment with anti-PD-1 IL-7 SS3 (● gray) or IgG1m (▲ black) containing the IgG4m isotype. D. (Left panel) Jurkat PD1+CD127+ cells after treatment with anti-PD-1 IL-7 WT or anti-PD-1 IL-7 SS2 constructed with IgG4m (● gray) or IgG1m (▲ black) isotypes. pSTAT5 signaling. (Right panel) pSTAT5 signaling after treatment of anti-PD-1 IL-7 SS2 with IgG4m isotype (● gray) or anti-PD-1 IL-7 SS2 with IgG1m (▲ black). An anti-PD-1 IL-7 mutant bifunctional molecule enhances T cell activation in vitro. Promega PD-1/PD-L1 bioassay: (1) effector T cells (PD-1, Jurkat stably expressing NFAT-induced luciferase) and (2) activated target cells (in an antigen-independent manner). CHO K1 cells stably expressing PDL1 and surface proteins designed to activate the cognate TCR at ) were co-cultured. Luminescence is quantified after addition of BioGlo™ luciferin and represents T cell activation. Serial molar concentrations of anti-PD1 antibody +/- recombinant IL-7 (rIL-7) or anti-PD1 IL7 bifunctional molecule were tested. Each dot represents the EC50 of one experiment. A. NFAT activation of anti-PD-1 IL-7 WT bifunctional molecules containing the IgG4m isotype (● gray) or anti-PD-1 (▴) or anti-PD-1+rIL-7 (○). B. NFAT activation of anti-PD-1 IL-7 D74E IgG4m (●), PD-1 IL-7 D74E IgG1m (▲ dotted line), and anti-PD-1 alone (black ▲). C. Anti-PD-1 IL-7 W142H bifunctional molecule containing IgG4m (●), PD-1 IL-7 W142H bifunctional molecule containing IgG1m (▲ dotted line), and anti-PD-1 alone (black ▲). NFAT activation. D. NFAT activation of anti-PD-1 IL-7 SS2 bifunctional molecules with IgG4m (●) and anti-PD-1 alone (black triangles). Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1m or IgG4m isotypes. Mice were injected intravenously with a single dose containing IgG fused to IL-7 wild-type or mutant IL-7. Concentrations of drug in serum were assessed by ELISA at multiple time points after injection. A. Anti-PD-1 IL-7 WT bifunctional molecule containing IgG4m (solid gray line), anti-PD-1 IL-7 WT bifunctional molecule containing IgG1m (dashed gray line), and anti-PD-1 IL-7 WT bifunctional molecule containing IgG1m PD-1 IL-7 D74E bifunctional molecule (▲ dashed black line), anti-PD-1 IL-7 W142H bifunctional molecule containing IgG4m (○ solid black line), anti-PD-1 IL-7 containing IgG1m Pharmacokinetics of the W142H bifunctional molecule (solid dashed black line), anti-PD-1 IL-7 SS3 with IgG4 (solid solid line), and anti-PD-1 IL-7 SS3 with IgG1m (dashed line). B. Pharmacokinetics of anti-PD-1 IL-7 D74E, D74Q, W142H, D74E+W142H mutant bifunctional molecules including IgG1m. Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecule constructed with IgG1 N298A+K444A isotype. Mice were injected intravenously with a single dose containing anti-PD-1 IL7 D74E bifunctional molecules with isotype IgG1N298A (■) or isotype IgG1m+K444A mutant isotype (●). Antibody concentrations were assessed by ELISA at multiple time points after injection. Linker length does not significantly affect pharmacokinetics but reduces stimulation of IL-7R signaling. A. Pharmacokinetics of anti-PD-1 IL-7 WT bifunctional molecules constructed with different linkers (GGGGS), (GGGGS)2, (GGGGS)3). B. Different linkers (GGGGS), (GGGGS)2. Pharmacokinetics of the anti-PD-1 IL-7 D74 bifunctional molecule constructed in (GGGGS) 3). C. Pharmacokinetics of anti-PD-1 IL-7 W142H bifunctional molecules constructed with different linkers ((GGGGS)2, (GGGGS)3). Mice were injected intravenously with a single dose containing IgG fused to IL-7 wild-type or mutant IL-7. Concentrations of IgG fused to IL-7 were assessed by ELISA at multiple time points post-injection. D. pSTAT5 signaling of anti-PD-1 IL-7 bifunctional molecules constructed with no linker or with GGGGS, (GGGGS)2, (GGGGS)3 linkers. Anti-PD-1 IL-7 mutations preferentially target PD-1+CD127+ cells over PD-1-CD127+ cells. Jurkat cells expressing CD127+ or Jurkat cells co-expressing CD127+ and PD-1+ were stained with 45 nM anti-PD-1 IL-7 bifunctional molecule and anti-IgG-PE (Biolegend, clone HP6017). clarified in Data represent the ratio of mean fluorescence on PD-1+CD127+ Jurkat cells to mean fluorescence obtained on PD1− cells CD127+ Jurkat cells. In this assay, anti-PD-1 IL-7 WT bifunctional IgG1m, anti-PD-1 IL-7 D74E bifunctional IgG1m, anti-PD-1 IL-7W142H bifunctional IgG1m, anti-PD-1 IL -7 SS2 bifunctional molecule IgG4m, anti-PD-1 IL-7 SS3 bifunctional molecule IgG1m were tested. In co-culture assays, anti-PD-1 IL-7 variants preferentially target PD-1+ CD127+ cells over PD-1- CD127+ cells. A. Expression was analyzed by flow cytometry of human CD127 and human PD-1 on CHO cells transduced with CD127 receptor alone or with both CD127 and PD-1 receptors. B. Binding of anti-PD-1 IL-7 variants to CHO cells expressing CD127+ or co-expressing CD127+ and PD-1+ in a co-culture assay. Cells were stained with cell proliferation dyes (CPDe450 or CPDe670) and then co-cultured in a 1:1 ratio before incubation with different concentrations of anti-PD-1 IL-7 bifunctional molecules. Development was performed with anti-IgG-PE (Biolegend, clone HP6017) and analyzed by flow cytometry. The binding EC50 (nM) of each construct to each cell type (CHO PD-1+ CD127+ (white histograms) and CHO PD-1- CD127+ (black histograms)) was calculated and reported. Histograms represent the mean +/- SD of three independent experiments. In this assay, irrelevant mAb IL7 WT (isotype control) molecule IgG4m, anti-PD-1 IL-7 W142H bifunctional molecule IgG1m, anti-PD-1 IL-7 SS2 bifunctional molecule IgG4m, anti-PD-1 IL- Seven SS3 bifunctional molecules IgG1m were tested. These contain a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. Anti-PD-1 IL-7 mutations preferentially activate pSTAT5 signaling into PD-1+ CD127+ cells over PD-1- CD127+ cells in co-culture assays. A. Expression was analyzed by flow cytometry of human CD127, human PD-1, and human CD132 on U937 cells transduced with CD127 receptor alone or with CD127 receptor and PD-1 receptor. B. pSTAT5 activity of anti-PD-1 IL-7 mutants in co-culture assays with U937 cells expressing CD127+ or U937 cells co-expressing CD127+ and PD-1+. Cells were stained with cell proliferation dyes (CPDe450 or CPDe670), co-cultured at a 1:1 ratio, and then incubated with different concentrations of anti-PD-1 IL-7 bifunctional molecules (15 min, 37° C.). Cells were then fixed, permeabilized and stained with AF647-labeled anti-pSTAT5 (clone 47/Stat5 (pY694). pSTAT5 activation EC50 (nM) was calculated for each construct and each cell type (CHO PD- 1+ CD127+ (white histograms) and CHO PD-1− CD127+ (black histograms).Histograms represent the mean +/- SD of four independent experiments.In this assay, rIL-7 (recombinant human IL -7 cytokine), irrelevant mAb IL7 WT (isotype control) molecule IgG4m, anti-PD-1 IL-7 D74E bifunctional molecule IgG1m, anti-PD-1 IL-7 W142H bifunctional molecule IgG1m, anti-PD-1 IL -7 SS2 bifunctional IgG4m, anti-PD-1 IL-7 SS3 bifunctional IgG1m were tested, which contain a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. Anti-PD-1 IL-7 W142H mutation preferentially activates pSTAT5 signaling into PD-1+ CD127+ human T cells and synergistically increases proliferation of PD-1+ CD127+ exhausted human T cells. Human PBMC were stimulated with CD3/CD28 coating (3 μg/mL OK3 and CD28.2 antibodies) to induce PD-1 expression, pSTAT5 activity and proliferation were It was evaluated with molecular IgG1m. A. Left graph. Representative expression of human CD127, human PD-1 on activated human T cells (CD3+ population) analyzed by flow cytometry; A. Right graph. Preincubation with anti-PD-1 competitive antibody (200 μg/mL) was followed by incubation with recombinant IL-7 or anti-PD-1 IL-7 W142H mutant molecules. IL-7R signaling pSTAT5 was quantified by flow cytometry after fixation and staining with AF647-labeled anti-pSTAT5 (clone 47/Stat5(pY694)). pSTAT5 activation (EC50) was calculated for the conditions used for the isotype control and for the anti-PD-1 competitive antibody. Data represent differences in doubling change between these two conditions; n=5 different donors tested in independent experiments. B. Proliferation of exhausted human PD-1+ T cells with isotype control, anti-PD-1+ isotype IL7 W142H IgG1m, or anti-PD-1 IL-7 W142H bifunctional IgG1m (3 nM). Proliferation was measured 5 days after restimulation with αCD3/PD-L1 recombinant protein-coated plates. Proliferation was quantified by flow cytometry using the click-it EDU assay (geometric mean and click-it EDU+ cells %); n=4 independent T cell donors were tested in independent experiments. All constructs tested contain a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. 1 is a schematic representation of various molecules used in Examples 8 and 9. FIG. The anti-PD-1 IL7 W142H mutation exhibits high binding efficiency to PD-1 and antagonizes PDL1 binding. A. PD-1 binding ELISA assay. Human recombinant PD-1 (rPD1) protein was immobilized and antibody was added at different concentrations. Development was performed with an anti-human Fc antibody conjugated to peroxidase. Colorimetry was determined at 450 nm using TMB substrate. Anti-PD-1 with one (anti-PD-1*1 ▲ gray) or two anti-PD-1 arms (anti-PD-1*2 ◆) were tested as controls. IL7 variant (anti-PD-1*2 IL7 W142H*2 black), (anti-PD-1*2 IL7 W142H*1 black), (anti-PD-1*1 IL7 W142H*2 gray), (anti-PD Bifunctional molecules including -1*1 IL7 W142H*1 ▼gray) were also tested. B. Antagonistic ability to block PD-1/PD-L1 measured by ELISA. PD-L1 was immobilized and conjugated antibody + biotinylated recombinant human PD-1 was added. This conjugate was treated with a fixed concentration of PD1 (0.6 μg/mL) and different concentrations of anti-PD1*2 IL7 W142H*1 (solid line), anti-PD1*2 IL7 W142H*2 (o dashed line), anti-PD-1*. 1 (grey ▲ dashed gray line), anti-PD1*1 IL7 W142H*2 (grey ● solid gray line), or anti-PD1*1 IL7 W142H*1 (grey ▼ solid gray line). All tested constructs contain a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. Anti-PD-1 IL7 molecules constructed with monovalent or bivalent anti-PD-1 and one IL-7 W142H cytokine activate pSTAT5 with high efficacy. A. PD-1/CD127 binding of the anti-PD-1 IL-7 W142H bifunctional molecule. PD-1 recombinant protein was immobilized, then different concentrations of the bifunctional molecule and a fixed amount of CD127 recombinant protein (histidine tagged, Sino reference 10975-H08H) were added. Development was performed with a mixture of anti-histidine antibody conjugated to biotin and streptavidin conjugated to peroxidase. Colorimetry was determined at 450 nm using TMB substrate. Anti-PD1*2 IL7 W142H1*1 (■) or anti-PD1*2 IL7 W142H*2 (● gray) was tested. B. pSTAT5 signaling assay with anti-PD-1*2 scaffold fused to IL-7 W142*1 cytokine. Human PBMC isolated from peripheral blood of healthy volunteers were incubated with anti-PD1*2 IL7 WT*2 (▼) or anti-PD1*2 IL7 W142H*1 (▪ dashed line) for 15 minutes. Cells were then fixed, permeabilized and stained with anti-CD3-BV421 and anti-pSTAT5 AF647 (clone 47/Stat5(pY694)). Data were obtained by calculating the % MFI pSTAT5+ cells relative to the CD3+ population. C. pSTAT5 signaling assay after treatment with anti-PD-1*1 IL7 W142H*1 (●), anti-PD-1*2 IL7WT*2 (■) or anti-PD1*2 IL7 W142H*1 (A). All W142H constructs tested contain a GGGGSGGGGSGGGGS linker between the IgG1m and Fc domains and the IL-7 domain. A single bivalent anti-PD-1 IL7 molecule significantly enhances T cell proliferation in vivo. One dose (34 nM/kg) of anti-PD-1 IL-7 W142H molecules (anti-PD-1*2 IL7 W142H*1, anti-PD-1*1 IL7 W142H*1, anti-PD-1*1 IL7 W142H*2) or isotype control were injected intraperitoneally. Blood was collected on day 4 and T cells were stained with anti-CD3, anti-CD8, anti-CD4 and ki67 proliferation markers. The percentage of KI67 in CD3 CD4+ and CD3 CD8+ populations was quantified. Statistical significance (*p<0.05) was calculated using one-way NOVA for multiple comparisons with control mice, n=2-8 mice per group in two independent experiments. Anti-PD-1*2 IL7*1, anti-PD-1*1 IL7*1, anti-PD-1*1 IL7*2 synergistically activate TCR signaling. Promega PD-1/PD-L1 bioassay: (1) effector T cells (PD-1, Jurkat stably expressing NFAT-induced luciferase) and (2) activated target cells (in an antigen-independent manner). CHO K1 cells stably expressing PDL1 and surface proteins designed to activate the cognate TCR at ) were co-cultured. Luminescence is quantified after addition of BioGlo™ luciferin and reflects T cell activation. A. Anti-PD1*2 (● black) and anti-PD-1*2 IL7 W142H*1 (○ white) were added at serial concentrations. An isotype antibody was used as a negative control for activation (■). B. Combination of anti-PD1*1 + isotype IL7 W142H*2 control (○ white dashed line), anti-PD-1*1 IL7 W142H*2 (● gray), anti-PD-1*1 IL7 W142H*1 (○ gray) was added at serial concentrations. All W142H constructs tested contain the GGGGSGGGGSGGGGGS linker between the IgG1m and Fc domains and the IL-7 domain. Anti-PD-1*2 IL7*1, anti-PD-1*1 IL7*1, anti-PD-1*1 IL7*2 W142H mutation favors PD-1+ CD127+ cells over PD-1- CD127+ cells binds to and activates pSTAT5 signaling. U937 cells expressing CD127+ or cells co-expressing CD127+ and PD-1+ were stained with cell proliferation dyes (CPDe450 or CPDe670) and co-cultured at a 1:1 ratio followed by different concentrations of anti-PD-1 IL. Incubated with -7 bifunctional molecules. After incubation, staining with anti-human IgG PE and pSTAT5 activation was quantified by flow cytometry. A. Binding EC50 (nM) was calculated for each cell type and each construct. B. pSTAT5 EC50 (nM) was calculated for each cell type and each construct. After treatment with bifunctional molecules, cells were then fixed, permeabilized and stained with AF647-labeled anti-pSTAT5 (clone 47/Stat5 (pY694). pSTAT5 activation. EC50 (nM) was determined for each construct and each Cell types U937 PD-1+ CD127+ (white histograms) and U937 PD-1- CD127+ (black histograms) were calculated, n=2 independent experiments.In this assay, anti-PD-1*2 IL7 W142*1, Anti-PD-1*1 IL7 W142*1 and anti-PD-1*1 IL7 W142*2 were tested, which contain the IgG1m isotype and the GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. Pharmacokinetics of anti-PD-1*2 IL7*1, anti-PD-1*1 IL7*1, anti-PD-1*1 IL7*2 W142H mutant molecules after intraperitoneal injection. Humanized PD1 mice received 1 dose (34 nM/kg) of anti-PD-1*2 IL7 IL7*2 IgG4m (△), anti-PD-1*2 IL7 W142H*1 IgG1m (▼), anti-PD-1*1 IL7 W142H*1 IgG1m (● gray) or anti-PD-1*1 IL7 W142H*2 IgG1m (○ gray) was injected intraperitoneally. Drug concentrations in serum were assessed by ELISA up to 48 hours post-injection.

INTRODUCTION The molecule according to the invention is bifunctional, as it is a combination that links the specific effects of human interleukin-7 variants with the targeting of specific targets expressed on immune cells.

As known to those skilled in the art, tumor cells may not be adequately cleared by T cells due to a phenomenon called T cell exhaustion observed in many cancers. For example, as described by Jiang, Y., Li, Y., and Zhu, B (Cell Death Dis vol. 6, e1792 (2015)), exhausted T cells in the tumor microenvironment are susceptible to inhibitory receptors. It can lead to overexpression in the body, effector cytokine production and decreased cytolytic activity, leading to failure of cancer elimination and immune evasion of the cancer in general. Restoration of exhausted T cells is therefore an envisioned clinical strategy for cancer therapy.

Programmed cell death protein 1 (PD1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell membrane protein-3 (TIM3), expressed on the surface of immune cells, especially T cells, are known in the art. and lymphocyte activation gene 3 protein (LAG3) are known. An immunosuppressive environment is induced, inter alia, by the interaction of such molecules with their counterparts expressed on the surface of tumor cells. More specifically, PD-1 is one of the major inhibitory receptors regulating T cell exhaustion. Indeed, T cells with high PD-1 expression show reduced ability to eliminate cancer cells. Anti-PD1 therapeutic compounds, particularly anti-PD1 antibodies, are in clinical use in cancer therapy to block the inhibitory effects of the PD1-PDL1 interaction (PD1 on T cells and PDL1 on tumor cells) and T cell exhaustion. there is However, anti-PD1 antibodies are not always efficient enough to allow 're'activation of exhausted T cells.

The inventors have found that bifunctional molecules comprising an IL-7 variant according to the invention and a binding moiety that blocks immunosuppressive interactions (checkpoint inhibitors) are surprisingly necessary for T cell activation. It was demonstrated to synergistically activate the NFAT pathway, a major pathway. Indeed, we observed a synergistic activation of T cells by TCR signaling. More specifically, we showed that a bifunctional IL-7 variant-anti-PD-1 molecule is associated with better activation of T cells, especially exhausted T cells, compared to anti-PD-1 alone. .

The present inventors herein describe novel bifunctional molecules and i) exhaustive factors (binding proteins) expressed on the surface of T cells, such as PD1, CTLA-4, BTLA, TIGIT, LAG3 and TIM3. ) and ii) interaction with the IL7 receptor (on the IL7 variant side) on the same T cell, leading to activation of this unexpected NFAT pathway (TCR signaling), leading to the activation of T cells, especially if not otherwise. It has been shown to have a positive effect on activating exhausted T cells, which are thought to be unable to eliminate tumor cells. This effect has never been disclosed before.

Additionally, the use of IL-7 variants in bifunctional molecules is important for increasing pharmacokinetics in vivo. Furthermore, by decreasing the affinity of the IL-7 variant for its receptor, the bifunctional molecule preferentially binds to target immune cells relative to others and is specific for such cells. Not only is the ability to provide an effect increased, but so is the ability to exploit synergistic effects associated with the action of two parts of a bifunctional molecule on the same immune cell. More specifically, a bifunctional molecule comprising a binding moiety that targets an IL-7 variant and an exhaustion factor is responsible for the accumulation of IL-7 in T cell infiltrates and relocalization of IL-7 on T cells. It is thought that it will be possible to The accumulation of IL-7 near these T cells is of particular interest in the context of exhausted T cells, which require high doses of IL-7 to activate or reactivate these T cells.

Surprisingly, we found that bifunctional molecules with an IgG1 heavy chain constant domain enhanced the activity of IL-7 variants (pStat5 signaling, synergistic effect, and CD127 binding). This enhancement is specific to the IL-7 mutant and has not been observed with wild-type IL-7. Additionally, the use of a linker (GGGGS) ₃ between the antibody and IL-7 maximizes the activity of IL-7 variants (pStat5 signaling and CD127 binding).

The bifunctional molecules of the invention have, in particular, one or several of the following advantages:
- Bifunctional molecules specifically localize IL-7 variants to the vicinity of immune cells, such as T cells or PD-1+ cells, especially within tumors, resulting in higher levels of IL-7 Allows targeting of cells in need.
- Mutations of IL-7 variants reduce the affinity of IL-7 variants for IL-7R compared to IL-7 wild type without complete or significant loss of their intrinsic biological activity .
- IL-7 variant mutations improve in vivo pharmacokinetics and pharmacology, especially compared to bifunctional molecules containing IL-7 wild type. More specifically, by improving the pharmacokinetics and pharmacodynamics of the molecule, bifunctional molecules are able to reach target cells and act on targets expressed on the surface of immune cells. Become.
- The bifunctional molecule according to the invention exhibits a synergistic activity of IL7 variants (NFAT signaling).
- The bifunctional molecule according to the invention has the highest selective activity against PD-1(+) cells over PD-1(-) cells compared to an antibody comprising wild-type IL7.
- Bifunctional molecules, including mutant IL-7 W142H molecules, selectively and synergistically cis-activate PD-1(+)CD127(+) exhausted T cells.
- The IL-7 variant has one or two IL-7 molecules and one or two antigen-binding fragments while maintaining the ability to bind a target (e.g. PD-1) and activate the IL7R pathway can be included in some structures of bifunctional molecules with In particular, bifunctional molecules with one or two IL7 W142H variants have good pharmacokinetic properties in vivo.
- The inventors have surprisingly found that constructs containing a single IL-7 variant have improved properties compared to constructs containing two IL7 variants, both in terms of activity and pharmacokinetics It is shown that.

DEFINITIONS Certain terms are defined herein below in order to make the invention easier to understand. Additional definitions are set forth throughout the detailed description.

Unless otherwise defined, all technical, notational, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. ing. In some cases, terms having commonly understood meanings are also defined herein for clarity and/or ease of reference. The inclusion of such definitions herein should not be construed as representing a necessarily different meaning than is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and widely employed by those skilled in the art using conventional methodology.

As used herein, the terms "wild-type interleukin-7," "wt-IL-7," and "wt-IL7" refer to mammalian endogenous secretory glycoproteins, particularly wild-type functional IL-7 has substantial amino acid sequence identity with mammalian IL-7 and has substantially equivalent biological activity, e.g., in a standard bioassay or assay of IL-7 receptor binding affinity. 7 polypeptides, derivatives and analogs thereof. For example, wt-IL-7 can be i) a naturally occurring or naturally occurring IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, iii) a biologically active fragment of an IL-7 polypeptide. or iv) an amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of a biologically active IL-7 polypeptide. IL-7 may contain or lack its peptide signal. Alternative names for this molecule are "pre-B cell growth factor" and "lymphopoietin-1". Preferably, the term "wt-IL-7" refers to human IL-7 (wth-IL7). For example, the human wt-IL-7 amino acid sequence is approximately 152 amino acids (without a signal peptide), Genbank accession number NP_000871.1, and the gene maps to chromosome 8q12-13. Human IL-7 is described, for example, in UniProtKB-P13232.

As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain an antigen-binding site. Immunoglobulin molecules may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. . The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Unless specifically indicated otherwise, the term "antibody" includes intact immunoglobulins as well as "antibody fragments" or "antigen-binding fragments" (Fab, Fab', F(ab')2, Fv etc.), single chain (scFv), variants thereof, molecules comprising antibody portions, diabodies, linear antibodies, single chain antibodies, and glycosylation variants of antibodies, amino acid sequence variants of antibodies, as required. It includes any other modified construction of the immunoglobulin molecule that contains a specific antigen recognition site. Preferably, the term antibody refers to humanized antibodies.

"Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in an antibody conformation. The CDRs of antibody heavy chains are typically referred to as "HCDR1," "HCDR2," and "HCDR3." The framework regions of antibody heavy chains are typically referred to as "HFR1," "HFR2," "HFR3," and "HFR4."

An "antibody light chain", as used herein, refers to the smaller of the two types of polypeptide chains present in antibody conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes. The CDRs of antibody light chains are typically referred to as "LCDR1," "LCDR2," and "LCDR3." The framework regions of antibody light chains are typically referred to as "LFR1," "LFR2," "LFR3," and "LFR4."

As used herein, an "antigen-binding fragment" of an antibody is a portion of an antibody, i.e., the structure of an antibody of the invention that, presumably in its native form, exhibits antigen-binding ability to a specific antigen. means the molecule corresponding to the portion of In particular, such fragments exhibit the same or substantially the same antigen-binding specificity for their antigen as compared to that of the corresponding four-chain antibody. Advantageously, antigen-binding fragments have similar binding affinities as the corresponding four-chain antibodies. However, antigen-binding fragments having reduced antigen-binding affinities compared to the corresponding four-chain antibodies are also encompassed within the invention. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. Such antigen-binding fragments can also be referred to as "functional fragments" of antibodies. Antigen-binding fragments of antibodies are fragments containing hypervariable domains, or parts thereof, called CDRs (Complementary Determining Regions).

The term "humanized antibody," as used herein, refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences (e.g., non-human It is intended to refer to a chimeric antibody that contains minimal sequence derived from an antibody). A “humanized form” of an antibody, eg, a non-human antibody, also refers to an antibody that has undergone humanization. A humanized antibody generally has residues from one or more CDRs replaced with at least one non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. A human immunoglobulin (recipient antibody) that has been replaced with residues from the CDRs. Additional framework region modifications may be made within the human framework sequences. Preferably, humanized antibodies have a T20 humanity score of greater than 80%, 85%, or 90%. "Humanity" quantifies the humanity of the antibody variable regions, eg, as described in Gao S H, Huang K, Tu H, Adler A S., BMC Biotechnology. 2013:13:55. Use the web-based tool to calculate the T20 score of an antibody sequence using the T20 score analyzer or the T20 Cutoff Human Databases: http://abAnalyzer.lakepharma.com can be measured by

By "chimeric antibody" is meant an antibody produced by combining genetic material, preferably derived from a non-human source, such as a mouse, with genetic material derived from a human. Such antibodies are derived from both human and non-human antibodies joined by chimeric regions. Chimeric antibodies generally contain constant domains from a human and variable domains from another mammalian species, and when used in therapeutic treatment reduce the risk of reaction to foreign antibodies from non-human animals.

As used herein, the terms "crystallizable fragment region", "Fc region", or "Fc domain" are synonymous and refer to antibodies that interact with cell surface receptors called Fc receptors. refers to the tail region of the The Fc region or domain is typically composed of two identical domains derived from the second and third constant domains (ie, the CH2 and CH3 domains) of the two heavy chains of an antibody. Part of the Fc domain refers to the CH2 or CH3 domain. Optionally, the Fc region or domain may optionally comprise all or part of the hinge region between CH1 and CH2. Optionally, the Fc domain is from IgG1, IgG2, IgG3, or IgG4, optionally having IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3.

In the context of IgG antibodies, IgG isotypes each have three CH regions. Thus, the 'CH' domains in the context of IgG are as follows: 'CH1' refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index, as with Kabat. “CH2” refers to positions 231-340 according to the EU Index as in Kabat, and “CH3” refers to positions 341-447 according to the EU Index as in Kabat.

By "amino acid change" or "amino acid modification" herein is meant a change in the amino acid sequence of a polypeptide. "Amino acid modifications" include substitutions, insertions and/or deletions in the polypeptide sequence. By "amino acid substitution" or "substitution," as used herein, is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. "Amino acid insertion" or "insertion" means the addition of an amino acid at a particular position in a parent polypeptide sequence. "Amino acid deletion" or "deletion" means removal of an amino acid at a particular position in a parent polypeptide sequence. Amino acid substitutions may be conservative. Conservative substitutions replace a given amino acid residue with another residue having a side chain (“R group”) with similar chemical properties (e.g., charge, bulk, and/or hydrophobicity) That is. As used herein, "amino acid position" or "amino acid position number" are used interchangeably and refer to the position of a particular amino acid in an amino acid sequence, generally designated by the single letter code for the amino acid. The first amino acid in the amino acid sequence (ie, starting from the N-terminus) should be considered to be position 1.

Conservative substitutions replace a given amino acid residue with another residue having a side chain (“R group”) with similar chemical properties (e.g., charge, bulk, and/or hydrophobicity) That is. In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. Conservative substitutions and corresponding rules are well described in the state of the art. For example, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the table below.

As used herein, "sequence identity" between two sequences is described by the parameters "sequence identity," "sequence similarity," or "sequence homology." For purposes of the present invention, the "percentage of identity" between two sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal fashion over a comparison window. Sequence alignments can be performed by methods well known in the art, for example, using the Needleman-Wunsch global alignment algorithm. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. Once a global alignment is obtained, the percentage identity is obtained by dividing the total number of aligned identical amino acid residues by the total number of residues contained in the longest sequence between sequences (A) and (B). be able to. Sequence identity is typically determined using sequence analysis software. To compare two amino acid sequences, for example the protein provided by EMBL-EBI or available at Initial settings: (I) Matrix: BLOSUM62, (ii) Gap open: 10, (iii) Gap extension: 0.5, (iv) Output format: Pairs, ( With v) endgap penalty: false, (vi) endgap open: 10, (vii) endgap extension: 0.5 can be used.

Alternatively, sequence identity can be determined using the sequence analysis software Clustal Omega, which typically uses the HHalign algorithm and its default settings as its core alignment engine. This algorithm, with default settings, is described in Soding, J. (2005) Protein homology detection by HMM-HMM comparison, Bioinformatics 21:951-960.

The terms "derived from" and "derived from," as used herein, have a structure that is derived from the structure of a parent compound or protein, whose structure is sufficiently similar to those disclosed herein that one of ordinary skill in the art would expect, based on the similarity, to exhibit the same or similar properties, activity, and utility as the claimed compounds; refers to the compound that will be

A "pharmaceutical composition", as used herein, is an active substance, such as one comprising a bifunctional molecule according to the invention together with optional other chemical ingredients such as physiologically suitable carriers and excipients. refers to one or more preparations of The purpose of pharmaceutical compositions is to facilitate the administration of active substances to living organisms. The compositions of the invention may be in a form suitable for any conventional route of administration or use. In one embodiment, a "composition" typically includes an active agent, such as a compound or composition, and a pharmaceutically acceptable carrier, adjuvants, diluents, binders, stabilizers, buffers, Combinations with inert (eg, detectable substances or labels) or active naturally occurring or non-naturally occurring carriers such as salts, lipophilic solvents, preservatives, or adjuvants are contemplated. An "acceptable vehicle" or "acceptable carrier" as referred to herein is any known compound or compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions. combination.

An "effective amount" or "therapeutically effective amount" as used herein, either alone or in combination with one or more other active agents, provides a therapeutic effect in a subject. It refers to the amount of active agent required to treat, for example, the amount of active agent required to treat a target disease or disorder or to produce a desired effect. "Effective amount" means the characteristics of the subject to be treated, including the substance, disease and its severity, age, physical condition, size, sex, and weight, duration of treatment, nature of concomitant therapy (if applicable). , the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. Such factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred to use the maximum dose of the individual components or combinations thereof, ie, the highest safe dose according to sound medical judgment.

As used herein, the term "pharmaceutical" refers to any substance or composition that has curative or preventive properties against a disorder or disease.

The term "treatment" refers to any act aimed at improving the state of health of a patient, such as curing, preventing, preventing, and ameliorating disease or symptoms of disease. The term refers to both curative and/or prophylactic treatment of disease. A curative treatment is defined as a treatment that results in a cure or treatment that alleviates, ameliorate, and/or eliminates, reduces, and/or stabilizes the disease or symptoms of the disease or the suffering it causes directly or indirectly. Prophylactic treatment includes both treatments that result in prevention of disease, as well as treatments that reduce and/or delay the progression and/or onset of disease or the risk of its occurrence. In certain embodiments, such terms refer to the amelioration or eradication of a disease, disorder, infection, or symptoms associated therewith. In other embodiments, the term refers to minimizing the spread or progression of cancer. Treatment according to the invention does not necessarily imply 100% or complete cure. Rather, there are varying degrees of treatment that those skilled in the art recognize as having potential benefit or therapeutic effect. Preferably, the term "treatment" refers to applying or administering a composition comprising one or more active agents to a subject with a disorder/disease.

As used herein, the term "disorder" or "disease" refers to the effects on the body due to genetic or developmental errors, infections, poisons, nutritional deficiencies or biases, toxicity, or unfavorable environmental factors. The failure of an organ, part, structure, or system to function properly. Preferably, such terms refer to health disorders or diseases, such as diseases that interfere with normal physical or mental functioning. More preferably, the term disorder refers to immune and/or inflammatory diseases affecting animals and/or humans, such as cancer.

"Immune cells" as used herein are defined, for example, as white blood cells (white blood cells) derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) and cells involved in innate and adaptive immunity such as natural killer T cells (NKT) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells) In particular, immune cells can be selected in a non-exhaustive list that includes B cells, T cells, especially CD4+ and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells, and monocytes. "T cells" as used herein include, for example, CD4+ T cells, CD8+ T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells, and Inhibitory T cells are included.

As used herein, the terms "T effector cells", "Teff" or "effector cells" refer to a group of immune cells comprising several T cell types that actively respond to stimuli such as co-stimulation. describe. The term specifically includes T cells that function to eliminate antigens (eg, by producing cytokines that modulate the activation of other cells or by cytotoxic activity). This term specifically includes CD4+ cells, CD8+ cells, cytotoxic T cells and helper T cells (Th1 and Th2).

As used herein, the term “regulatory T cells,” “Treg cells,” or “Treg” refers to cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. refers to a subpopulation of T cells that Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naive CD4 cells.

The term "exhausted T cells" refers to a population of T cells that are in a state of dysfunction (ie, "exhaustion"). T cell exhaustion is characterized by progressive loss of function, altered transcriptional profiles, and persistent expression of inhibitory receptors. Exhausted T cells lose their ability to produce cytokines, high proliferation, and cytotoxicity, which ultimately leads to their elimination. Exhausted T cells typically present higher levels of CD43, CD69, and inhibitory receptors, along with lower expression of CD62L and CD127.

The term "immune response" refers to the selective damage, destruction, or Refers to the action of e.g. lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, leading to elimination from the body .

The term "antagonist," as used herein, refers to a substance that blocks or reduces the activity or functionality of another substance. In particular, the term binds to a cell receptor (e.g. PD-1) as a reference substance (e.g. PD-L1 and/or PD-L2), which is responsible for its normal biological effects (e.g. immune It refers to antibodies that prevent all or part of the production of an inhibitory microenvironment). Antagonist activity of humanized antibodies according to the invention can be assessed by competitive ELISA.

The term "agonist" as used herein refers to a substance that activates the function of an activated receptor. In particular, the term refers to an antibody that binds to a cell activating receptor as a reference substance and has at least partially the same effect as the biologically natural ligand (e.g. induces an activating effect of the receptor). .

Pharmacokinetics (PK) refers to the movement of drugs within the body and pharmacology (PD) refers to the body's biological response to drugs. PK describes drug exposure by characterizing absorption, distribution, bioavailability, metabolism, and excretion as a function of time. PD describes drug response in terms of biochemical or molecular interactions. PK and PD analyzes are used to characterize drug exposure, predict and assess changes in dosage, estimate elimination and absorption rates, and determine relative bioavailability/bioequivalence of formulations. to characterize intra- and inter-subject variability, understand concentration-effect relationships, and establish margins of safety and efficacy characteristics. By "enhanced PK" is meant that one of the above characteristics is enhanced, eg, increased half-life of the molecule, in particular the molecule exhibits a longer serum half-life when injected into a subject.

As used herein, the term "isolated" means that the material being described (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially free from other materials with which they are associated in nature. separated or concentrated relative to them. In particular, an "isolated" antibody is one that has been identified, separated and/or recovered from a component of its natural environment.

The term "and/or", as used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" shall be construed as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually presented. should.

The term "a" or "an" refers to one or more of the elements it modifies, unless the context clearly indicates that either one of the elements or more than one of the elements is described. (eg, "reagent" can mean one or more reagents).

The term “about,” as used herein in reference to any and all values (including the lower and upper limits of numerical ranges), up to +/-10% (e.g., +/-0.5%, +/- -1%, +/-1.5%, +/-2%, +/-2.5%, +/-3%, +/-3.5%, +/-4%, +/-4.5%, +/-5 %, +/-5.5%, +/-6%, +/-6.5%, +/-7%, +/-7.5%, +/-8%, +/-8.5%, +/-9%, +/-9.5%) means any value with an acceptable deviation range. Use of the term "about" at the beginning of a string of values qualifies each of the values (ie, "about 1, 2, and 3" refers to about 1, about 2, and about 3). Further, when a list of values is provided herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate and fractional values thereof. (eg, 54%, 85.4%).

IL-7 Mutants The present disclosure provides an interleukin 7 mutant (IL-7m), as well as two entities comprising a first entity comprising an interleukin 7 variant (IL-7m) and a second entity comprising a binding moiety. Provide functional molecules.

The terms "interleukin-7 variant", "mutant IL-7", "IL-7 variant", "IL-7 variant", "IL-7m" or "IL-7v" are used herein used synonymously in the literature. A "variant" or "mutant" of an IL-7 protein is defined as an amino acid sequence that is altered by one or more amino acids. Variants may have "conservative" or "non-conservative" modifications. Such modifications can include amino acid substitutions, deletions, and/or insertions. Preferably, modifications are substitutions, especially conservative substitutions. Variant IL-7 proteins encompassed within the present invention are specifically IL-7 proteins that do not retain substantially equivalent biological properties (e.g., activity, binding capacity, and/or structure) as compared to wild-type IL-7. -7 on proteins. An IL-7 protein mutant or variant contains at least one mutation. In particular, at least one mutation reduces the affinity of IL-7m for IL-7R, but does not lead to loss of IL-7R recognition. Thus, an IL-7 mutant or variant has the ability to activate IL-7R, e.g., as measured by pStat5 signaling, e.g., as disclosed in Bitar et al., Front. Immunol., 2019, 10 et al. Hold. Biological activity of IL-7 protein can be measured using an in vitro cell proliferation assay or by measuring P-Stat5 in T cells by ELISA or FACS. Preferably, an IL-7 variant according to the invention has a biological property (e.g. activity, binding capacity and/or structure) that is at least 2 min compared to wild-type IL-7, preferably wth-IL7. 1/5, 1/10, 1/20, 1/30, 1/40, 1/50, 1/100, 1/250, 1/500, 750 minutes 1, 1,000, 1, 2,500, 5,000, or 8,000 times less. More preferably, the IL-7 variant exhibits reduced binding to the IL-7 receptor, but retains the ability to activate the IL-7R. For example, binding to IL-7 receptor may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60% compared to wild-type IL-7 and activating IL-7R. retain at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the ability to convert to wild-type IL-7. Preferably, IL-7m is a variant of human wild-type IL-7, eg, as set forth in SEQ ID NO:1.

In one embodiment, an IL-7 variant according to the invention has at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60% Preferably, it retains at least 80%, 90%, 95%, even more preferably 99% biological activity compared to wild-type IL-7.

In one aspect, the IL-7 variant or mutant is characterized by i) comparing the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) to the affinity of wt-IL-7 for the IL-7R. and ii) differ from wt-IL-7 in at least one amino acid mutation that improves the pharmacokinetics of the IL7 variant compared to wt-IL7. More particularly, IL-7 variants or mutants further retain the ability to activate IL-7R, particularly through pStat5 signaling.

In another embodiment, a bifunctional molecule comprising an IL-7 variant or mutant i) adjusts the affinity of the bifunctional molecule for the IL-7 receptor (IL-7R) to a bifunctional molecule comprising wt-IL-7. ii) comparing the pharmacokinetics of bifunctional molecules comprising IL-7 variants or mutants to bifunctional molecules comprising wt-IL-7. differs from wt-IL-7 in at least one amino acid mutation that improves More particularly, bifunctional molecules comprising IL-7 variants or mutants further retain the ability to activate IL-7R, particularly through pStat5 signaling. For example, binding of bifunctional molecules comprising IL-7 variants or mutants to the IL-7 receptor is at least 10%, 20%, 30% greater than bifunctional molecules comprising wild-type IL-7. %, 40%, 50%, 60% and reduce the ability to activate IL-7R by at least 90%, 80%, 70% compared to bifunctional molecules comprising wild-type IL-7. %, 60%, 50%, 40%, 30%, or 20%.

According to the present invention, IL-7m exhibits reduced affinity for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R. In particular, IL-7m exhibits reduced affinity for CD127 and/or CD132 compared to the affinity of wth-IL-7 for CD127 and/or CD132, respectively. Preferably, IL-7m exhibits reduced affinity for CD127 compared to the affinity of wth-IL-7 for CD127.

Preferably, the at least one amino acid mutation reduces the affinity of IL-7m for IL-7R, in particular for CD132 or CD127, by at least 10-fold compared to the affinity of wt-IL-7 for IL-7R. , 100th, 1000th, 10000th, or 100000th. Such affinity comparisons can be performed by any method known to those skilled in the art, such as ELISA or Biacore.

Preferably, the at least one amino acid mutation reduces the affinity of IL-7m for IL-7R, but in particular the biological activity of IL-7m, as measured by pStat5 signal, compared to IL-7 wt. not be reduced by

Alternatively, at least one amino acid mutation reduces the affinity of IL-7m for IL-7R but significantly increases the biological activity of IL-7m compared to IL-7 wt, particularly as measured by pStat5 signal. does not decrease.

Additionally or alternatively, IL-7m can be used to control IL-7 variants or mutants or IL-7 variants compared to wild-type IL-7 or bifunctional molecules comprising wild-type IL-7, respectively. Improves the pharmacokinetics of bifunctional molecules containing In particular, IL-7m according to the invention improves the pharmacokinetics of IL-7 variants by at least 10-fold, 100-fold or 1000-fold compared to wth-IL-7. In particular, IL-7m according to the invention enhances the pharmacokinetics of bifunctional molecules comprising IL-7 variants or mutants by at least 10-fold, 100-fold compared to bifunctional molecules comprising wth-IL-7. , or 1000 times better. Pharmacokinetic profile comparisons can be performed using methods known to those of skill in the art, for example, by injecting drugs in vivo and performing serum drug dosage ELISAs at multiple time points, as shown in Example 2. It can be implemented by any method.

As used herein, the terms "pharmacokinetics" and "PK" are used interchangeably and refer to the fate of a compound, substance, or drug administered to a living organism. Pharmacokinetics includes, inter alia, release (i.e. release of a substance from a composition), absorption (i.e. entry of a substance into the blood circulation), distribution (i.e. dispersion or diffusion of a substance throughout the body) metabolism (i.e. transformation or degradation), and excretion (ie removal or clearance of substances from organisms). The two stages of metabolism and excretion can also be grouped together under the heading elimination. Those skilled in the art are familiar with, among other Various pharmacokinetic parameters can be monitored, such as Scheff et al., Pharm Res. 2011 May;28(5):1081-9).

Accordingly, improved pharmacokinetics through the use of IL-7m, particularly in bifunctional molecules, refers to an improvement in at least one of the parameters referred to above. Preferably, the enhancement refers to an enhancement of the elimination half-life of the bifunctional molecule, ie an increase in half-life duration or Cmax.

In certain embodiments, at least one mutation in IL-7m enhances elimination half-life of a bifunctional molecule comprising IL-7m compared to a bifunctional molecule comprising IL-7 wt.

In one embodiment, IL-7m is a 152 amino acid wild-type human IL-7 (wth-IL-7) protein, such as that disclosed in SEQ ID NO: 1, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity. Preferably, the IL-7m exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

In particular, at least one mutation occurs at amino acid positions 74 and/or 142 of IL-7. Additionally or alternatively, at least one mutation occurs at amino acid positions 2 and 141, 34 and 129, and/or 47 and 92. These positions refer to the amino acid positions shown in SEQ ID NO:1.

In particular, at least one mutation is C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, Q11E, Y12F, M17L, Q22E, K81R, D74E, An amino acid substitution or group of amino acid substitutions selected from the group consisting of D74Q, and D74N, or any combination thereof. Such mutations refer to the amino acid positions shown in SEQ ID NO:1. Thus, for example, mutation W142H represents substitution of histidine for tryptophan in wth-IL7 to obtain IL-7m with histidine at amino acid position 142. Such variants are described, for example, in SEQ ID NO:5.

In one embodiment, IL-7m comprises a set of substitutions to disrupt disulfide bonds between C2 and C141, between C47 and C92, and between C34-C129. In particular, IL-7m forms disulfide bonds between C2 and C141 and between C47 and C92; between C2 and C141 and between C34-C129; or between C47 and C92 and between C34-C129. contains two sets of permutations to destroy For example, cysteine residues may be substituted with serine to prevent disulfide bond formation. Thus, the amino acid substitutions are C2S-C141S and C47S-C92S (referred to as "SS2"), C2S-C141S and C34S-C129S (referred to as "SS1"), and C47S-C92S and C34S-C129S (referred to as "SS3"). ). Such mutations refer to the amino acid positions shown in SEQ ID NO:1. Such IL-7m is specifically described in the sequences shown in SEQ ID NOs: 2-4 (SS1, SS2, and SS3, respectively). Preferably, IL-7m comprises the amino acid substitutions C2S-C141S and C47S-C92S. Even more preferably, IL-7m exhibits the sequence shown in SEQ ID NO:3.

In another embodiment, IL-7m comprises at least one mutation selected from the group consisting of W142H, W142F, and W142Y. Such IL-7m is specifically described in the sequences shown in SEQ ID NOS:5-7, respectively. Preferably, IL-7m contains the mutation W142H. Even more preferably, IL-7m exhibits the sequence shown in SEQ ID NO:5.

In another embodiment, IL-7m comprises at least one mutation selected from the group consisting of D74E, D74Q and D74N, preferably D74E and D74Q. Such IL-7m is specifically described in the sequences shown in SEQ ID NOs: 12-14, respectively. Preferably, IL-7m contains the mutation D74E. Even more preferably, IL-7m exhibits the sequence shown in SEQ ID NO:12.

In another embodiment, IL-7m comprises at least one mutation selected from the group consisting of Q11E, Y12F, M17L, Q22E, and/or K81R. Such mutations refer to the amino acid positions shown in SEQ ID NO:1. Such IL-7m are specifically described in the sequences shown in SEQ ID NOs: 8, 9, 10, 11, and 15, respectively.

In one embodiment, IL-7m is i) W142H, W142F, or W142Y, and/or ii) D74E, D74Q, or D74N, preferably D74E or D74Q, and/or iii) C2S-C141S and C47S-C92S, At least one mutation consisting of C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

In one embodiment, IL-7m has a W142H substitution and i) D74E, D74Q, or D74N, preferably D74E or D74Q, and/or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or at least one mutation consisting of C47S-C92S and C34S-C129S.

In one embodiment, IL-7m has a D74E substitution and i) W142H, W142F, or W142Y, and/or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S and at least one mutation consisting of -C129S.

In one embodiment, IL-7m consists of at least one of the mutations C2S-C141S and C47S-C92S and i) W142H, W142F, or W142Y, and/or ii) D74E, D74Q, or D74N, preferably D74E or D74Q. including one permutation.

In one embodiment, IL-7m comprises i) D74E and W142H substitutions and ii) mutations C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

The IL-7m protein may contain or lack its peptide signal. Variants of IL-7 may also include altered polypeptide sequences of IL-7 (eg, oxidized, reduced, deaminated, or truncated forms).

In one aspect, the IL-7 variant or mutant used in the present invention is recombinant IL-7. The term "recombinant" as used herein means that the polypeptide is derived from a recombinant expression system, i.e. from host cell (e.g., microbial or insect or plant or mammalian) culture, or IL-7m It means obtained or derived from a transgenic plant or animal that has been genetically engineered to contain a nucleic acid molecule that encodes a polypeptide. Preferably, the recombinant IL-7 is human recombinant IL-7m (eg, human IL-7m produced in a recombinant expression system).

In one embodiment, IL-7m has the sequence shown in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Preferably, the bifunctional molecule according to the invention comprises IL-7 variants comprising or consisting of the amino acid sequences shown in SEQ ID NOs:2-15. Even more preferably, the bifunctional molecule according to the invention comprises an IL-7 variant comprising or consisting of the amino acid sequence shown in SEQ ID NO:3, 5 or 12.

In one embodiment, the present invention reduces immunogenicity compared to wild-type IL-7 protein, particularly by removing T-cell epitopes within IL-7 that may stimulate an immune response. IL-7 variants that exhibit and bifunctional molecules comprising IL-7 variants are provided. Examples of such IL-7 are described in WO2006061219.

The present invention also provides any fusion protein comprising an IL-7 variant or variant as disclosed herein, and any conjugate comprising an IL-7 variant or variant as disclosed herein. Regarding. IL-7 variants or mutants may be N-terminally or C-terminally fused. IL-7 variants or variants may be peptides, proteins (e.g. antibodies, fragments and derivatives thereof, antibody mimetics, cytokines or cytokine receptors, tumor or viral antigens, albumin or albumin binding proteins), polymers (e.g. PEG ), chemical compounds such as drugs (eg, anticancer or antiviral agents), carbohydrates, and nucleic acid molecules (eg, siRNA, shRNA, antisense, gapmers).

A non-exhaustive list of molecules that can be conjugated or fused to IL-7 variants or variants include antibodies such as anti-CD19, anti-calreticulin, anti-tumor antigens: IL-15 or IL-15R, etc. the Fc region or part thereof of an immunoglobulin, albumin, an albumin-binding polypeptide, Pro/Ala/Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), IL-7, such as polyethylene glycol (PEG), long unstructured hydrophilic sequences of amino acids (XTEN), hydroxyethyl starch (HES), albumin-binding small molecules, and combinations thereof. Domains that extend the half-life of the variant; as well as fibronectin binding peptides may be included.

Particular examples of fusion proteins or conjugates comprising IL-7 are e.g. 19046313, WO 18215937, WO 18201047, WO 18064611, WO 17216223, US Patent Application 2018319858, WO 17158436, WO 16200219, WO 05063820.

In certain aspects, the IL-7 variant or mutant may be included in a bifunctional molecule comprising a binding moiety.

Binding Moieties Bifunctional molecules according to the present invention comprise an IL-7 variant or mutant as disclosed herein and an additional or second entity comprising a binding moiety.

It is understood that the binding moieties included in the bifunctional molecule are not interleukins, in particular neither IL-7 nor IL-7R.

As used herein, the expression "binding moiety" relates to any moiety capable of binding to a target, such as peptides, polypeptides, proteins, fusion proteins and antibodies. In particular, binding moieties include antibodies or antigen-binding fragments thereof as well as antibody mimetics or antibody mimetics. Targets of binding moieties are defined in more detail herein below.

In one embodiment, the binding moiety is selected from the group consisting of antibodies or fragments thereof and antibody mimetics or antibody mimetics. Those skilled in the art of biochemistry are familiar with antibody mimetics or antibody mimetics as discussed in Gebauer and Skerra, 2009, Curr Opin Chem Biol 13(3):245-255. . Examples of antibody mimetics include: Affibodies (also called trinectins; Nygren, 2008, FEBS J 275:2668-2676; CTLDs (also called tetranectins; Innovations Pharmac. Technol. (2006), 27-30); adnectins (also called monobodies; Meth, Mol. Biol., 352 (2007), 95-109); anticalins (Drug Discovery Today (2005)). ), 10, 23-33); DARPins (ankyrin; Nat. Biotechnol. (2004), 22, 575-582); Avimer (Nat. Biotechnol. (2005), 23, 1556-1561). pp); microbodies (FEBS J, (2007), 274, 86-95); aptamers (Expert. Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains ( J. Pharmacol. Exp. Ther. (2006) 318:803-809); Affilin (Trends. Biotechnol. (2005) 23:514-522); Mol. Biol. 383(5):1058-68), alfabody (Desmet, J. et al., 2014, Nature Communications. 5:5237), fynomer (Grabulovski D. 2007, J Biol Chem. 282(5):3196-3204), and Affimer (Avacta Life Sciences, Wetherby, UK).

Thus, binding moieties are preferably immunoglobulins, scFvs, or VHHs, Fabs, single domain antibodies, and antibody mimetics, preferably affibodies, CTLDs, adnectins, anticalins, DARPins, avimers, micro It can be selected from the group consisting of antibodies or antibody fragments thereof, such as bodies, aptamers, Kunitz domains, affilins, affitins, alphabodies, finomers and affimers.

Preferably the binding moiety is an antibody or antibody fragment thereof. Even more preferably, the binding portion is a human, humanized, or chimeric antibody, or antigen-binding fragment thereof.

Targets of Binding Moieties According to the present invention, binding moieties specifically bind to targets expressed on the surface of immune cells, in particular to targets expressed only or specifically on immune cells. In particular, the binding moieties are not directed against targets expressed on tumor cells.

With respect to the "binding capacity" of a binding moiety, the terms "bind" or "binding" refer to peptides, polypeptides, proteins, fusion proteins that recognize and contact another peptide, polypeptide, protein, or molecule. , molecules, and antibodies (including antibody fragments and antibody mimetics). In one embodiment, the term refers to an antigen-antibody type interaction. The terms "specific binding,""specificallybinds,""specificfor,""selectivelybinds," and "selective for," with respect to a particular target, mean that the binding moiety is It means that it recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in the sample. For example, an antibody that specifically (or preferentially) binds to an antigen may bind to this antigen, e.g., with higher affinity, avidity, more readily, and/or It is an antibody that binds for a longer period of time. Preferably, the term "specific binding" refers to contact between antibody and antigen with a binding affinity equal to or lower than ^10-7M . In certain embodiments, the antibody binds with an affinity equal to or less than 10 ⁻⁸ M, 10 ⁻⁹ M, or 10 ⁻¹⁰ M.

As used herein, the term "target" refers to carbohydrates, lipids, peptides, polypeptides, expressed on the outer surface of immune cells, specifically recognized or targeted by a binding moiety according to the invention. Refers to protein, antigen, or epitope. With respect to expression of a target on the surface of an immune cell, the term "expressed" refers to a target such as a carbohydrate, lipid, peptide, polypeptide, protein, antigen, or epitope that is present or presented on the outer surface of the cell. point to The term "specifically expressed" means that the target is expressed on immune cells but not substantially expressed on other cell types, such as tumor cells.

In one embodiment, the target is specifically expressed by immune cells of a healthy subject or a subject suffering from disease, particularly cancer. This may indicate that the target exhibits a higher level of expression in immune cells than in other cells, or that the ratio of immune cells expressing the target to all immune cells is Means higher than the ratio for other cells. Preferably, the expression level or ratio is 2, 5, 10, 20, 50 or 100 fold higher. The expression level or ratio may be more specifically for certain types of immune cells, such as T cells, more specifically CD8+ T cells, effector T cells, or exhausted T cells, or in certain circumstances, e.g. , can be determined for a subject suffering from a disease such as cancer or an infectious disease.

"Immune cells" as used herein are defined, for example, as white blood cells (white blood cells) derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) Cells involved in innate and adaptive immunity, such as cells, and natural killer T cells (NKT)), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells) point to In particular, immune cells are selected in a non-exhaustive list including B cells, T cells, especially CD4 ⁺ T cells and CD8 ⁺ T cells, NK cells, NKT cells, APC cells, macrophages, dendritic cells and monocytes. be able to.

Preferably, the binding moiety specifically binds to target-expressing immune cells selected from the group consisting of B cells, T cells, natural killers, dendritic cells, monocytes, and innate lymphocytes (ILCs).

Even more preferably, the immune cells are T cells. "T cells" or "T lymphocytes" as used herein include, for example, CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T regulatory cells (T regulator), T helper type 17 T cells, and inhibitory T cells. In a very specific embodiment, the immune cells are exhausted T cells.

The target may be a receptor expressed on the surface of immune cells, particularly T cells. The receptor may be an inhibitor receptor. Alternatively, the receptor may be an activating receptor.

In one aspect, the target is PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3 , CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8. Such targets are described in more detail in Table D below.

Thus, in this embodiment the binding moiety is PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA to a target selected from the group consisting of -1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8 Binds specifically.

In certain embodiments, the immune cell is an exhausted T cell and the target of the binding moiety is an exhaustion factor expressed on the surface of exhausted T cells. T cell exhaustion is a condition in which T cells progressively lose function, proliferative capacity, and cytotoxic capacity, ultimately leading to their elimination. T cell exhaustion can be triggered by several factors such as persistent antigen exposure or inhibitory receptors such as PD-1, TIM3, CD244, CTLA-4, LAG-3, BTLA, TIGIT, and CD160. . Preferably, such exhaustive factors are selected from the group consisting of PD-1, TIM3, CD244, CTLA-4, LAG-3, BTLA, TIGIT and CD160.

In preferred embodiments, the binding moiety has antagonist activity against the target.

Numerous antibodies against PD-1, TIM3, CD244, CTLA-4, LAG-3, BTLA, TIGIT, and CD160 have already been described in the art.

Some anti-PD-1 have already been clinically approved and others are still in clinical development. For example, anti-PD1 antibodies can be selected from the group consisting of: Pembrolizumab (Keytrudalambrolizumab, also known as MK-3475), Nivolumab (Opdivo, MDX-1106, BMS-936558 , ONO-4538), pidilizumab (CT-011), cemiplimab (Libtayo), camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (Tisleizumab), PDR001 (spartalizumab), MK-3477 , SCH-900475, PF-06801591, JNJ-63723283, genolimzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103 (HX-008), MEDI- 0680 (also known as AMP-514), MEDI0608, JS001 (see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI-1110 (see WO 2014/194302), AGEN2034 (see WO 2017/040790), MGA012 (see WO 2017/19846), or IBI308 (see WO 2017/024465, International See Publication 2017/025016, WO 2017/132825, and WO 2017/133540), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3 described in WO 2006/121168. , and 5F4. Target PD-1, such as RG7769 (Roche), XmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda), and XmAb23104 (Xencor) Bifunctional or bispecific molecules are also known.

In certain embodiments, the anti-PD1 antibody is pembrolizumab (Keytruda lambrolizumab, also known as MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538). good.

Antibodies against TIM3 and bifunctional or bispecific molecules targeting TIM3, such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367, RG7769 (Roche) are known. . In some embodiments, the TFM-3 antibody is WO2013006490, WO2016/161270, WO2018/085469, or WO2018/129553, WO2011/155607 , U.S. Patent No. 8,552,156, EP 2581113, and U.S. Patent Application No. 2014/044728.

Antibodies against CTLA-4 and bifunctional or bispecific molecules targeting CTLA-4, such as ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), MEDI5752 (AstraZeneca) is known. Anti-CTLA-4 antibodies are disclosed in WO 18025178, WO 19179388, WO 19179391, WO 19174603, WO 19148444, WO 19120232, WO 19056281, Publication No. 19023482, WO 18209701, WO 18165895, WO 18160536, WO 18156250, WO 18106862, WO 18106864, WO 18068182, WO 18035710, WO 18025178, WO 17194265, WO 17106372, WO 17084078, WO 17087588, WO 16196237, WO 16130898, WO 16015675 , WO12120125, WO09100140 and WO07008463.

Also, antibodies to LAG-3 and bifunctional or bispecific antibodies targeting LAG-3 such as BMS-986016, IMP701, MGD012, or MGD013 (bispecific PD-1 and LAG-3 antibodies) molecules are known. Anti-LAG-3 antibodies are also disclosed in WO2008132601, EP2320940, WO19152574.

Antibodies against BTLA are also known in the art, such as hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mab19A7, or hu Mab4C7. Antibody TAB004 against BTLA is currently in clinical trials in subjects with advanced malignancies. Anti-BTLA antibodies include WO08076560, WO10106051 (e.g., BTLA8.2), WO11014438 (e.g., 4C7), WO17096017, and WO17144668 (e.g., 629.3).

BMS-986207 or AB154, BMS-986207 CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057 as disclosed in WO19232484 , CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA .9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5, CHA.9.560.6, CHA.9.560 .7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4, CHA.9.547.6, CHA.9.547.7 , CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA Antibodies to TIGIT are also known, such as .9.541.7 and CHA.9.541.8. The anti-TIGIT antibody is WO 16028656, WO 16106302, WO 16191643, WO 17030823, WO 17037707, WO 17053748, WO 17152088, WO 18033798, WO 18102536, WO 18102746, WO 18160704, WO 18200430, WO 18204363, WO 19023504, WO 19062832, WO 19129221 , WO19129261, WO19137548, WO19152574, WO19154415, WO19168382, WO19215728.

CL1-R2 CNCM I-3204 as disclosed in WO06015886 or other as disclosed in WO10006071, WO10084158, WO18077926. Antibodies to CD160 are also known, such as

In a preferred embodiment, the binding portion of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for PD-1, CTLA-4, BTLA, TIGIT, LAG3 and TIM3.

In another specific embodiment, the target is PD-1 and the binding moiety of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for PD-1. Thus, in certain embodiments, a binding portion comprised in a bifunctional molecule according to the invention is an anti-PD1 antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-PD1 antibody or antigen-binding antibody thereof. Fragments. Preferably, the binding moiety is an antagonist of PD-1. Bifunctional molecules, therefore, combine the effects of IL-7 variants or mutants on the IL-7 receptor and block the inhibitory effects of PD-1, and are useful in T cells, particularly exhausted T cells, and more. Specifically, synergistic effects can be demonstrated on the activation of TCR signaling.

In another specific embodiment, the target is CTLA-4 and the binding portion of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for CTLA-4. Thus, in certain embodiments, a binding moiety comprised in a bifunctional molecule according to the invention is an anti-CTLA-4 antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-CTLA-4 antibody or It is an antigen-binding fragment thereof. Preferably, the binding moiety is an antagonist of CTLA-4. Thus, bifunctional molecules combine the effects of IL-7 variants or mutants on the IL-7 receptor with blocking of the inhibitory effect of CTLA-4, and are useful in T cells, particularly exhausted T cells, and more. Specifically, synergistic effects can be demonstrated on the activation of TCR signaling.

In another specific embodiment, the target is BTLA and the binding portion of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for BTLA. Thus, in certain embodiments, a binding portion comprised in a bifunctional molecule according to the invention is an anti-BTLA antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-BTLA antibody or antigen-binding antibody thereof. Fragments. Preferably, the binding moiety is an antagonist of BTLA. Thus, bifunctional molecules combine the effects of IL-7 variants or mutants on the IL-7 receptor with blocking of the inhibitory effect of BTLA to target T cells, particularly exhausted T cells, more particularly can exhibit synergistic effects on the activation of TCR signaling.

In another specific embodiment, the target is TIGIT and the binding portion of the bifunctional molecule is a TIGIT-specific antibody, fragment or derivative thereof, or antibody mimetic. Thus, in certain embodiments, a binding portion comprised in a bifunctional molecule according to the invention is an anti-TIGIT antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-TIGIT antibody or antigen-binding antibody thereof. Fragments. Preferably, the binding moiety is an antagonist of TIGIT. Thus, bifunctional molecules combine the effects of IL-7 variants or mutants on the IL-7 receptor with blocking of the inhibitory effects of TIGIT to target T cells, particularly exhausted T cells, more particularly can exhibit synergistic effects on the activation of TCR signaling.

In another specific embodiment, the target is LAG-3 and the binding portion of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for LAG-3. Thus, in certain embodiments, a binding moiety comprised in a bifunctional molecule according to the invention is an anti-LAG-3 antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-LAG-3 antibody or It is an antigen-binding fragment thereof. Preferably, the binding moiety is an antagonist of LAG-3. Thus, bifunctional molecules combine the effects of IL-7 variants or mutants on the IL-7 receptor with blocking of the inhibitory effects of LAG-3 and are useful in T cells, particularly exhausted T cells, and more. Specifically, synergistic effects can be demonstrated on the activation of TCR signaling.

In another specific embodiment, the target is TIM3 and the binding moiety of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for TIM3. Thus, in certain embodiments, a binding portion comprised in a bifunctional molecule according to the invention is an anti-TIM3 antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-TIM3 antibody or antigen-binding fragment thereof. Fragments. Preferably, the binding moiety is an antagonist of TIM3. Thus, bifunctional molecules combine the effects of IL-7 variants or mutants on the IL-7 receptor with blocking of the inhibitory effect of TIM3 to target T cells, particularly exhausted T cells, more particularly can exhibit synergistic effects on the activation of TCR signaling.

Fc Domains In certain aspects of the present disclosure, bifunctional molecules comprise an IL-7 variant or mutant, a binding moiety, and an Fc domain. Where the binding moiety is an antibody, particularly an IgG immunoglobulin, the Fc domain may be part of the binding moiety. However, bifunctional molecules may have other structures, including Fc domains. For example, a bifunctional molecule may comprise an Fc domain linked to an antibody derivative or diabodies such as scFv.

One approach to improving the pharmacokinetics of a bifunctional molecule according to the invention is to increase its half-life serum persistence, allowing for higher circulating levels, less frequent dosing, and reduced doses. become. This need can be met, for example, by including an Fc domain or part thereof in the bifunctional molecule according to the invention.

Thus, in one embodiment the bifunctional molecule, in particular the binding moiety, according to the invention comprises an Fc domain or part thereof.

In particular, the binding moiety according to the invention is at least part of an immunoglobulin constant region (Fc), typically of a mammalian immunoglobulin, even more preferably of a chimeric, human or humanized immunoglobulin. immunoglobulin constant region (Fc). Binding moieties may comprise immunoglobulin constant regions, or constant region fragments, analogs, variants, mutants, or derivatives. As is well known to those of skill in the art, the choice of IgG isotype for the heavy chain constant domain is of central concern as to whether a particular function is required and the need for a suitable in vivo half-life. be.

In preferred embodiments, the Fc domain or fragment thereof comprised in the binding portion comprises a heavy chain constant domain derived from a human immunoglobulin heavy chain, eg, IgG1, IgG2, IgG3, IgG4, or other classes. In a further aspect, the human constant domain is selected from the group consisting of IgG1, IgG2, IgG2, IgG3 and IgG4. Preferably, the binding moiety comprises an IgG1 heavy chain constant domain or an IgG4 heavy chain constant domain.

In one embodiment, the binding portion comprises a truncated Fc region or a fragment of that Fc region. In such Fc fragments the constant region comprises the CH2 or CH3 domain. In another embodiment, the constant region comprises a CH2 domain and a CH3 domain. Alternatively, the constant region may comprise all or part of the hinge region, CH2 domain and/or CH3 domain. In some embodiments, the constant region contains CH2 and/or CH3 domains derived from human IgG4 or IgG1 heavy chains.

Preferably, the constant region includes all or part of the hinge region. The hinge region may be derived from an immunoglobulin heavy chain, eg, IgG1, IgG2, IgG3, IgG4, or other class. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4. More preferably, the hinge region is derived from human or humanized IgG1 or IgG4 heavy chains.

The IgG1 hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of immunoglobulins. These same cysteines allow efficient and consistent disulfide bond formation between the Fc moieties. Accordingly, preferred hinge regions of the present invention are derived from IgG1, more preferably human IgG1. In some embodiments, the first cysteine within the human IgG1 hinge region is mutated to another amino acid, preferably serine.

The hinge region of IgG4 is known to be inefficient at forming interchain disulfide bonds. However, a suitable hinge region of the invention may be derived from an IgG4 hinge region and preferably contains mutations that enhance the correct formation of disulfide bonds between heavy chain derived portions (Angal S et al. (1993) Mol. Immunol., 30:105-8). More preferably, the hinge region is derived from human IgG4 heavy chain.

For bifunctional molecules that target cell surface molecules, especially those on immune cells, it is necessary to abrogate effector function. It may also be desirable to genetically engineer the Fc region to either reduce or increase the effector functions of the antibody.

In certain embodiments, amino acid modifications may be introduced into the Fc region to generate Fc region variants. In certain embodiments, Fc region variants have some, but not all, effector function. Such antibodies may be useful, for example, in applications where the half-life of the antibody in vivo is important, but certain effector functions are unnecessary or detrimental. Numerous substitutions or deletions that alter effector function are known in the art.

In one embodiment, the constant region contains mutations that reduce affinity for Fc receptors or reduce Fc effector function. For example, the constant region may contain mutations that eliminate glycosylation sites within the constant region of IgG heavy chains. Preferably, the CH2 domain contains mutations that eliminate glycosylation sites within the CH2 domain.

In certain embodiments, the Fc domain is modified to increase binding to FcRn, thereby increasing the half-life of the bifunctional molecule. In another or additional embodiment, the Fc domain is modified to decrease binding to FcγRs, thereby decreasing ADCC or CDC, or to increase binding to FcγRs, thereby increasing ADCC or CDC. It is

Amino acid alterations near the junction of the Fc and non-Fc portions can dramatically increase the serum half-life of Fc fusion proteins, as shown in WO 01/58957. Thus, the junction regions of the proteins or polypeptides of the invention may contain alterations, preferably within about 10 amino acids of the junction, compared to the naturally occurring sequences of immunoglobulin heavy chain and erythropoietin. . Such amino acid changes can lead to increased hydrophobicity. In one embodiment the constant region is derived from an IgG sequence in which the C-terminal lysine residue has been replaced. Preferably, the C-terminal lysine of the IgG sequence is replaced with a non-lysine amino acid such as alanine or leucine to further increase serum half-life.

In one embodiment, the constant region may contain CH2 and/or CH3 with one or any combination of the mutations listed in Table E below.

In certain embodiments, the bifunctional molecule, preferably the binding moiety, is optionally T250Q/M428L; M252Y/S254T/T256E+H433K/N434F; E233P/L234V/L235A/G236A+A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; N297A optionally in combination with M252Y/S254T/T256E, and a human IgG1 heavy chain constant domain or IgG1 Fc domain with a substitution or combination of substitutions selected from the group consisting of L234A/L235A.

In another aspect, the binding portion is a human IgG4 heavy chain constant domain, optionally with a substitution or combination of substitutions selected from the group consisting of S228P; L234A/L235A, S228P+M252Y/S254T/T256E, and K444A. or contains a human IgG4 Fc domain. Even more preferably, the bifunctional molecule, preferably the binding moiety, comprises an IgG4 Fc region with S228P that stabilizes IgG4.

All subclasses of human IgG have a C-terminal lysine residue (K444) of the antibody heavy chain that is susceptible to cleavage in circulation. This cleavage in the blood can impair or reduce the biological activity of the bifunctional molecule by releasing IL-7 linked to IgG. To circumvent this problem, the K444 amino acid of the IgG domain can be replaced with alanine to reduce proteolytic cleavage. This is a commonly used mutation in antibodies. Accordingly, in one embodiment, when the binding moiety is an antibody, the antibody comprises at least one additional amino acid substitution consisting of K444A.

In one embodiment, when the binding moiety is an antibody, the antibody has an additional Contains cysteine residues.

In one embodiment the binding moiety comprises an antibody. In such embodiments, such antibodies comprise the heavy chain constant domain of SEQ ID NO:39 or 52 and/or the light chain constant domain of SEQ ID NO:40, particularly the heavy chain constant domain of SEQ ID NO:39 or 52 and SEQ ID NO:40. light chain constant domains, such as those disclosed in Table F below.

In a preferred embodiment, the binding moiety comprises the heavy chain constant domain of SEQ ID NO:52 and/or the light chain constant domain of SEQ ID NO:40, in particular the heavy chain constant domain of SEQ ID NO:52 and the light chain constant domain of SEQ ID NO:40. including anti-hPD-1 antibodies with

Peptide Linker In certain embodiments, the bifunctional molecule according to the invention further comprises a peptide linker connecting the binding moiety and IL-7m. Peptide linkers are typically of sufficient length and length to ensure that IL-7m and the binding moieties between which the linker connects have sufficient spatial freedom to perform their function. Flexible.

In aspects of the disclosure, the binding moiety is preferably linked to IL-7 by a peptide linker. As used herein, the term "linker" refers to a sequence of at least one amino acid that connects IL-7m and a binding moiety. Such linkers may be useful in preventing steric hindrance. Linkers are usually 3 to 44 amino acid residues in length. Preferably, the linker has 3-30 amino acid residues. In some embodiments, the linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, It has 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues.

A linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to whom the bifunctional molecule is administered. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies, as described in WO96/34103 and WO94/04678. Another example is a polyalanine linker sequence. _Further preferred examples of _{linker sequences are :} Gly/Ser linker. Preferably, the linker is selected from the group consisting of (Gly4Ser) ₄ , (Gly4Ser) ₃ and (Gly3Ser2) ₃ . Even more preferably, the linker is (GGGGS) ₃ .

In one embodiment, the linker included in the bifunctional molecule is selected in the group consisting of (Gly4Ser) ₄ , (Gly4Ser) ₃ , (Gly4Ser) ₂ , Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2) ₃ ; Preferably, it is (Gly4Ser) ₃ . Preferably, the linker is selected from the group consisting of (Gly4Ser) ₄ , (Gly4Ser) ₃ and (Gly3Ser2) ₃ .

Bifunctional Molecules The present invention provides, inter alia, bifunctional molecules comprising IL-7m, a binding moiety, optionally an Fc fragment and optionally a peptide linker as described herein above. .

In particular, the bifunctional molecule comprises or consists of a binding moiety as disclosed herein above and IL-7m, wherein the binding moiety preferably comprises a binding moiety as disclosed herein above. It is covalently conjugated (eg, by gene fusion or chemical coupling) to IL-7 via a peptide linker.

In particular, the conjugation of IL-7m with the binding moiety is direct or indirect (ie via a linker) covalent bond and/or chemical, enzymatic or genetic. Conjugation can be performed by any acceptable means of coupling known in the art, taking into account the chemical nature of the binding moieties. Thus, in this regard, coupling is by one or more covalent, ionic, hydrogen, hydrophobic, or van der Waals bonds that are cleavable or uncleavable in a physiological medium or within a cell. can be implemented.

In particular, chemical conjugation involves attaching an exposed sulfhydryl group (Cys), an affinity tag (e.g., 6-histidine, Flag-tag, Strep-tag, SpyCatcher, etc.) to either a binding moiety or IL7-m, or clicking Incorporation of unnatural amino acids or compounds for chemical conjugation can be performed.

In a preferred embodiment, conjugation is obtained by gene fusion (ie by expressing in a suitable system a nucleic acid construct as a gene fusion encoding the binding moiety and IL-7).

In one aspect, the invention features a fusion protein comprising a first portion comprising an immunoglobulin (Ig) chain, particularly an Fc domain, and a second portion comprising interleukin-7 (IL-7).

In one embodiment, the invention relates to bifunctional molecules comprising a binding moiety fused to IL-7m. In particular in such fusion molecules the binding moiety is an antibody and the C-terminus of an antibody chain, such as a light or heavy chain, preferably a heavy chain, even more preferably a heavy or light chain, comprises IL-7m to, preferably to the N-terminus of IL-7m, optionally via a peptide linker.

In a particular aspect, the invention provides a bifunctional molecule comprising an antibody or antigen-binding fragment thereof and IL-7m, wherein IL-7m is preferably linked to the C-terminus of the heavy chain of said antibody ( For example, it relates to a bifunctional molecule linked to the (C-terminal end of the heavy chain constant domain).

Preferably, the heavy chain of the antibody, preferably the C-terminus of the heavy chain, is genetically fused to the N-terminus of IL-7m via a flexible (Gly ₄ Ser) ₃ linker. At the fusion junction, the C-terminal lysine residue of the antibody heavy chain can be mutated to alanine (ie, K444A) to reduce proteolytic cleavage.

In one embodiment, a bifunctional molecule according to the invention comprises one or more IL-7m molecules. Preferably, a bifunctional molecule according to the invention may comprise 1, 2, 3 or 4 IL-7m molecules. In particular, the bifunctional molecule may contain only one molecule of IL-7 linked to only one light or heavy chain of the antibody. Preferably, the bifunctional molecule may comprise only one IL-7m molecule, preferably linked to only one heavy chain of the antibody, more preferably linked to the C-terminus of the Fc domain of the antibody. . A bifunctional molecule may also comprise two IL-7m molecules linked to either the light or heavy chain of an antibody. The bifunctional molecule may also comprise two IL-7m molecules, the first molecule linked to the light chain of the antibody and the second molecule linked to the heavy chain of the antibody. .

In one embodiment, the bifunctional molecule according to the invention is
(a) a binding moiety that specifically binds to a target expressed on the surface of immune cells, conjugated thereto, such as those described herein above;
(b) IL-7m exhibiting at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein such The IL-7 variant i) reduces the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R, and ii) IL-7m containing at least one amino acid mutation that enhances the pharmacokinetics of a bifunctional molecule comprising an IL-7 variant compared to a bifunctional molecule comprising wth-IL-7
comprising or consisting of

In particular, the at least one amino acid mutation is as described herein above in the "IL-7 Variants" paragraph.

Preferably, the bifunctional molecule according to the invention is
(a) a binding moiety that specifically binds to a target expressed on the surface of immune cells, conjugated thereto, such as those described herein above;
(b) IL-7m exhibiting at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein such IL-7 variants are (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F, or W142Y, (iii) D74E, D74Q or D74N, preferably D74E or D74Q, iv) at least one mutation selected from the group consisting of Q11E, Y12F, M17L, Q22E, and/or K81R, or any combination thereof, IL-7m
comprising or consisting of

More preferably, such variants i) reduce the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R and ii) improve the pharmacokinetics of bifunctional molecules comprising IL-7 variants compared to bifunctional molecules comprising wth-IL-7. More preferably, such variants i) reduce the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R ii) retain the ability to activate IL-7R, and iii) enhance the pharmacokinetics of bifunctional molecules comprising IL-7 variants compared to bifunctional molecules comprising wth-IL-7. Let

In certain embodiments, the immune cell surface expressed target is a T cell surface expressed exhaustion factor.

Preferably the binding moiety is an antibody or antibody fragment thereof.

Preferably, the binding moiety is conjugated to IL-7m by gene fusion and the bifunctional molecule optionally comprises at least one molecule connecting the N-terminus of IL-7m to the C-terminus of the heavy chain of the antibody. comprising one peptide linker, wherein the peptide linker is preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more Preferably, it is (GGGGS)3.

Preferably, the bifunctional molecule according to the invention is
(a) an antibody or antibody fragment thereof, such as those described herein above, that specifically binds to a target expressed on immune cell surfaces, preferably on T cells;
(b) IL-7m exhibiting at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein such IL-7 variants have amino acid substitutions (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F, or W142Y, (iii) D74E , D74Q, or D74N, preferably D74E or D74Q, iv) Q11E, Y12F, M17L, Q22E, and/or K81R, or any combination thereof, and
(c) optionally selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, preferably (GGGGS)3 A fusion protein comprising or consisting of a peptide linker.

Preferably, the antibody is PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, consisting of CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8, preferably PD-1, TIM3, CD244, LAG -3, BTLA, TIGIT, and an antibody against a target selected from the group consisting of CD160.

Preferably, the antibody or antibody fragment thereof has an IgG1 Fc domain or an IgG4 Fc domain.

In one aspect, the antibody or antibody fragment thereof is optionally K444A, T250Q/M428L; M252Y/S254T/T256E+H433K/N434F; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A+M252Y/S254T/T256E; N297A in combination with T256E, and an IgG1 Fc domain with a substitution or combination of substitutions selected from the group consisting of L234A/L235, even more preferably IgG1 with mutation N297A, such as those described herein above. It has an Fc domain.

Surprisingly, we found that bifunctional molecules with an IgG1 heavy chain constant domain reduced IL-7 variant activity (pStat5 signal, synergistic effect) compared to the same molecule with an IgG4 heavy chain constant domain. , and CD127 binding). This enhancement is IL-7 variant specific and has not been observed with wild-type IL-7. In addition, using long linkers such as (GGGGS) ₃ between the antibody and IL-7 maximizes the activity of IL-7 variants (pStat5 signaling and CD127 binding).

Thus, more particularly, the present invention relates to bifunctional molecules, antibodies or antibody fragments thereof, such as those described hereinabove, targeting antibodies expressed on the surface of immune cells, preferably on T cells. and more preferably the target is PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1 , LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8, preferably PD -1, TIM3, CD244, LAG-3, BTLA, TIGIT, and CD160, wherein the antibody or antibody fragment thereof is optionally T250Q/M428L; L234V/L235A/G236A+A327G/A330S/P331S;E333A;S239D/A330L/I332E;P257I/Q311;K326W/E333S;S239D/I332E/G236A;N297A;L234A/L235A;N297A+M252Y/S254T/T256E;K322A; and K444A, preferably N297A optionally in combination with M252Y/S254T/T256E, and an IgG1 Fc domain having a substitution or combination of substitutions selected from the group consisting of L234A/L235, and even more Preferably, it has an IgG1 Fc domain with mutation N297A, such as those described above. Preferably, the antibody or fragment thereof is linked to IL-7 or a variant thereof by a linker selected from the group consisting of (GGGGS) ₃ , (GGGGS) ₄ and (GGGS) ₃ , more preferably by (GGGGS) ₃ . Concatenated. Preferably, the IL-7 variant is selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q and D74N containing groups of amino acid substitutions that are More preferably, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N. include. Even more preferably, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, and D74E.

In another aspect, the antibody or antibody fragment thereof comprises an IgG4 Fc domain, optionally with a substitution or combination of substitutions selected from the group consisting of K444A, S228P; L234A/L235A, S228P+M252Y/S254T/T256E, Preferably, it has an IgG4 Fc domain with mutation S228P, such as those described above.

In a particular embodiment, the bifunctional molecule according to the invention is
(a) an antibody or antibody fragment thereof, such as those described herein above, that specifically binds to a target expressed on the surface of an immune cell, preferably on a T cell, more preferably a target PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, the group consisting of NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8, preferably PD-1, TIM3, CD244, LAG-3, an antibody or antibody fragment thereof selected from the group consisting of BTLA, TIGIT, and CD160;
(b) IL-7m exhibiting at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein such IL-7 variants have amino acid substitutions (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F, or W142Y, (iii) D74E , D74Q, or D74N, preferably D74E or D74Q, iv) Q11E, Y12F, M17L, Q22E, and/or K81R, or any combination thereof, and
(c) optionally a peptide selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGS, GGG, GGS and (GGGS)3, preferably (GGGGS)3 A fusion protein comprising or consisting of a linker.

In a preferred embodiment of this aspect, the antibody or antibody fragment thereof is optionally T250Q/M428L; M252Y/S254T/T256E+H433K/N434F; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; IgG1 Fc domain with a substitution or combination of substitutions selected from the group consisting of N297A in combination with M252Y/S254T/T256E, and L234A/L235, even more preferably IgG1 with mutation N297A, such as those described above. It has an Fc domain.

Alternatively, the bifunctional molecule according to the invention is
(a) an antibody or antibody fragment thereof, such as those described herein above, that specifically binds to a target expressed on the surface of an immune cell, preferably on a T cell, more preferably the target PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, the group consisting of NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8, preferably PD-1, TIM3, CD244, LAG-3, an antibody or antibody fragment thereof selected from the group consisting of BTLA, TIGIT, and CD160;
(b) IL-7m exhibiting at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein such IL-7 variants comprise the amino acid substitution W142H, W142F, or W142Y, preferably W142H, IL-7m, and
(c) optionally selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, preferably (GGGGS)3 A fusion protein comprising or consisting of a peptide linker.

Preferably, the antibody or antibody fragment thereof has an IgG1 Fc domain or an IgG4 Fc domain, optionally with substitutions as detailed above.

In a preferred embodiment of this aspect, the antibody or antibody fragment thereof is optionally T250Q/M428L; M252Y/S254T/T256E+H433K/N434F; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; IgG1 Fc domain with a substitution or combination of substitutions selected from the group consisting of N297A in combination with M252Y/S254T/T256E, and L234A/L235, even more preferably IgG1 with mutation N297A, such as those described above. It has an Fc domain.

Alternatively, the bifunctional molecule according to the invention is
(a) an antibody or antibody fragment thereof, such as those described herein above, that specifically binds to a target expressed on the surface of an immune cell, preferably on a T cell, more preferably the target is , PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D , LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8, preferably PD-1, TIM3, CD244, LAG-3, BTLA an antibody or antibody fragment thereof selected from the group consisting of: , TIGIT, and CD160;
(b) IL-7m exhibiting at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein such IL-7 variants comprise the amino acid substitutions D74E, D74Q, or D74N, preferably D74E, IL-7m, and
(c) optionally selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, preferably (GGGGS)3 It comprises or consists of a peptide linker.

Preferably, the antibody or antibody fragment thereof has an IgG1 Fc domain or an IgG4 Fc domain, optionally with substitutions as detailed above.

In a preferred embodiment of this aspect, the antibody or antibody fragment thereof is optionally T250Q/M428L; M252Y/S254T/T256E+H433K/N434F; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; IgG1 Fc domain with a substitution or combination of substitutions selected from the group consisting of N297A in combination with M252Y/S254T/T256E, and L234A/L235, even more preferably IgG1 with mutation N297A, such as those described above. It has an Fc domain.

Alternatively, the bifunctional molecule according to the invention is
(a) an anti-PD1 antibody or antibody fragment thereof that specifically binds to PD-1;
(b) IL-7m exhibiting at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein such IL-7 variants include the amino acid substitutions D74E, W142H, and/or C2S-C141S+C47S-C92S, IL-7m, and
(c) optionally selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, preferably (GGGGS)3 It comprises or consists of a peptide linker.

Preferably, the antibody or antibody fragment thereof has an IgG1 Fc domain or an IgG4 Fc domain, optionally with substitutions as detailed above.

Preferably, the C-terminus of the heavy chain of the antibody is genetically fused to the N-terminus of IL-7m via a flexible linker, preferably (Gly ₄ Ser) ₃ . At the fusion junction, the C-terminal lysine residue (ie, K444) of the antibody heavy chain can be mutated to alanine to reduce proteolytic cleavage.

Optionally, the bifunctional molecule may further comprise additional moieties such as other cytokines or other binding moieties.

In certain aspects, the molecule has a dimeric Fc domain linked to a single IL-7 variant and a single antigen-binding domain. In another specific embodiment, the molecule has a dimeric Fc domain in which a single IL-7 variant and two antigen binding domains are linked. An antigen-binding domain binds any target specifically expressed on the surface of an immune cell, as disclosed herein. More specifically, the targets are PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1 , TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8, more particularly It can be selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, LAG3 and TIM3. In a very particular embodiment, the antigen binding domain binds PD-1.

In certain embodiments, the molecule comprises a first monomer and a second monomer, wherein the first monomer is covalently attached to the first Fc chain, optionally via a peptide linker. The first Fc chain is covalently linked to the IL-7 variant, optionally via a peptide linker, and the second monomer is preferably antigen-binding. a complementary second Fc chain that lacks the sex domain and/or IL-7 variant, wherein the first and second Fc chains form a dimeric Fc domain. Optionally, the dimeric Fc domain is a heterodimeric Fc domain. More particularly, the molecule comprises a first monomer and a second monomer, the first monomer optionally attached to the N-terminus of the first heterodimeric Fc chain. comprising an antigen-binding domain covalently linked via a linker, wherein the first heterodimeric Fc chain is covalently linked at its C-terminus to an IL-7 variant, optionally via a peptide linker and the second monomer comprises a complementary second heterodimeric Fc chain that lacks the antigen-binding domain. Optionally, the second monomer comprises a complementary second heterodimeric Fc chain lacking the IL-7 variant, preferably lacking any other molecule. Optionally, the second monomer is a complementary second monomer optionally covalently linked via a peptide linker to the IL-7 variant at the N-terminus of the C-terminus of the Fc chain. Contains a heterodimeric Fc chain. Even more particularly, the molecule comprises a first monomer and a second monomer, wherein the first monomer is C-terminally attached to the N-terminus of the first heterodimeric Fc chain. The first heterodimeric Fc chain comprises an antigen-binding domain optionally covalently linked via a peptide linker, wherein the first heterodimeric Fc chain is C-terminal to the N-terminus of the IL-7 variant, optionally through the peptide linker. and the second monomer lacks the antigen-binding domain and the IL-7 variant, preferably lacks any other molecule, a complementary second heterodimeric Fc Including chains. Such a molecule is shown, for example, as "construct 3" in FIG.

In another particular embodiment, the molecule comprises a first monomer and a second monomer, wherein the first monomer is C-terminally N-terminally of the first heterodimeric Fc chain wherein the first heterodimeric Fc chain is C-terminally to the N-terminus of the IL-7 variant, optionally with the peptide linker and the second monomer lacks an antigen-binding domain and is optionally linked to an IL-7 variant at the N-terminus of the C-terminus of the Fc chain, optionally via a peptide linker. a complementary second heterodimeric Fc chain covalently linked together. Such a molecule is shown, for example, as "construct 4" in FIG.

Optionally, a complementary second heterodimeric Fc chain is covalently linked at its C-terminus to the N-terminus of the IL-7 variant, optionally via a peptide linker.

In additional embodiments, the molecule comprises a first monomer and a second monomer, wherein the first monomer is covalently linked to the first Fc chain, optionally via a peptide linker. comprising linked antigen binding domains, wherein the first Fc chain optionally lacks the IL-7 variant and the second monomer comprises a complementary second Fc chain lacking the antigen binding domain wherein the second Fc chain is covalently linked to the IL-7 variant, optionally via a peptide linker, the first and second Fc chains forming a dimeric Fc domain. Optionally, the dimeric Fc domain is a heterodimeric Fc domain. More particularly, the molecule comprises a first monomer and a second monomer, the first monomer optionally attached to the N-terminus of the first heterodimeric Fc chain. comprising an antigen-binding domain covalently linked via a linker, wherein the first heterodimeric Fc chain lacks the IL-7 variant and the second monomer lacks the antigen-binding domain comprising a complementary second heterodimeric Fc chain, the second heterodimeric Fc chain covalently linked at its C-terminus to the IL-7 variant, optionally via a peptide linker. Even more particularly, the molecule comprises a first monomer and a second monomer, wherein the first monomer is C-terminally attached to the N-terminus of the first heterodimeric Fc chain. an antigen-binding domain optionally covalently linked via a peptide linker, wherein the first heterodimeric Fc chain lacks the IL-7 variant and the second monomer is antigen-binding a complementary second heterodimeric Fc chain lacking a sex domain, the second heterodimeric Fc chain sharing the C-terminus to the N-terminus of the IL-7 variant, optionally via a peptide linker connected by a bond.

In another particular aspect, the molecule comprises a first monomer and a second monomer, the first monomer shared to the first Fc chain, optionally via a peptide linker. A first Fc chain is covalently linked to an IL-7 variant, optionally via a peptide linker, and a second monomer comprises an antigen-binding domain linked by a bond, and a second monomer is an IL- It lacks the 7 variant and comprises a complementary second Fc chain linked to an antigen binding domain, the first and second Fc chains forming a dimeric Fc domain. Such a molecule is shown, for example, in Figure 17 as "construct 2". Optionally, the dimeric Fc domain is a heterodimeric Fc domain. More particularly, the molecule comprises a first monomer and a second monomer, the first monomer optionally attached to the N-terminus of the first heterodimeric Fc chain. comprising an antigen-binding domain covalently linked via a linker, wherein the first heterodimeric Fc chain is covalently linked at its C-terminus to an IL-7 variant, optionally via a peptide linker wherein the second monomer comprises a complementary second heterodimeric Fc chain lacking the IL-7 variant, and optionally a peptide linker at the N-terminus of the second heterodimeric Fc chain comprising an antigen-binding domain covalently linked via More specifically, the molecule comprises a first monomer and a second monomer, the first monomer optionally having its C-terminus to the N-terminus of the first heterodimeric Fc chain. comprising an antigen-binding domain optionally covalently linked via a peptide linker, wherein the first heterodimeric Fc chain is C-terminally to the N-terminus of the IL-7 variant, optionally via a peptide linker wherein the second monomer comprises a complementary second heterodimeric Fc chain lacking the IL-7 variant, and the C-terminus of the second heterodimeric Fc chain is optionally covalently linked via a peptide linker to the N-terminus of the antigen-binding domain.

Linkers, if present, can be selected from among the linkers disclosed herein.

Preferably, the two monomers each comprise one Fc chain, and the Fc chains can form a dimeric Fc domain.

In one aspect, the dimeric Fc-fusion protein is a homodimeric Fc-fusion protein. In another aspect, the dimeric Fc-fusion protein is a heterodimeric Fc-fusion protein.

More specifically, the Fc domain is a heterodimeric Fc domain. Heterodimeric Fc domains are constructed by altering the amino acid sequence of each monomer. Heterodimeric Fc domains differ on each chain by amino acid variants in the constant region such that heterodimer formation is facilitated and/or purification of the heterodimer is easier than homodimers. It depends. There are a number of mechanisms that can be used to generate the heterodimers of the invention. Additionally, as one skilled in the art will appreciate, such mechanisms can be combined to ensure a high degree of heterodimerization. Amino acid variants that lead to the production of heterodimers are therefore termed "heterodimerization variants". Heterodimerization variants include steric variants (e.g. "knobs and holes" or "skew" variants as described below, and "charge pair" variants as described below). , as well as the "pi variant", which allows the homodimer to be separated and purified from the heterodimer. WO 2014/145806, which is incorporated herein by reference in its entirety, describes a "knob and hole" variant, an "electrostatic steering" variant, or a "charge pair" variant, a pie variant, and Useful mechanisms for heterodimerization have been disclosed, including additional common Fc variants. Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997, 270:26; U.S. Patent No. 8,216,805, Merchant et al., Nature Biotech. Vol.: 677 (1998). All of these documents are incorporated herein by reference in their entirety. For "electrostatic steering" see Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010). This document is incorporated herein by reference in its entirety. See US Patent Application Publication No. 2012/0149876 for pyrvariants. This document is incorporated herein by reference in its entirety.

Thus, in a preferred embodiment, the heterodimeric Fc domain comprises a first Fc chain and a complementary second Fc chain, based on the "knob and hole" technology. For example, the first Fc chain is the "knob" or K chain, meaning that the first Fc chain contains substitutions that characterize the knob chain, and the second Fc chain is the "whole" or H chain, meaning that the second Fc chain contains substitutions that characterize the whole chain. Vice versa, the first Fc chain is the "whole" or H chain, meaning that the first Fc chain contains the substitutions that characterize the whole chain, and the second Fc chain is the " The "knob chain" or K chain, which means that the second Fc chain contains the substitutions that characterize the knob chain. In a preferred embodiment, the first Fc chain is the "whole" or H chain and the second Fc chain is the "knob" or K chain.

Examples of bifunctional molecular structures according to the invention are provided in FIG.

Optionally, the heterodimeric Fc domains comprise one heterodimeric Fc chain containing substitutions as shown in the table below and the other heterodimeric Fc chain containing substitutions as shown in the table below. A heterodimeric Fc chain may be included.

In a preferred embodiment, the first Fc chain is the "whole" or H chain, comprising the substitutions T366S/L368A/Y407V/Y349C, and the second Fc chain is the "knob" or K chain, containing the substitutions T366W/ Including S354C.

Optionally, the Fc chain may further comprise additional substitutions.

In one aspect, bifunctional molecules according to the invention comprise heterodimers of Fc domains comprising "knob into holes" modifications, such as those described above. Preferably such Fc domain is an IgG1 Fc domain or an IgG4 Fc domain such as those disclosed above, even more preferably an IgG1 Fc domain comprising the mutation N297A such as those disclosed above.

For example, the first Fc chain is the "whole" or H chain, containing the substitutions T366S/L368A/Y407V/Y349C and N297A, and the second Fc chain is the "knob" or K chain, containing the substitutions T366W/ Including S354C and N297A. More particularly, the second Fc chain may comprise or consist of SEQ ID NO:75 and/or the first Fc chain may comprise or consist of SEQ ID NO:77.

More specifically, the IL7 variants according to the invention are linked to the knob and/or whole chains of heterodimeric Fc domains. Thus, a bifunctional molecule according to the invention comprises i) a single IL7 variant linked to either the whole strand or the knob strand of the Fc domain, or ii) one linked to the whole strand of the Fc domain. and one may contain two IL7 variants linked to the knob chain. Preferably, the bifunctional molecule according to the invention comprises a single IL7 variant linked to the whole strand of the Fc domain.

In a first aspect, the bifunctional molecule comprises an IL7 variant linked to the C-terminus or N-terminus of the knob chain Fc domain. Optionally, such Fc domains are not linked to an antigen binding domain. Alternatively, such Fc domains are linked to antigen binding domains.

In a second embodiment, the bifunctional molecule comprises an IL7 variant linked to the C-terminus of the whole chain Fc domain. Preferably, such an Fc domain is linked at its N-terminus to an antigen binding domain.

Optionally, the bifunctional molecule comprises a single IL7 variant linked to the C-terminus of the whole strand of the Fc domain, wherein the bifunctional molecule is linked to the N-terminus of the whole strand of the Fc domain. contains only a single antigen-binding domain that is labeled In such embodiments, the knob chain domain lacks the IL7 variant and lacks or lacks the antigen binding domain.

More particularly, the bifunctional molecule preferably comprises a single IL7 variant linked at its N-terminus to the C-terminus of the whole strand of the Fc domain, optionally via a linker, wherein bifunctional The sex molecule contains only a single antigen-binding domain linked at the N-terminus of the whole chain of the Fc domain, and an IL7 variant and a knob chain lacking the antigen-binding domain.

Therefore, the object of the present invention is to provide, from N-terminus to C-terminus, the antigen-binding domain (or at least part thereof corresponding to the heavy chain), the Fc chain (knob Fc chain or whole Fc chain), preferably the Fc domain. It relates to polypeptides containing whole chains and IL7 variants. Complementary chains include IL7 variants and complementary Fc chains lacking antigen-binding domains, preferably the knob chain of the Fc domain.

In another particular embodiment, the bifunctional molecule comprises a single IL7 variant linked at its N-terminus to the C-terminus of the whole strand of the Fc domain, optionally via a linker, wherein two The functional molecule comprises an antigen-binding domain linked at the N-terminus of the whole chain of the Fc domain and a knob chain comprising an antigen-binding domain lacking an IL7 variant and having its C-terminus linked to the N-terminus of the knob chain. including.

Therefore, the object of the present invention is to provide, from N-terminus to C-terminus, the antigen-binding domain (or at least part thereof corresponding to the heavy chain), the Fc chain (knob Fc chain or whole Fc chain), preferably the Fc domain. It relates to polypeptides containing whole chains and IL7 variants. The complementary chain comprises, from N-terminus to C-terminus, an antigen binding domain (or at least a portion thereof corresponding to the heavy chain) and a complementary Fc chain lacking an IL7 variant, preferably the knob chain of the Fc domain.

In another specific embodiment, the bifunctional molecule comprises a single IL7 variant linked to the N-terminus or C-terminus of the knob strand, optionally via a linker, wherein the bifunctional molecule comprises , which contains the antigen-binding domain linked at the N-terminus of the whole chain of the Fc domain, which lacks the IL7 variant.

Optionally, the antigen binding domain may be a Fab domain, Fab', single chain variable fragment (scFV) or single domain antibody (sdAb). An antigen-binding domain preferably comprises a heavy chain variable region (VH) and a light chain variable region (VL). When the antigen binding domain is Fab or Fab', the molecule further comprises a heavy chain constant domain and a light chain constant domain (ie CH and CL).

When the antigen binding domain is Fab or Fab', the bifunctional molecule may further comprise an IL-7 variant linked to the C-terminus of the VL domain of the antigen binding domain.

A bifunctional molecule according to the invention may comprise one or two antigen binding domains. Optionally, one antigen-binding domain may be linked to the N-terminus of the knob Fc chain and one antigen-binding domain may be linked to the N-terminus of the whole Fc chain. Alternatively, a single antigen-binding domain is linked to the N-terminus of either the knob Fc chain or the whole Fc chain. Preferably, the IL-7 variant is linked to an Fc chain that is linked to the antigen binding domain. In certain embodiments, the antigen-binding domain targets PD-1.

For example, an antigen-binding domain targeting PD-1 may be derived from an anti-PD1 antibody selected from the group consisting of: pembrolizumab (Keytruda lambrolizumab, also known as MK-3475); ), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pidilizumab (CT-011), cemiplimab (Libtayo), camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 ), PDR001 (spartalizumab), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, gonolimuzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK- 103 (HX-008), MEDI-0680 (also known as AMP-514), MEDI0608, JS001 (see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)). , BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM- 0001 (Armo), STI-1110 (see WO 2014/194302), AGEN2034 (see WO 2017/040790), MGA012 (see WO 2017/19846), or IBI308 ( see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540), the monoclonal antibody 5C4 described in WO 2006/121168; 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4. Bifunctional or bispecific molecules targeting PD-1 include RG7769 (Roche), XmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda). ), and XmAb23104 (Xencor). In particular, antigen-binding domains targeting PD-1 comprise the six CDRs or VH and VL of anti-PD1 antibodies selected in this list. Such antigen binding domains may in particular be Fab or svFc domains derived from this antibody. In a preferred embodiment, the antigen-binding domain targeting PD-1 is pembrolizumab (Keytrudalambrolizumab, also known as MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538) The six CDRs or VH and VL of an anti-PD1 antibody selected from may be, for example, Fab or scFc domains.

In a particular embodiment, the antigen-binding domain targeting PD-1 is derived from an antibody disclosed in WO2020/127366. The disclosure of this document is incorporated herein by reference.

Therefore, an antigen-binding domain is
(i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and
(ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3;
- the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO:51, optionally with substitutions, additions, deletions and any thereof at any position of SEQ ID NO:51 except position 3 having one, two, or three modifications selected from a combination of
- the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO:53, optionally with substitutions, additions, deletions at any position other than positions 13, 14 and 16 of SEQ ID NO:53 , and any combination thereof, with 1, 2, or 3 modifications selected from
- the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 54, wherein X1 is D or E and X2 is T, H, A, Y, N, E, and S preferably in the group consisting of H, A, Y, N, E, optionally substituted at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 54, having 1, 2, or 3 modifications selected from additions, deletions, and any combination thereof;
- the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 63, wherein X is G or T and optionally 5, 6, 10, 11 and SEQ ID NO: 63 having 1, 2, or 3 modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 16;
- the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 66, optionally one, two, or selected from substitutions, additions, deletions, and any combination thereof has three modifications,
- the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with substitutions, additions, deletions at any position other than positions 1, 4 and 6 of SEQ ID NO: 16 , and any combination thereof.

In one aspect, the antigen-binding domain is
(i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and
(ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3;
- the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO:51, optionally with substitutions, additions, deletions and any thereof at any position of SEQ ID NO:51 except position 3 having one, two, or three modifications selected from a combination of
- the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO:53, optionally with substitutions, additions, deletions at any position other than positions 13, 14 and 16 of SEQ ID NO:53 , and any combination thereof, with 1, 2, or 3 modifications selected from
- the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 54, wherein X1 is D and X2 consists of T, H, A, Y, N, E, and S from the group, preferably in the group consisting of H, A, Y, N, E, or X1 is E and X2 consists of T, H, A, Y, N, E, and S any selected from the group, preferably in the group consisting of H, A, Y, N, E and S, optionally other than positions 2, 3, 7 and 8 of SEQ ID NO:54 having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, at any position of
- the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 63, wherein X is G or T and optionally 5, 6, 10, 11 and SEQ ID NO: 63 having 1, 2, or 3 modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 16;
- the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 66, optionally one, two, or selected from substitutions, additions, deletions, and any combination thereof has three modifications,
- the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with substitutions, additions, deletions at any position other than positions 1, 4 and 6 of SEQ ID NO: 16 , and any combination thereof.

In another embodiment, the antigen-binding domain comprises (i) CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:55, 56, 57, 58, 59, 60, 61, or 62 and (ii) a light chain comprising the CDR1 of SEQ ID NO:64 or SEQ ID NO:65, the CDR2 of SEQ ID NO:66, and the CDR3 of SEQ ID NO:16.

In another aspect, the antigen-binding domain is
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:55; and (ii) CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:56; and (ii) CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:57; and (ii) CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:58; and (ii) CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:59, and (ii) CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16 light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:60, and (ii) CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16 light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:61; and (ii) CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:62; and (ii) CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:55; and (ii) CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:56; and (ii) CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:57; and (ii) CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:58, and (ii) CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16 light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:59; and (ii) CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:60, and (ii) CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16 light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:61; and (ii) CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. light chain; or
(i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:62; and (ii) CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66, and CDR3 of SEQ ID NO:16. It comprises or consists essentially of a light chain.

In one aspect, the anti-PD1 antibody or antigen-binding fragment according to the invention comprises framework regions, in particular the heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3 and HFR4, and the light chain variable region framework regions ( LFR) includes LFR1, LFR2, LFR3, and LFR4.

Preferably, an anti-PD1 antibody or antigen-binding fragment according to the invention comprises human or humanized framework regions. A "human acceptor framework", for the purposes of this specification, is a light chain variable domain (VL) framework or heavy chain derived from a human immunoglobulin framework or a human consensus framework, as defined below. A framework comprising the amino acid sequence of a variable domain (VH) framework. A human acceptor framework derived from a human immunoglobulin framework or a human consensus framework may contain that same amino acid sequence or may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 1 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence. A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.

In particular, the anti-PD1 antibody or antigen-binding fragment comprises heavy chain variable region framework regions (HFRs) HFR1, HFR2, HFR3 and HFR4 comprising the amino acid sequences of SEQ ID NOS: 41, 42, 43 and 44, respectively; optionally 1, 2 or 1 selected from substitutions, additions, deletions and any combination thereof at any position of HFR3, i.e. SEQ ID NO: 43, except positions 27, 29 and 32 Has 3 modifications. Preferably, the anti-PD1 antibody or antigen-binding fragment comprises HFR1 of SEQ ID NO:41, HFR2 of SEQ ID NO:42, HFR3 of SEQ ID NO:43, and HFR4 of SEQ ID NO:44.

Alternatively or additionally, the anti-PD1 antibody or antigen-binding fragment comprises light chain variable region framework regions (LFRs) LFR1, LFR2, LFR3, and LFR4, optionally having 1, 2, or 3 modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the humanized anti-PD1 antibody or antigen-binding fragment comprises LFR1 of SEQ ID NO:45, LFR2 of SEQ ID NO:46, LFR3 of SEQ ID NO:47, and LFR4 of SEQ ID NO:48.

The VL and VH domains of an anti-hPD1 antibody comprised in a bifunctional molecule according to the invention are preferably in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino-terminus to carboxy-terminus). It may comprise four framework regions separated by three complementarity determining regions that are operably linked.

In another aspect, the antigen-binding domain comprises or consists essentially of:
(a) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein X1 is D or E and X2 is from the group consisting of T, H, A, Y, N, E and S, preferably , H, A, Y, N, E, optionally 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73 of SEQ ID NO: 17 , 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112, substitutions, additions, deletions, and a heavy chain variable region (VH) having 1, 2 or 3 modifications selected from any combination of
(b) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, and optionally 3, 4, 7, 14, 17, 18, 28, 29 of SEQ ID NO:26 , 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 at any position other than substitutions, additions, deletions, and any combination thereof Light chain variable region (VL) with 1, 2 or 3 modifications
including.

In another aspect, the antigen-binding domain is
(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25;
(b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28
comprising or consisting essentially of

In another aspect, the antigen-binding domain comprises or consists essentially of any of the following combinations of heavy chain variable regions (VH) and light chain variable regions (VL).

In a very particular embodiment, the antigen binding domain comprises or consists essentially of the heavy chain variable region (VH) of SEQ ID NO:24 and the light chain variable region (VL) of SEQ ID NO:28.

In certain embodiments, the bifunctional molecule is
(a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36, optionally SEQ ID NOs: 29, 30, 31, respectively; , 32, 33, 34, 35, or 36 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, one or two selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 , or a heavy chain with three modifications, the substitutions corresponding to the whole or knob strand, preferably the whole strand, more particularly as disclosed in Table G (Table 7), in particular either T363S/L365A/Y4047V/Y346C or T363W/S351C, preferably T363S/L365A/Y4047V/Y346C, in SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36; a heavy chain that is N294A in any of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36;
(b) comprises or consists of the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:38, optionally 3, 4, 7, 14, 17, 18, 28, 29, 33 of SEQ ID NO:37 or SEQ ID NO:38; one selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105; Including light chains with two or three modifications.

In another aspect, the bifunctional molecule comprises or consists of any of the following combinations of heavy (CH) and light (CL) chains:

The heavy chain corresponds to a whole chain or a knob chain, preferably a whole chain, more particularly as disclosed in Table G (Table 7), in particular SEQ ID NOs: 29, 30, 31, 32, 33, especially any of T366S/L368A/Y407V/Y349C or T366W/S354C, preferably T366S/L368A/Y407V/Y349C, in 34, 35 or 36, optionally SEQ ID NO: 29, 30, 31, 32, 33 , 34, 35 or 36, the position of the substitution being designated according to EU numbering.

Thus, in one aspect, the bifunctional molecule according to the invention is
(a) an anti-human PD-1 antigen-binding domain comprising (i) one heavy chain with a first Fc chain and (ii) one light chain,
(b) IL-7 variants, and
(c) comprising or consisting of a complementary second Fc chain, the IL-7 variant preferably having its N-terminus to the C-terminus of the first Fc chain and/or to the N-terminus of the second Fc chain It is covalently linked to the terminus or C-terminus, optionally via a peptide linker.

The IL-7 variant can be any IL-7 variant as disclosed above.

The first and second Fc chains may be as disclosed above. Preferably, the Fc chain is an Fc chain, preferably derived from an IgG1 antibody or an IgG4 antibody.

Anti-human PD-1 antigen binding domains are as disclosed above.

In one aspect, the bifunctional molecule comprises a single anti-human PD-1 antigen binding domain (only one). Preferably, the bifunctional molecule is a single anti-human molecule selected from the group consisting of anti-human PD-1 Fab, anti-human PD-1 Fab', anti-human PD-1 scFV and anti-human PD-1 sdAb. Contains the PD-1 antigen-binding domain.

A bifunctional molecule comprises one or two IL-7 variants, preferably a single IL-7 variant.

A bifunctional molecule may comprise a light chain comprising or consisting of SEQ ID NO:37 or 38.

The bifunctional molecule may comprise a heavy chain comprising or consisting of any of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35 and 36, the Fc chain optionally comprising: It is modified to promote heterodimerization of the Fc chains to form heterodimeric Fc domains. More specifically, the heavy chain corresponds to a whole or knob chain, preferably a whole chain, more specifically as disclosed in Table G (Table 7), especially SEQ ID NOs: 29, 30. , either T366S/L368A/Y407V/Y349C or T366W/S354C, preferably T366S/L368A/Y407V/Y349C, and optionally N297A Including certain substitutions, the positions of substitution are specified according to EU numbering.

In a very particular aspect, the bifunctional molecule comprises a light chain comprising or consisting of SEQ ID NO:38 and a heavy chain comprising or consisting of SEQ ID NO:35, wherein the Fc chain is optionally an Fc chain are modified to promote heterodimerization of Fc domains to form heterodimeric Fc domains.

In a very particular embodiment, the bifunctional molecule comprises a first monomer of SEQ ID NO: 75 and optionally via a linker at the N-terminus to an antigen binding domain (especially of SEQ ID NO: 79) and at the C-terminus It may comprise a second monomer comprising the Fc chain SEQ ID NO:77, optionally linked by a linker to any IL-7 variant as disclosed herein. More particularly, the bifunctional molecule comprises a first monomer of SEQ ID NO: 75, a second monomer of SEQ ID NO: 83 and SEQ ID NO: 37, 38 or 80, preferably SEQ ID NO: 38 or 80 contains a third monomer of

In another very particular embodiment, the bifunctional molecule comprises the first monomer of SEQ ID NO: 77 and, optionally at the N-terminus, to the antigen binding domain (particularly of SEQ ID NO: 79), optionally via a linker, and the C It may comprise a second monomer comprising the Fc chain SEQ ID NO: 75 optionally linked by a linker at the end to any IL-7 variant as disclosed herein. More particularly, the bifunctional molecule comprises a first monomer of SEQ ID NO: 77, a second monomer of SEQ ID NO: 82 and SEQ ID NO: 37, 38 or 80, preferably SEQ ID NO: 38 or 80 contains a third monomer of

In another very particular embodiment, the bifunctional molecule is a first monomer of SEQ ID NO: 75, optionally linked at its N-terminus to an antigen binding domain (particularly of SEQ ID NO: 79) by a linker; and optionally linked at the N-terminus to an antigen binding domain (especially of SEQ ID NO: 79) by a linker and at the C-terminus to any IL-7 variant as disclosed herein, optionally via a linker A second monomer comprising the Fc chain SEQ ID NO:77 may be included. More particularly, the bifunctional molecule comprises a first monomer of SEQ ID NO: 81, a second monomer of SEQ ID NO: 83 and SEQ ID NO: 37, 38 or 80, preferably SEQ ID NO: 38 or 80 contains a third monomer of

In another very particular embodiment, the bifunctional molecule is a first monomer of SEQ ID NO: 77, optionally linked at its N-terminus to an antigen binding domain (particularly of SEQ ID NO: 79) by a linker; and optionally linked at the N-terminus to an antigen binding domain (especially of SEQ ID NO: 79) by a linker and at the C-terminus to any IL-7 variant as disclosed herein, optionally via a linker A second monomer comprising the Fc chain SEQ ID NO:75 may be included.

Preparation of Bifunctional Molecules - Nucleic Acid Molecules Encoding IL-7 Variants or Mutants, or Fusion Proteins and Bifunctional Molecules Comprising IL-7 Variants or Mutants, Recombinant Expression Vectors and Hosts Containing Those Nucleic Acid Molecules Cells encoding IL-7 variants or mutants, fusion proteins or bifunctional molecules, particularly for producing IL-7 variants or mutants, fusion proteins or bifunctional molecules according to the invention in mammalian cells A nucleic acid sequence or group of nucleic acid sequences is subcloned into one or more expression vectors. Such vectors are commonly used to transfect mammalian cells. Basic techniques for producing molecules containing antibody sequences are described in Coligan et al. (eds), Current protocols in immunology, pp. 10.19.1-10.19.11 (Wiley Interscience, 1992) (the content of which is incorporated herein by reference). (incorporated herein), and in WH Freeman and Company, "Antibody engineering: a practical guide" (1992), interspersed with the corresponding text, related to the production of the molecule. .

In general, such methods
(1) transfecting a suitable host cell with a polynucleotide encoding an IL-7 variant or mutant, fusion protein, or recombinant bifunctional molecule of the invention or a variant thereof, or a vector containing the polynucleotide; or transforming,
(2) culturing the host cells in a suitable medium, and
(3) Optionally, isolating or purifying the protein from the culture medium or host cells.

The present invention provides a nucleic acid encoding an IL-7 variant or mutant, fusion protein, or bifunctional molecule as disclosed above, a vector comprising a nucleic acid of the invention, preferably an expression vector, a vector of the invention, or Genetically Engineered Host Cells Directly Transformed with Sequences Encoding IL-7 Variants or Mutants, Fusion Proteins, or Recombinant Bifunctional Molecules, and Methods for Producing Proteins of the Invention by Recombinant Techniques further relates to

Nucleic acids, vectors, and host cells are described in more detail herein below.

Nucleic Acid Sequences The present invention also relates to nucleic acid molecules encoding IL-7 variants or mutants, fusion proteins or bifunctional molecules as defined above, or IL-7 variants or mutants as defined above. , fusion proteins, or bifunctional molecules. A nucleic acid encoding an IL-7 variant or mutant, fusion protein, or bifunctional molecule disclosed herein can be amplified by any technique known in the art, such as PCR. Such nucleic acids can be readily isolated and sequenced using conventional procedures.

In particular, nucleic acid molecules encoding bifunctional molecules as defined above are
- a first nucleic acid molecule encoding a binding moiety as disclosed herein, and
- comprising a second nucleic acid molecule encoding IL-7m, preferably human IL-7m.

In a very particular embodiment, the nucleic acid molecule encoding the binding moiety is a variable heavy domain having the sequence set forth in SEQ ID NO:73 and/or a variable light domain having the sequence set forth in SEQ ID NO:74. contains chain domains.

In one embodiment, the second nucleic acid molecule is operably linked to the first nucleic acid, optionally via a nucleic acid encoding a peptide linker. By operably linked is intended that the nucleic acids encode a protein fusion. Thus, in certain aspects, a nucleic acid encodes a fusion protein comprising a binding moiety, optionally a peptide linker, and an IL-7 variant disclosed herein. Preferably, in such nucleic acid molecules, where the binding moiety comprises an Fc domain, the N-terminus of the IL-7 variant is fused to the C-terminus of the heavy chain constant domain, preferably via a peptide linker.

In one embodiment, the nucleic acid molecule is an isolated, particularly non-naturally occurring nucleic acid molecule.

In one aspect, the nucleic acid encodes IL-7m having the amino acid sequences set forth in SEQ ID NOs:2-15.

Vectors In another aspect, the invention relates to a vector comprising a nucleic acid molecule or group of nucleic acid molecules as defined above.

As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell. The term "vector" encompasses plasmids, viruses, cosmids and artificial chromosomes. In general, genetically engineered vectors contain an origin of replication, a multiple cloning site, and a selectable marker. Vectors are themselves generally nucleotide sequences, generally DNA sequences, containing an insert (transgene), and larger sequences that serve as the "backbone" of the vector. Modern vectors may include transgene inserts and other additional backbone features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags. Vectors, called expression vectors (expression constructs), are specifically for expressing transgenes in target cells and generally have regulatory sequences.

Nucleic acid molecules encoding bifunctional molecules, fusion proteins, binding moieties, or IL-7 variants can be cloned into vectors and then transformed into host cells by those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. Nucleic acid sequences of the bifunctional molecules, fusion proteins, binding moieties, or IL-7 variants described herein and appropriate regulatory elements for transcription/translation using methods known to those of skill in the art. can be constructed.

Accordingly, the invention also provides a recombinant vector comprising a nucleic acid molecule encoding a bifunctional molecule, fusion protein, binding moiety, or IL-7 variant according to the invention. In one preferred embodiment, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretory signal peptide, and optionally at least one drug resistance gene for screening. The expression vector may further include a ribosome binding site for translation initiation, a transcription terminator, and the like.

Suitable expression vectors typically include (1) a prokaryotic DNA element encoding a bacterial origin of replication and an antibiotic resistance marker to provide for growth and selection of the expression vector in bacterial hosts; (2) a promoter, etc.; eukaryotic DNA elements that control transcription initiation; (3) DNA elements that control transcript processing, such as transcription termination/polyadenylation sequences.

Expression vectors can be introduced into host cells using a variety of techniques, including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like. Preferably, transfected cells in which the expression vector has stably integrated into the host cell genome are selected and grown to produce stable transformants.

Host Cells In another aspect the invention relates to a host cell comprising a vector or a nucleic acid molecule or group of nucleic acid molecules as defined above, eg for the purpose of producing a bifunctional molecule.

As used herein, the term "host cell" refers to vectors, exogenous nucleic acid molecules, and polynucleotides encoding bifunctional molecules, fusion proteins, binding moieties, or IL-7 variants according to the invention. It is intended to include any individual cell or cell culture that can be or has been a recipient of. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, bacteria, yeast cells, fungal cells, plant cells, and insect and mammalian cells such as mice, rats, rabbits, macaques. , or animal cells such as human.

Suitable host cells are especially eukaryotic hosts which provide suitable post-translational modifications such as glycosylation. Preferably, such suitable eukaryotic host cells are fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe; ); plant cells such as tobacco; and mammalian cells such as BHK cells, 293 cells, CHO cells, NSO cells, and COS cells.

Preferably, the host cells of the invention are selected from the group consisting of CHO cells, COS cells, NSO cells and HEK cells.

Thus, host cells stably or transiently express bifunctional molecules, fusion proteins, binding moieties and/or IL-7 variants according to the invention. Such expression methods are known to those skilled in the art.

Also provided herein are methods for producing IL-7 variants or mutants, fusion proteins, or bifunctional molecules. The method includes culturing host cells containing nucleic acids encoding bifunctional molecules, fusion proteins, binding moieties, and/or IL-7 variants as provided above under conditions suitable for their expression. and optionally recovering the bifunctional molecule, fusion protein, binding moiety, and/or IL-7 variant from the host cell (or host cell culture medium). In particular, in the case of recombinant production of bifunctional molecules, nucleic acid molecules encoding bifunctional molecules, e.g. Insert into multiple vectors. IL-7 variants or mutants, fusion proteins, bifunctional molecules are then isolated and/or purified by any method known in the art. Such methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (such as salt precipitation), centrifugation, osmotic cell lysis, sonication, ultracentrifugation, molecular sieve chromatography or gel chromatography. chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and combinations thereof. For example, as described by Coligan, bifunctional molecular isolation techniques can include affinity chromatography with protein A sepharose, size exclusion chromatography, and ion exchange chromatography, among others. Protein A is preferably used for the isolation of the bifunctional molecules of the invention.

PHARMACEUTICAL COMPOSITIONS AND METHODS OF ADMINISTRATION THEREOF It relates to pharmaceutical compositions containing either groups of molecules, vectors and/or host cells, preferably as active ingredients or compounds. The formulations can be sterile and, if desired, do not adversely interact with the bifunctional molecules of the invention, nucleic acids, vectors, and/or host cells of the invention and do not produce undesired toxicological effects. It can be mixed with adjuvants such as pharmaceutically acceptable carriers, excipients, salts, antioxidants, and/or stabilizers, which do not provide any. Optionally, the pharmaceutical composition may further comprise additional therapeutic agents.

In particular, pharmaceutical compositions according to the present invention can be administered in any conventional manner, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration. can be formulated for any route of administration. To facilitate administration, the bifunctional molecules as described herein can be formulated into pharmaceutical compositions for in vivo administration. Means for making such compositions are described in the art (see, eg, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Inc., 21st Ed. (2005)).

Pharmaceutical compositions are prepared by mixing a bifunctional molecule of desired purity with optional pharmaceutically acceptable carriers, excipients, antioxidants, and/or stabilizers to prepare lyophilized formulations or It can be prepared in the form of an aqueous solution. Suitable such carriers, excipients, antioxidants and/or stabilizers are well known in the art and can be found, for example, in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980). Are listed.

Either the bifunctional molecule or its encoding nucleic acid may be conjugated with a chaperone agent to facilitate delivery. Chaperone agents are naturally occurring substances such as proteins (e.g., human serum albumin, low-density lipoproteins, or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or lipids. It may be a substance that Chaperone agents may also be recombinant or synthetic molecules, such as synthetic polymers, eg synthetic polypeptides.

Pharmaceutical compositions according to the invention may be formulated to release the active ingredient (e.g., the bifunctional molecule of the invention) substantially immediately after administration, or at any predetermined time or period after administration. good. In some aspects, the pharmaceutical composition includes a time-release delivery system, a delayed release delivery system, such that delivery of the composition occurs prior to and for a time sufficient to cause sensitization of the site to be treated. Release and sustained release delivery systems can be used. Means known in the art can be used to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed release of the composition. Such systems can avoid repeated administrations of the compositions, thereby increasing convenience to the subject and the physician.

Those skilled in the art will appreciate that the formulations of the invention may be isotonic with human blood, ie, the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by vapor pressure or ice-freezing type osmometers.

Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. Prevention of microbial presence can be ensured both by sterilization procedures (eg, by microfiltration) and/or the inclusion of various antibacterial and antifungal agents.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect.

Subjects, Regimens, and Administration The present invention is directed to therapeutic agents for use as a medicament, or for use in treating disease, or for administration to a subject, or for use as a medicament, as disclosed herein. IL-7 variants or mutants, fusion proteins, or bifunctional molecules of; nucleic acids or vectors, host cells, or pharmaceutical compositions, nucleic acids, vectors, or host cells encoding them. The invention also relates to a method for treating a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or bifunctional molecule. Examples of treatments are described in more detail in the "Methods and Uses" section herein below.

The subject to be treated is a human, especially a human in the prenatal stage, a neonate, a child, an infant, an adolescent, or an adult, especially an adult at least 30, 40, preferably at least 50, even more preferably at least 60. , and even more preferably at least 70 years of age.

In certain aspects, the subject may be immunosuppressed or immunocompromised.

Bifunctional molecules or pharmaceutical compositions as disclosed herein, depending on the type of disease or site of disease to be treated, using conventional methods known to those skilled in the medical arts. can be administered, for example, orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or Administration can be via an implanted reservoir. Preferably, the bifunctional molecule or pharmaceutical composition is subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intratumoral, intrasternal, intrathecal, intralesional, and intracranial. It is administered by injection or infusion techniques.

The form of the pharmaceutical composition, the route of administration and the dosage of the pharmaceutical composition or the bifunctional molecule according to the invention can be determined by the person skilled in the art depending on the type and severity of the infection, the age, weight, size of the patient, especially the patient. , gender, and/or general health. The compositions of the invention can be administered in several ways depending on whether local or systemic treatment is desired.

Uses in Treating Disease The bifunctional molecules, nucleic acids, vectors, host cells, compositions and methods of the invention have numerous in vitro and in vivo utilities and applications. In particular, any of the IL-7 variants or variants, fusion proteins, bifunctional molecules, nucleic acid molecules, groups of nucleic acid molecules, vectors, host cells or pharmaceutical compositions provided herein are used in therapeutic methods to: and/or may be used for therapeutic purposes.

The invention also provides IL-7 variants or mutants, fusion proteins, or bifunctional molecules, for use in the treatment of disorders and/or diseases in a subject and/or for use as a medicament or vaccine. or a pharmaceutical composition comprising them. The present invention also provides IL-7 variants or mutants, fusion proteins, or bifunctional molecules described herein for treating diseases and/or disorders in a subject; nucleic acids or vectors encoding them; Alternatively, it relates to the use of pharmaceutical compositions containing them. Finally, the invention provides a method of treating a disease or disorder in a subject comprising a therapeutically effective amount of a pharmaceutical composition, or IL-7 variant or mutant, fusion protein, or bifunctional molecule, or encoding thereof. A method comprising the step of administering to a subject a nucleic acid or vector that

In one embodiment, the present invention provides a method of treatment of a disease and/or disorder selected from the group consisting of cancer, infectious disease, and chronic viral infection in a subject in need thereof, comprising: 2, administering an effective amount of an IL-7 variant or mutant, fusion protein, or bifunctional molecule, or pharmaceutical composition as defined above. Examples of such diseases are more specifically described herein below.

In one aspect, a method of treatment comprises the steps of: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of an IL-7 variant, mutant, fusion protein described herein. , or administering either a bifunctional molecule, nucleic acid, vector, or pharmaceutical composition.

A subject in need of treatment may be a human having, at risk of having, or suspected of having a disease. Such patients can be identified by routine physical examination.

In another aspect, the bifunctional molecules disclosed herein are administered to a subject, e.g., in vivo, to enhance immunity, preferably to treat disorders and/or diseases. can be done. Thus, in one aspect, the invention provides a method of modifying an immune response in a subject, comprising administering to the subject a bifunctional molecule, nucleic acid, vector or pharmaceutical composition of the invention, such that the immune response in the subject is modified. A method is provided comprising the step of administering an agent. Preferably the immune response is enhanced, augmented, stimulated or upregulated. Bifunctional molecules or pharmaceutical compositions can be used to enhance immune responses, such as T cell activation, in subjects in need of treatment. In certain embodiments, bifunctional molecules or pharmaceutical compositions can be used to reduce T cell exhaustion or reactivate exhausted T cells.

The present invention provides, inter alia, a method of enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a bifunctional molecule, nucleic acid, as described herein, such that the immune response in the subject is enhanced. A method is provided comprising administering the vector or a pharmaceutical composition comprising it. In certain embodiments, bifunctional molecules or pharmaceutical compositions can be used to reduce T cell exhaustion or reactivate exhausted T cells.

Bifunctional molecules according to the invention target CD127+ immune cells, in particular CD127+ T cells. Such cells have the following specific areas of interest: resident lymphoid cells in lymph nodes (predominantly paracortical, including occasional cells in follicles), tonsils (interfollicular region), Spleen (mainly white pulp periarteriolar lymphocytic sheath (PALS) and some scattered cells in red pulp), thymus (mainly in the medulla; or in the cortex), bone marrow (with scattered GALT (gut-associated lymphoid tissue, mainly in the interfollicular region and lamina propria) in the gastrointestinal tract (stomach, duodenum, jejunum, ileum, cecum, rectum), MALT in the gallbladder (mucosa-associated lymphoid tissue) can be seen in The bifunctional molecules of the invention are therefore of particular interest to treat diseases that lie in or involve these areas, especially cancer.

Cancer In another embodiment, the present invention provides an IL-7 variant or mutation disclosed herein in the manufacture of a medicament for treating cancer, e.g., for inhibiting the growth of tumor cells in a subject. Uses of the body, fusion protein, or bifunctional molecule, or pharmaceutical composition are provided.

The term "cancer" as used herein is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

Thus, in one embodiment, the invention provides a method of treating cancer, e.g., inhibiting tumor cell growth, in a subject, comprising administering to the subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition according to the invention. A method is provided comprising the step of administering an agent. In particular, the invention relates to treatment of a subject using bifunctional molecules such that cancerous cell growth is inhibited.

In one aspect of the disclosure, the cancer to be treated is associated with exhausted T cells.

Any suitable cancer cancer that can be treated with those provided herein can be a cancer of the hematopoietic system or a solid cancer. Such cancers include carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, medium Including dermoma, melanoma, gastric cancer, urethral cancer, environmentally induced cancer and any combination of such cancers. Additionally, the present invention includes refractory or recurrent malignancies. Preferably, the cancer to be treated or prevented is metastatic or non-metastatic, melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial cancer, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric or gastroesophageal cancer and cervical cancer.

In certain aspects, the cancer is a hematological or solid tumor. Such cancers can be selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndromes, acute myelogenous leukemia.

In certain aspects, the cancer is a virally induced cancer or a cancer associated with immunodeficiency. Such cancers include Kaposi's sarcoma (e.g. Kaposi's sarcoma associated with herpesvirus); squamous cell carcinoma and oropharyngeal carcinoma of the cervix, anus, penis and vulva (e.g. associated with human papillomavirus) ); B-cell non-Hodgkin lymphoma (NHL), including diffuse large B-cell lymphoma, Burkitt lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary exudative lymphoma, classic Hodgkin Lymphomas and lymphoproliferative disorders (e.g. associated with Epstein-Barr virus (EBV) and/or Kaposi's sarcoma herpesvirus); hepatocellular carcinoma (e.g. associated with hepatitis B and/or C virus); Merkel cell carcinoma (eg, associated with Merkel cell polyomavirus (MPV)); and cancers associated with human immunodeficiency virus infection (HIV) infection.

Preferred cancers for treatment typically include cancers responsive to immunotherapy. Alternatively, preferred cancers for treatment are cancers that are not responsive to immunotherapy.

Infectious Diseases Bifunctional molecules, nucleic acids, groups of nucleic acids, vectors, host cells or pharmaceutical compositions of the invention can be used to treat patients exposed to particular toxins or pathogens. Accordingly, aspects of the present invention preferably include administering to the subject a bifunctional molecule according to the present invention, or a pharmaceutical composition comprising the same, such that the subject is treated for an infectious disease. Methods of treating infectious diseases are provided.

Any suitable infectious disease may be treated with the bifunctional molecules, nucleic acids, groups of nucleic acids, vectors, host cells or pharmaceutical compositions provided herein.

Some examples of pathogenic viruses that cause infections treatable by the methods of the invention include HIV, hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV -II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, Including vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria that cause infections treatable by the methods of the present invention are chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococcus, Klebsiella, Proteus. , Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulinum, Bacillus anthracis, Plague, Leptospira, and Lyme disease bacteria.

Some examples of pathogenic fungi causing infections treatable by the methods of the present invention include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucor (mucor, absidia, rhizophus), Sporothrix Includes Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites that cause infections treatable by the methods of the present invention are Entamoeba histolytica, Bal antidium coli, Naegleria fowleri, Acanthamoeba (Acanthamoeba) species, Giardia lambia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei ( Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

Combination therapy The bifunctional molecule according to the invention can be combined with several other possible strategies for overcoming immune evasion mechanisms, using substances in clinical development or already on the market (see Antonia et al., Immuno-oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. Such combinations with bifunctional molecules according to the invention are
1- reversing the inhibition of adaptive immunity (blocking the T cell checkpoint pathway);
2- switching adaptive immunity (promoting T cell co-stimulatory receptor signaling using agonist molecules, especially antibodies),
3- improving the function of innate immune cells;
4- activating the immune system (enhancing immune-cell effector function), e.g. through vaccine-based strategies,
can be clearly useful for

Accordingly, combination therapy for treating a disease or disorder using any of the bifunctional molecules described herein or a pharmaceutical composition comprising same and a suitable second agent is also herein provided. In aspects, the bifunctional molecule and the second agent can be present in the unique pharmaceutical compositions described above. Alternatively, as used herein, the term "combination therapy" or "combination therapy" refers to these two agents in a sequential fashion (e.g., the bifunctional molecules described herein and an additional or secondary administration of two suitable therapeutic agents), ie, each therapeutic agent is administered at a different time, and the administration of at least two of these therapeutic agents or agents is in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent can be effected by any suitable route. The substances can be administered by the same route or by different routes. For example, a first agent (e.g., bifunctional molecule) can be administered orally and an additional therapeutic agent (e.g., anticancer agent, antiinfective agent; or immune modulator) is administered intravenously. be able to. Alternatively, the substances of the combination selected may be administered by intravenous injection while the other substances of the combination may be administered orally.

In embodiments, the additional therapeutic agent is an alkylating agent, an angiogenesis inhibitor, an antibody, an antimetabolite, an antimitotic agent, an antiproliferative agent, an antiviral agent, an Aurora kinase inhibitor, a proapoptotic agent (e.g., Bcl -2 family inhibitors), activators of the death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell induction) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinases ( BTK) inhibitor, cyclin-dependent kinase inhibitor, cell cycle inhibitor, cyclooxygenase-2 inhibitor, DVD, leukemia virus oncogene homolog (ErbB2) receptor inhibitor, growth factor inhibitor, heat shock protein (HSP)- 90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunological agents, inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors agents, mammalian targets of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids Epitopes or neoepitopes derived from tumor antigens such as plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, etc. Selections can be made in a non-exhaustive list, including one or more combinations.

For example, additional therapeutic agents may include chemotherapy, radiation therapy, targeted therapy, antiangiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes derived from tumor antigens, myeloid checkpoint inhibitors, other It can be selected in the group consisting of immunotherapy and HDAC inhibitors.

The invention also provides a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapy. It relates to a method comprising administering an agent.

Specific examples of additional or second therapeutic agents are provided in WO2018/053106, pages 36-43.

In preferred embodiments, the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents, cell therapeutic agents (eg, CAR-T cells), antibiotics and probiotics.

Combination therapy can also rely on combining administration of the bifunctional molecule with surgery.

Kits Any of the bifunctional molecules or compositions described herein may be included in kits provided by the invention. The present disclosure provides, inter alia, kits for use in enhancing immune responses and/or treating diseases or disorders (eg, cancer and/or infectious diseases).

In the context of the present invention, the term "kit" means two or more components, one of which corresponds to the bifunctional molecule, nucleic acid molecule, vector or cell of the invention, packaged in a container, recipient or vice versa. ) means A kit can be described herein as a set of products and/or materials sufficient to accomplish a certain goal that can be marketed as a single unit. Kits of the invention are in suitable packaging.

Specifically, the kit according to the invention comprises:
- an IL-7 variant or mutant, fusion protein, or bifunctional molecule as defined above,
- a nucleic acid molecule or group of nucleic acid molecules encoding said IL-7 variant or mutant, fusion protein or bifunctional molecule;
- a vector containing the nucleic acid molecule or group of nucleic acid molecules, and/or
- may comprise a cell containing the vector or the nucleic acid molecule or group of nucleic acid molecules.

Thus, a kit may contain, in suitable container means, a pharmaceutical composition of the invention, and/or an IL-7 variant or mutant, a fusion protein, or a bifunctional molecule, and/or a host cell, and/or an agent of the invention. It may include vectors encoding nucleic acid molecules and/or nucleic acid molecules or related reagents of the invention. In some embodiments, means for obtaining a sample from an individual and/or means for assaying a sample may be provided. The compositions contained in the kits according to the invention may be particularly formulated in syringe-compatible compositions.

In some embodiments, the kit further comprises an additional agent for treating cancer or an infectious disease, wherein the additional agent is an IL-7 variant or mutant, fusion protein, or Bifunctional molecules, or may be combined with other components, or provided separately in a kit. In particular, the kits described herein may include one or more additional therapeutic agents such as those described in "Combination Therapy" hereinabove. The kit may be tailored to the particular cancer for the individual and include each of the second cancer therapies for the individual described herein above.

Instructions for use of the bifunctional molecules or pharmaceutical compositions described herein generally include dosages, dosing schedules, intended routes of administration for treatment, methods for reconstituting the bifunctional molecule. Includes information on means and/or means for diluting the bifunctional molecule of the invention. Instructions provided in kits of the invention are typically written instructions on a label or package insert (eg, a paper sheet included with the kit in the form of a leaflet or instruction manual).

Example 1. Fc-Fused IL-7 Mutants Modulate IL-7R Binding and pSTAT5 Signaling, Improving In Vivo Pharmacokinetics
To obtain IL-7 variants, amino acids involved in the interaction of IL7 to CD127 were replaced with amino acids with similar properties and properties. Several mutations were generated: Q11E, Y12F, M17L, Q22E, D74E, D74Q, D74N, K81R, W142H, W142F and W142Y.

Substitutions C2S-C141S+C34S-C129S (mutations designated <<SS1>>), or C2S-C141S+C47S-C92S (mutations designated "SS2"), or C47S-C92S+C34S-C129S (mutations designated "SS3") The IL-7 disulfide bond was disrupted by replacing the cysteine residue with a serine residue, leading to a mutation named ).

A single amino acid substitution in the IL7 sequence did not modify its ability to bind to the PD-1 receptor (Figures 1A, 1B and 1C). However, these mutations modify its biological activity, as shown by CD127 binding and pSTAT5 signaling in ex vivo T cell assays (Figures 2 and 3 and Table 1). ) and Table 4 (Table 13)). Mutations D74E and W142H are the most efficient mutations to reduce both IL-7 binding to CD127 and activation of pStat5 in T lymphocytes (Figures 2A, 2B and 3A, Figure 3B and Table 1). 1 (Table 10) and Table 5 (Table 14)). In another experiment, the effect of disulfide bond disruption was analyzed (Fig. 2C). At high concentrations (10 μg/ml), SS2 or SS3 were able to activate pStat5 in T lymphocytes with a 3 log deviation from IL-7WT (Fig. 2C and Table 4).

To confirm the binding capacity of these mutations, a Biacore assay was performed to determine the KD (equilibrium dissociation constant between the receptor and its antigen, see Table 2). Mutations SS2 and W142H have lower affinities for CD127 with KDs close to 7-57 nM. The SS3 mutation has the lowest affinity for CD127 with a KD close to 3 μM. Affinities for the CD132 receptor were also evaluated as shown in Table 3 (Table 12). In this experiment, IgG4 alone was used as the baseline KD affinity because CD127 dimerizes with CD132 in the absence of IL-7. IL-7 mutant W142H binds CD132, but with a 5-fold higher affinity compared to IgG IL-7WT. The data show that mutation W142H reduces binding to CD127 and redirects IL-7 binding to the CD132 receptor, which is associated with loss of pSTAT5 activation in T cells, as shown in FIG. guide. In contrast, we observed that the SS2 mutation lost the ability to bind the CD132 receptor in the conditions tested, indicating that the SS2 mutation preferentially binds CD127 over the CD132 receptor. , suggesting that it leads to a decrease in pSTAT5 activity in T cells (Fig. 3).

To determine the pharmacokinetics/pharmacology of anti-PD-1 IL-7 in vivo, mice were injected intravenously with a single dose of IgG-IL-7 (34.4 nM/kg). Plasma drug concentrations were analyzed by an ELISA specific for human IgG. Figure 3 and Table 5 show that the IgG4 IL-7 WT molecules have a rapid distribution, as the Cmax obtained (maximum concentration 15 minutes after injection) is 30-fold lower than the theoretical concentration. indicates All W142Y, F, H mutations tested showed better distribution characteristics with Cmax 5-10 fold higher than IL-7WT (Figure 3A and Table 5). The W142H mutation shows the highest Cmax. The anti-PD-1 IL-7 D74E mutation also showed good Cmax. Mutants SS2 and SS3 show the best PK characteristics with Cmax 7-13 fold higher than IL-7WT and good linearity characteristic curves. At the same time, the AUC (area under the curve) was determined (Table 5 and Figure 4D), which gives insight into the extent of drug exposure and its clearance rate from the body. These data show that AUC is increased with IL-7 mutations, implying that IL-7 mutations have improved drug exposure. As depicted in Figure 4D, we observed that drug exposure correlated with mutant IL-7 potency (as measured by pSTAT5 EC50). Taken together, IL-7 affinity correlates with product pharmacokinetics. Reducing the affinity of IL-7 for their receptors CD127 and CD132 enhances the absorption and distribution of IL-7 bifunctional molecules in vivo.

Example 2: Addition of cysteine at the C-terminal domain of IgG reduces the flexibility of the IL7 molecule and improves its pharmacokinetics in vivo.
The addition of a cysteine at the C-terminal domain of IgG was also tested to create an additional disulfide bond, potentially limiting the flexibility of the IL-7 molecule. This mutation was named "C-IL-7". Figure 5. Addition of a disulfide bond in the IgG structure reduced pSTAT5 activity of IL-7 compared to the anti-PD-1 IL7 WT bifunctional molecule (Figure 5A) in an in vivo pharmacokinetic assay. It is shown to increase Cmax (5-fold) (Figure 5B).

Example 3: Anti-PD-1 IL-7 variants constructed with IgG1N298A isotype have better binding to IL-7R, better pSTAT5 signaling and better pharmacokinetic properties in vivo
Anti-PD-1 IL-7 bifunctional molecules of different isotypes were tested with IgG4m (S228P) or IgG1m (N298A or N297A depending on numbering scheme). The IgG4 isotype contains the S228P mutation to prevent Fab arm exchange in vivo and the IgG1 isotype contains the N298A mutation that suppresses IgG1 isotype binding to FcγR receptors that can reduce non-specific binding of immune cytokines. (mutations designated "IgG4m" or "IgG1N298A"). An anti-PD-1 IL-7 bifunctional molecule was then constructed with two different isotypes, an IgG1 isotype mutated at N297A (referred to as IgG1m) versus an IgG4 S288P isotype (referred to as IgG4m), such that the isotypic structure is It is shown to determine whether it modifies the biological activity of IL-7 and its pharmacokinetic properties.

Figures 6A and 6B show that anti-PD-1 IL7 bifunctional molecules constructed with IgG4m or IgG1m isotypes have the same binding properties to the PD-1 receptor, with isotypes VH and VL. and does not modify the affinity of anti-PD-1 antibodies for PD-1. However, we found that the IgG1m isotype unexpectedly showed binding of IL-7 D74, SS2 and, to a lesser extent, SS3 on CD127 (FIGS. 7A, 7B, 7C and 7D) and on human PBMCs. were observed to enhance pSTAT5 activation (FIGS. 8A, 8B and 8C) at . This increase in pSTAT5 signaling was confirmed for the SS2 mutation on another T cell line (Jurkat cells expressing PD-1 and CD127, see Figure 8D), but in a surprising manner, the IgG1m isotype The anti-PD-1 IL-7 WT bifunctional molecule did not modify the pSTAT5 activity, suggesting that the IgG1m isotype only enhances the activity of the IL-7 mutant. To determine the ability of bifunctional molecules comprising anti-PD1 antibodies and IL7 mutations to reactivate signaling mediated by reactive TCRs, an NFAT bioassay was performed. Results show FIG. 9A showing that bifunctional molecules are better than anti-PD1 or anti-PD1+rIL7 (as separate compounds) at activating TCR-mediated signaling (NFAT); This indicates a synergistic effect of bifunctional molecules on PD1+ T cells. We next evaluated the synergistic potency of bifunctional molecules comprising anti-PD-1 antibodies constructed with IgG4m vs. IgG1m isotypes and IL-7 mutations (including mutations D74E, W142H or SS2). (Fig. 9B, Fig. 9C, Fig. 9D). All mutations tested preserved a synergistic effect on the activation of NFAT signaling, and the level of activation increased pSTAT5 signaling, particularly for bifunctional molecules including IgG4m and IL-7 D74E. correlates with the ability of

Pharmacokinetic studies in mice show that IgG1 isotype does not modify drug exposure for IL7WT and SS3 molecules and minimal effect on W142H molecules (FIG. 10A). Collectively these data indicate that the optimized isotype (IgG1m) is sufficient to enhance the biological activity of the mutation while preserving good pharmacokinetics of the product in vivo. Along with the IgG1m isotype, other IL-7 mutations were tested: D74N, D74Q and D74E+W142H mutation combinations. No differences with the anti-PD-1 IL-7 D74E mutation were observed in pSTAT5 activation (Fig. 9B) and pharmacokinetics (Fig. 10B).

We specifically tested anti-PD-1 bifunctional molecules comprising IL-7 D74 mutations with different amino acid substitutions D74E, D74Q and D74N. These constructs contain the GGGGS linker and the IgG1N298A isotype. As detailed in Table 6 (Table 15), all constructs had similar binding efficiencies to PD-1, but binding to dual PD-1/CD127 was lower for the D74Q and D74N mutations. Decreased compared to the D74E mutant. This suggests that the substitutions Q and N slightly attenuate the affinity of the mutant for the CD127 receptor.

The double mutation D74E+W142H showed similar properties compared to W124H IgG1 and D74Q showed similar properties compared to the D74E mutation. We also constructed a bifunctional molecule with IgG1m isotype + YTE mutations (M252Y/S254T/T256E). This mutation has been described to increase the half-life of the antibody by increasing binding to the FcRn receptor. As shown in Figure 7D, YTE mutations do not modify pSTAT5 signaling of bifunctional molecules containing D74 or W142H mutations.

(Example 4: Mutation K444A to C-terminal lysine residue does not affect pharmacokinetics in vivo)
All subclasses of human IgG have a C-terminal lysine residue (K444) of the antibody heavy chain that can be cleaved during circulation. This cleavage in the blood can impair the biological activity of immune cytokines by releasing IL-7 bound to IgG. To circumvent this problem, the K444 amino acid in the IgG domain was replaced with alanine to reduce proteolytic cleavage. This is a commonly used mutation for antibodies. A similar curve was obtained between IgG WT IL-7 versus IgG K444A IL-7, as shown in FIG. This suggests that the mutation does not affect the pharmacokinetic properties of the drug.

Example 5: A linker between IgG antibodies does not modify pharmacokinetics in vivo, but enhances activation of pSTAT5 signaling.
Different linkers between the IgG Fc domain and IL-7m were tested to modify flexibility. Several conditions were tested (eg no linker, GGGGS, GGGGSGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS).

For Examples 1 and 2, a linker (G4S)3 between the C-terminal domain of Fc and the N-terminal domain of IL-7 was used for IgG4m-IL7 and IgG1m-IL-7 constructions, respectively. This linker allowed for high flexibility and enhanced IL7 activation signals. To reduce the affinity of IL7 for CD127 and improve pharmacokinetics, different constructs were tested with varying linker lengths (no linker, G4S, (G4S)2 or (G4S)3). . For comparison, IgG1m or IgG4m Fc IL-7 WT were also generated with different linkers.

Pharmacokinetic studies showed that the length of the linker affected the constructs tested: anti-PD-1 IL7 WT (Figure 12A), anti-PD-1 IL-7 D74 (Figure 12B) and anti-PD-1 IL-7 W142H (Figure 12C). ) has no effect on product distribution, absorption and excretion. However, linker length affects the activation of pStat5, as shown in Figure 12D. Indeed, anti-PD-1 IL7 constructed with the (G4S)3 linker was more potent in activating pSTAT5 signaling compared to anti-PD-1 IL-7 constructed with the (G4S)2 or G4S3 linker, Even more potent compared to anti-PD-1 IL-7 constructed without a linker. These data emphasize that the use of (G4S)3 linkers allows IL-7 flexibility without compromising the drug's pharmacokinetics in vivo.

(Example 6: Anti-PD-1 IL-7 variants allow preferential binding to PD-1+CD127+ cells over PD-1-CD127+ cells)
Next, we evaluated the ability of anti-PD-1 IL-7 bifunctional molecules to target PD-1+ T cells. Jurkat cells expressing CD127+ or Jurkat cells co-expressing CD127+ and PD-1+ were stained with 45 nM of the following bifunctional molecules: anti-PD-1 IL-7 WT, D74, W142H, SS2 and SS3. bottom. Binding was detected with anti-IgG-PE (Biolegend, clone HP6017) and analyzed by flow cytometry.

Results: Figure 13 shows that anti-PD-1 IL-7 WT and D74 mutants bind with similar efficacy to PD-1+/CD127+ versus PD-1-/CD127+ cells, while anti-PD-1 1 IL-7 mutants SS2, SS3 bind PD-1+/CD127+ vs. PD-1−/CD127+ cells with 2-3 fold higher efficacy. The anti-PD-1 IL-7 W142H bifunctional molecule exhibits intermediate efficacy, binding to PD-1+/CD127+ cells with 1.4-fold higher efficacy.

Next, to confirm the specific targeting of anti-PD-1 IL-7 mutations to PD-1+ T cells in a heterologous cell model, we next examined PD-1(+) cells and PD-1(-) cells were mixed and analyzed for their binding to each cell subset. In this assay, CHO cells co-expressing human CD127+ and human PD-1+ cells were co-cultured at a 1:1 ratio with CHO expressing only the human CD127+ receptor (Fig. 14A), followed by increasing doses of bifunctional cells. Anti-PD-1 IL-7 mutant D74E, W142H, SS2, and SS3 molecules, anti-PD-1 alone, or an irrelevant isotype IL-7 antibody were stained. Binding was revealed with anti-IgG-PE (Biolegend, clone HP6017) and analyzed by flow cytometry. Binding EC50 (nM) was determined for each construct and each PD-1(+) and PD-1(-) cell population (FIG. 14B). An irrelevant isotype IL-7 control is used as a negative control and shows equal binding to PD-1(+) and PD-1(-) cells. Although all bifunctional anti-PD-1 IL-7 molecules preferentially bind to PD-1(+) cells over PD-1(−) cells in this co-culture assay, we observed that IL-7 mutations enhance selective cis-binding of this molecule to PD-1+ cells. As shown in FIG. 14B, anti-PD-1 IL-7 W142H, SS2, and SS3 mutations reduced PD-1(−)CD127(+) cells compared to anti-PD-1 IL-7 wild type. anti-PD-1 IL-7 mutant showed similar strong binding to PD-1(+)CD127(+) cells as anti-PD-1 IL-7 wild type (EC50, about 300 pM). Notably, the anti-PD-1 IL-7 W142H and SS3 mutations exhibited the highest selective activity, with 62-fold and 311-fold binding between PD-1(+) and PD-1(-) cells, respectively. There was a double difference.

Taken together, these data indicate that IL-7 mutations not only allow better pharmacokinetics of the drug, but also preferential binding of IL-7 to PD-1+ cells, i. It shows that it enables the targeting of Since anti-PD-1 IL-7 concentrates IL-7 within the tumor microenvironment on PD-1+ CD127+ exhausted T cells over CD127+ naive T cells, this embodiment is useful for drug biology in vivo. subject to active activity.

Example 7: Anti-PD-1 IL-7 Mutant Bifunctional Molecules Preferentially Activate IL7R on PD-1+ Cells and Synergistically Promote Proliferation of Human Activated T Cells
IL-7R signaling activation (pSTAT5) was also tested in a co-culture model of mixed U937 PD-1(+)CD127(+) and PD-1(-)CD127(+) cells. U937 cells also expressed the endogenous CD132 receptor required for transduction of IL-7R signaling (Figure 15A). pSTAT5 signaling data show that PD-1 IL-7 mutations W142H, SS2, and SS3 have much higher selective activity in PD-1(+) cells than in PD-1(-) cells. there is 10- to 50-fold decreased activity in PD-1(-) cells for anti-PD-1 bifunctional molecules containing anti-IL7 mutations compared to anti-PD-1 bifunctional molecules containing IL-7 wild-type is observed (Fig. 15B). In PD-1(-) cells induced pSTAT5 activity was very low, whereas in PD-1(+) cells restoration of activity of anti-PD-1 bifunctional molecules containing IL-7 mutations To a similar extent as the recombinant IL-7 wild-type cytokine, the EC50 for pSTAT5 activity was close to 10 pM. In particular, the W142H mutation exhibits >450-fold greater binding/activity in PD1+ cells compared to PD-1− cells.

Since the anti-PD-1 IL-7 bifunctional molecule was designed to highly favorably target PD-1(+)CD127(+) exhausted T cells in particular, we decided to: We analyzed the ability of the anti-PD-1 IL-7 W142H bifunctional molecule to preferentially activate pSTAT5 signaling and proliferation into primary human exhausted T cells. To generate PD-1(+)CD127(+) exhausted T cells, human peripheral blood T cells were subjected to repeated stimulation in vitro (αCD3/αCD28) to mimic the chronic antigenic stimulation that occurs in the tumor microenvironment. .

To assess the targeted effects of bifunctional anti-PD-1 IL-7 molecules on PD-1(+) T cells, exhausted T cells were incubated with high concentrations of anti-PD-1 competitive antibodies and anti-PD-1 1 blocked the binding of the anti-PD-1 portion of the IL-7 bifunctional molecule. After incubation, exhausted T cells were treated with anti-PD-1 IL-7 W142H bifunctional molecule or recombinant IL-7 wild-type cytokine. pSTAT5 activation was then quantified by flow cytometry. The pSTAT5 activation ratio (EC50) between the two conditions (PD-1 blocked vs. unblocked isotype) was calculated and reported in Figure 16A. A non-targeted IL-7 recombinant cytokine was used as a negative control to give a ratio of 1 in this assay. This indicates that the activity of non-targeted IL-7 on PD-1(+) and PD-1(-) T cells was similar. Treatment with the anti-PD-1 IL-7 W142H molecule resulted in a significant difference in activity (2- to 4-fold less activity). This suggests that this molecule allows preferential cis-activation of IL-7R signaling to PD-1(+)-exhausted primary T cells over PD-1(-)-exhausted T cells. .

In addition, we show that specific cis-targeting of anti-PD-1 IL-7 W142H allows synergistic expansion of exhausted T cells in vitro, whereas two separate agents (anti-PD -1 antibody + isotype IL-7 W142H) combination induced a significant reduction in proliferation stimulation of exhausted T cells (Fig. 16B). Taken together, these data demonstrate that bifunctional molecules, including a mutant IL-7 W142H molecule and an anti-PD-1 antibody, selectively and synergistically cis-activate PD-1(+)CD127(+)-depleted T cells. support the benefits of

(Example 8: Anti-PD-1 IL-7 molecules with one IL-7 W142H cytokine and one or two anti-PD-1 arms promote cis-activation to PD-1+ IL-7R+ cells. , showed high efficacy in stimulating IL-7R T-cell proliferation in vivo, and a synergistic ability to reactivate TCR signaling)
Next, we studied the biology of multiple structures of bifunctional molecules containing one or two anti-PD-1 binding domains and one or two IL7 W142H mutations, as described in FIG. were designed and compared.

Construct 1 contains two anti-PD-1 antigen-binding domains and two IL-7 W142H variants (construct 1 is also called anti-PD-1*2 IL-7 W142H*2), and this molecule is Corresponds to the constructs tested in Examples 1-7. This molecule is also called BICKI-IL-7 W142H. In the Examples, a control molecule called BICKI-IL-7 WT corresponds to construct 1 but has wild-type IL-7.

Construct 2 contains two anti-PD-1 antigen-binding domains and a single IL-7 W142H variant (construct 2 is also called anti-PD-1*2 IL-7 W142H*1).

Construct 3 contains a single anti-PD-1 antigen binding domain and a single IL-7 W142H variant (construct 3 is also called anti-PD-1*1 IL-7 W142H*1). A control construct called anti-PD-1*1 is similar to construct 3 but lacks the IL-7 variant.

Construct 4 contains a single anti-PD-1 antigen binding domain and two IL-7 W142H variants (construct 4 is also called anti-PD-1*1 IL-W142H*2).

Constructs 2, 3, and 4 were engineered to have the IgG1 N298A isotype and amino acids in the Fc portion were modified to create knobs in heavy chain A CH2 and CH3 and holes in heavy chain B CH2 and CH3. sequence was mutated.

All anti-PD-1 IL7 constructs have high affinity for the PD-1 receptor as shown by ELISA assays (FIG. 18A and Table 7). Anti-PD-1 IL-7 molecules with two anti-PD-1 arms (anti-PD-1*2) have the same binding efficacy (equal EC50 ). Similarly, anti-PD-1 IL-7 molecules with one anti-PD-1 arm (anti-PD-1*1 IL7 W142H*1 and anti-PD-1*1 IL7 W142H*2) have IL-7. The EC50 of anti-PD-1 IL7 was equal to 86 and 111 nM, whereas the EC50 of anti-PD-1 was 238 nM, showing the same binding efficacy compared to anti-PD-1*1 without. These data indicate that IL-7 fusions do not appear to interfere with PD-1 binding, regardless of the construct tested.

Furthermore, the PD-L1/PD-1 antagonist bioassay (Fig. 18B) demonstrated that anti-PD-1 IL7 molecules with one or two anti-PD-1 arms showed binding to PD-L1 and PD-1 receptors. This indicates that it is highly effective in blocking Although one anti-PD-1 arm was removed from constructs 3 and 4, all anti-PD-1*1 IL7 constructs exhibit high antagonistic properties. EC50s for constructs 3 and 4 were calculated with only a 2.5-fold decreased activity compared to the anti-PD-1*2 IL7 construct (Table 8).

We then used Biacore and ELISA assays to assess the affinity of the different constructs for the CD127 receptor. Due to the removal of one IL-7 molecule from constructs 2 and 3, these molecules have lower binding capacity to the CD127 receptor and lower activation of pSTAT5 compared to IL-7 heterodimeric constructs. was expected. However, we found that the anti-PD-1*2 IL-7 W142H*1 molecule is anti-PD-1*2 IL-7 W142H*2 (BICKI-IL-7 W142H) against the CD127 receptor. and lower affinity compared to anti-PD-1 IL7 bifunctional molecules, including the IL-7 wild-type form (Figure 19A and Table 9). )). Surprisingly, the anti-PD-1*2 IL7 W142H*1 molecule exhibits high pSTAT5 activity similar to PD-1 IL7 bifunctional molecules, including the IL-7 wild-type form (FIG. 19B). Based on these observations, the monomeric form of IL-7 in combination with the W142H IL-7 mutation allows for optimal conformation of the IL-7 molecule to facilitate IL-7 signaling to human T cells. It is possible to hypothesize that Even in the presence of only one IL7, the molecule with the W142H IL-7 mutation shows as good an activating effect (pSTAT5) as the molecule with IL7 wt with two cytokines. This result is surprising in the context of IL-7 variants that have lower affinity for the receptor than wild-type IL-7.

Similar conclusions were drawn with an anti-PD-1 IL7 molecule constructed with one anti-PD-1 arm fused to one IL-7 W142H mutation. Similar and comparably high pSTAT5 activity was obtained (Figure 19C).

In vivo experiments were performed to determine the efficacy of various anti-PD-1 IL-7 constructs. One dose of anti-PD-1 IL-7 molecule was injected into mice at equimolar concentrations (34 nM/kg). Four days after treatment, CD4 and CD8 T cell proliferation was quantified by flow cytometry using the Ki67 marker. Figure 20 shows anti-PD-1 IL7 molecules with a single W142H mutation (anti-PD-1*2 IL-7 W142H*1 and anti-PD-1*1 IL-7 W142H*1) or single PD- An anti-PD-1 IL7 molecule with a monovalent and two IL7 W142H cytokines (anti-PD-1*1 IL7 W142H*2) showed high efficacy in promoting CD8 T cell proliferation and was more effective in promoting CD4 T cell proliferation. It has been shown to exhibit a low degree of efficacy.

To determine the ability of bifunctional molecules comprising anti-PD1 antibodies (monovalent or bivalent) and one or two IL7 mutant cytokines to reactivate TCR-mediated signaling, an NFAT bioassay was performed. . FIG. 21A shows that a bifunctional molecule constructed with two anti-PD-1 arms and one IL-7 cytokine enhances activation of NFAT compared to anti-PD-1 antibody alone. This indicates that the synergistic activity of drugs to enhance TCR-mediated signaling is preserved in anti-PD-1 IL-7 bifunctional molecules constructed with only one IL-7 cytokine. As seen in Figure 9A, treatment of cells with a combination of anti-PD1 and IL7 had no such synergistic effect.

In addition, we next evaluated the activity of anti-PD-1 IL-7 molecules designed with only one anti-PD-1 valency (anti-PD-1*1), anti-PD-1* 1 IL-7 W142H constructs (anti-PD-1*1 IL7 W142H*1 and *2) maintained synergistic activity, but combined PD-1*1 + isotype IL-7 W142H*2 treatment reduced TCR signaling It demonstrated less efficacy of transduction (NFAT activation) stimulation (FIG. 21B).

Finally, the specific cis-targeting and cis-activities of different anti-PD-1 IL-7 constructs were analyzed in co-culture assays. U937 PD-1+CD127+ cells were mixed with PD-1− CD127+ cells (1:1 ratio) and then incubated with increasing doses of different constructs. Binding and IL-7R signaling (pSTAT5) were quantified by flow cytometry. The EC50 (nM) of binding and pSTAT5 activation was determined for each construct and each PD-1+ and PD-1- cell population (Figures 22A and 22B). We found that various anti-PD-1 IL-7 mutant molecules (anti-PD-1*2 IL7 W142H*1, anti-PD-1*1 IL7 W142H*1, anti-PD-1*1 IL7 W142H* 2) substantially preferentially binds IL-7R and signals into PD-1+ cells, and in different representative structures significantly activates IL7R signaling pSTAT5 into PD-1+ cells. I confirmed that it will be changed.

(Example 9. Anti-PD-1 IL-7 molecules constructed with one or two anti-PD-1 arms and one or two IL7 W142H cytokines have good pharmacokinetic properties in vivo)
Pharmacokinetic studies of anti-PD-1 IL-7 bifunctional molecular constructs 2, 3, and 4, such as those described in Figure 17, were evaluated. Humanized PD1 KI mice were injected intraperitoneally with one dose of anti-PD-1 IL-7 molecule (34.4 nM/kg). Plasma drug concentrations were analyzed by an ELISA specific for human IgG (Figure 23). Also, the area under the curve was calculated (see Table 10). The area under the curve represents total drug exposure over time for each construct. Anti-PD-1*2 IL-7 W142H*1, anti-PD-1*1 IL-7 W142H*1, and anti-PD-1*1 IL-7 W142H*2 constructs were anti-PD-1*2 IL7WT* It showed highly favorably enhanced PK properties compared to 1. A 2.8- to 19-fold higher Cmax was observed compared to anti-PD-1*1 IL7WT*2. Importantly, the anti-PD-1*1 IL7 W142H*1 molecule, the anti-PD-1*1 IL7 W142H*2 molecule were found at high drug concentrations (11-15 nM) corresponding to sufficient PK values in vivo. are maintained for at least 96 hours, yet only 2 nM of anti-PD-1*2 IL7WT*2 molecules are detected in plasma. Residual drug concentration of anti-PD-1*2 IL-7 W142H*1 is 2.5 times that of anti-PD-1*2 IL7WT*2. Plasma drug exposure often correlates with in vivo efficacy. Here we demonstrate that all anti-PD-1 IL-7 W142H molecules constructed with one arm of anti-PD-1 allow long-term drug exposure after a single injection. ing. This suggests that such constructs will induce higher biological activity in vivo.

Also, certain molecules PD-1*2 IL7WT*2 with IL7 wild type have IL7 W142H even though they can show better PK compared to IL7 W142H (especially for intravenous injection). The molecule has other properties that are even better: better proliferation of T cells (CD4, CD8, as shown in Figure 20) and much better specific targeting of PD1+ cells compared to PD1- cells. It is also mentioned to show the reduction (10-50 fold as illustrated in Figure 15B).

Overall, multiple constructs of bifunctional molecules with mutated IL7 (especially W142H) are obtained that exhibit a sufficient PK (preferably at least 10 nM after 24 hours) that is more than sufficient for effective pharmaceutical use, They also indicate:
- having a substantial beneficial effect on LT proliferation,
- LT activation by pSTAT5 IL7R signaling to PD-1+ cells thanks to the surprising synergistic effect on T cells between the anti-PD1 part of the bifunctional molecule and the IL7 part of the bifunctional molecule high performance in terms of
- Higher specific targeting of PD1+ T cells than PD1- T cells (much higher than bifunctional molecules without mutated IL7) and PD-1 but not PD-1- T cells + cis-activate IL-7R signaling to exhausted primary T cells, which is a substantial advantage for tumor treatment, and
- have a potent antagonistic effect of the PD-1/PD-L1 interaction (as well as binding to PD1).

Materials and methods
PD1 binding ELISA
For the activity ELISA assay, recombinant hPD1 (Sino Biologicals Co., Beijing, China; reference 10377-H08H) was immobilized on plastic at 0.5 μg/ml in carbonate buffer (pH 9.2) and purified antibody was added. was used to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035-149) was added and revealed by conventional methods.

Affinity measurement using the Biacore method
Affinity evaluation by Biacore of IgG fused to IL-7 on its heavy chain for CD127 (A) or CD132 (B). CD127 (Sinobiological, 10975-H03H-50) was immobilized on CM5 biochips at 20 μg/ml and the indicated proteins were added at serial concentrations (0.35; 1.1; 3.3; 10; 30 nM). Affinities were analyzed using a two-state response model. To assess the affinity of IL-7 for CD132, CD127 was immobilized on CM5 biochips and each IL-7 construct was injected at a concentration of 30 nM. CD132 receptor (Sinobiological 10555-H08B) was added at different concentrations eg 31.25, 52.5, 125, 250, 500 nM. A steady-state affinity model was used for analysis.

CD127 binding ELISA
CD127 binding was assessed by a sandwich ELISA method. The targeted recombinant protein was immobilized by an antibody scaffold and then an IL-7 fusion antibody pre-incubated with CD127 recombinant protein (histidine tag, Sino ref 10975-H08H) was incubated. Development was performed with a mixture of biotin-conjugated anti-histidine antibody (MBL #D291-6) and peroxidase-conjugated streptavidin (JI 016-030-084). Colorimetry was determined at 450 nm using TMB substrate.

pSTAT5 Analysis PBMC isolated from peripheral blood of healthy human volunteers were incubated with recombinant IL-7 or IL-7-fused IgG for 15 minutes. To determine cis-activity, CD127+ PD-1+ transduced U937 cells were mixed with CD127+ only transduced U937 cells. Cells were mixed in a 1:1 ratio and treated with recombinant IL-7 or different IgG fused to IL-7 constructs described herein. Each cell subset was labeled with a cell proliferation dye (CPDe450 or CPDe670) prior to co-culture. Cells were then fixed, permeabilized and stained with AF647-labeled anti-pSTAT5 (clone 47/Stat5(pY694)). Data were obtained by calculating MFI pSTAT5 in the CD3+ T cell population.

Cell binding analysis
To determine cis binding of IgG fused to IL-7 molecules, CD127+ PD-1+ transduced U937 or CHO cells were mixed with CD127+ only transduced CHO or U937. Cells were mixed in a 1:1 ratio and treated with different IgGs fused to IL-7 constructs as described herein. Each cell subset was labeled with a cell proliferation dye (CPDe450 or CPDe670) prior to co-culture. After incubation for 20 minutes, binding of different IgG fusion molecules was detected with an anti-IgG-PE antibody (Biolegend, clone HP6017) and analyzed by flow cytometry.

Pharmacokinetics of IgG-fused IL-7 in vivo
To analyze the pharmacokinetics of the IL-7 immunocytokine, a single dose of the molecule was injected intraorbitally or intraperitoneally into BalbcRJ mice (females 6-9 weeks) or C57bl6JrJ mice (females 6-9 weeks). Drug concentrations in plasma were determined by ELISA using immobilized anti-human light chain antibody (clone NaM76-5F3) diluted serum containing Il67-fused IgG. Detection was with peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035-149) and revealed by conventional methods.

T cell activation assay using Promega cell-based bioassay
The ability of anti-PD-1 antibodies to restore T cell activation was tested using the Promega PD-1/PD-L1 kit (reference J1250). Two cell lines, (1) effector T cells (Jurkat stably expressing PD-1, NFAT-induced luciferase) and (2) activated target cells (stimulating cognate TCRs in an antigen-independent manner). CHO K1 cells stably expressing the designed PDL1 and surface proteins were used. When cells are co-cultured, the PD-L1/PD-1 interaction inhibits TCR-mediated activation, thereby blocking NFAT activation and luciferase activity. Addition of an anti-PD-1 antibody blocks the PD-1-mediated inhibitory signal, which leads to NFAT activation and luciferase synthesis and bioluminescence signal release. Experiments were performed according to the manufacturer's recommendations. Serial dilutions of PD-1 antibody were tested. After 4 hours of co-culture of PD-L1 + target cells, PD-1 effector cells and anti-PD-1 antibody, BioGlo™ luciferin substrate are added to the wells and the plates are plated using a Tecan™ luminometer. read.

In Vivo Growth A single dose (34 nM/kg) of the bifunctional molecule was injected intraperitoneally into subcutaneous MC38 tumor-bearing C57bl6JrJ mice (female 6-9 weeks). Four days after treatment, blood MC38 tumors were collected and T cells were stained with anti-CD3, anti-CD8, anti-CD4 and anti-ki67 antibodies to quantify proliferation by flow cytometry.

Antibodies and Bifunctional Molecules The following antibodies and bifunctional molecules were used in different experiments disclosed herein: Pembrolizumab (Keytrudra, Merck), Nivolumab (Opdivo, Bristol-Myers Squibb), and SEQ ID NO: an anti-PD1 humanized antibody comprising a variable heavy chain (VH) as defined in 24 and a variable light chain (VL) as defined in SEQ ID NO:28 or a heavy chain as defined in SEQ ID NO:71 and a light chain as defined in SEQ ID NO:72 A bifunctional molecule disclosed herein comprising an anti-PD1 chimeric antibody.

Construct 1 contains two anti-PD-1 antigen-binding domains and two IL-7 W142H variants (construct 1 is also called anti-PD-1*2 IL-7 W142H*2). This molecule corresponds to the constructs tested in Examples 1-7. This molecule is also called BICKI-IL-7 W142H. In particular, construct 1 comprises a variable heavy chain (VH) as defined in SEQ ID NO:24 and a variable light chain (VL) as defined in SEQ ID NO:28, or a heavy chain as defined in SEQ ID NO:71 and SEQ ID NO:71 72 includes anti-PD1 chimeric antibodies containing light chains as defined in 72. This molecule also includes IL7 variants such as those set forth in SEQ ID NO:5.

In the Examples, a control molecule called BICKI-IL-7 WT corresponds to construct 1 but has wild-type IL-7. It comprises a variable heavy chain (VH) as defined in SEQ ID NO:24 and a variable light chain (VL) as defined in SEQ ID NO:28. This molecule has the IgG4 S288P isotype.

Another control molecule is anti-PD1*2 (without any IL7). This molecule comprises a heavy chain as defined in SEQ ID NO:79 and a light chain as defined in SEQ ID NO:80.

Construct 2 contains two anti-PD-1 antigen-binding domains and a single IL-7 W142H variant (construct 2 is also called anti-PD-1*2 IL-7 W142H*1). In particular, construct 2 comprises a variable heavy chain (VH) as defined in SEQ ID NO:24 and a variable light chain (VL) as defined in SEQ ID NO:28. This molecule is in particular a heavy chain (hole) as defined in SEQ ID NO:83 or a heavy chain (knob) as defined in SEQ ID NO:81 and a light chain (knob) as defined in SEQ ID NO:80 bound to IL-7 W142H as defined in SEQ ID NO:80. Including chains.

Construct 3 contains a single anti-PD-1 antigen binding domain and a single IL-7 W142H variant (construct 3 is also called anti-PD-1*1 IL-7 W142H*1). In particular, construct 3 comprises a variable heavy chain (VH) as defined in SEQ ID NO:24 and a variable light chain (VL) as defined in SEQ ID NO:28. This molecule comprises a heavy chain bound to IL-7 W142H as defined in SEQ ID NO:83, an Fc region as defined in SEQ ID NO:75 and a light chain as defined in SEQ ID NO:80.

A control construct called anti-PD-1*1 is similar to structure 3 but lacks the IL-7 variant. Such controls include a variable heavy chain (VH) as defined in SEQ ID NO:24 and a variable light chain (VL) as defined in SEQ ID NO:28. This molecule comprises a heavy chain as defined in SEQ ID NO:81, an Fc region as defined in SEQ ID NO:75, and a light chain as defined in SEQ ID NO:80.

Construct 4 contains a single anti-PD-1 antigen binding domain and two IL-7 W142H variants (construct 4 is also called anti-PD-1*1 IL-W142H*2). In particular, construct 4 comprises a variable heavy chain (VH) as defined in SEQ ID NO:24 and a variable light chain (VL) as defined in SEQ ID NO:28. This molecule comprises a heavy chain bound to IL-7 W142H as defined in SEQ ID NO:83, an Fc region bound to IL-7 W142H as defined in SEQ ID NO:76, and a light chain as defined in SEQ ID NO:80. Including chains.

Constructs 2, 3, and 4 were engineered to have the IgG1 N298A isotype and amino acids in the Fc portion were modified to create knobs in heavy chain A CH2 and CH3 and holes in heavy chain B CH2 and CH3. sequence was mutated. Anti-PD-1 IL-7 and anti-PD-1*1 constructs were all constructed with IgG4 S288P isotype, anti-PD-1*2 construct (lacking IL-7) and anti-PD-1*2 IL7wt*2 ( BICKI-IL-7 WT), including IgG1N298A mutant isotypes.

Claims (24)

A bifunctional molecule comprising an interleukin 7 (IL-7) variant conjugated to a binding moiety,
- said binding moiety binds to a target specifically expressed on the immune cell surface;
- said IL-7 variant exhibits at least 75% identity with wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, said variant comprising: (i) W142H, W142F, or W142Y, (ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (iii) D74E, D74Q, or D74N, iv) Q11E, Y12F, M17L, Q22E, and/or K81R; or any combination thereof, wherein the amino acid numbering is as shown in SEQ ID NO: 1. wherein said at least one amino acid mutation i) reduces the affinity of said IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R; , ii) a bifunctional molecule that enhances the pharmacokinetics of a bifunctional molecule comprising said IL-7 variant compared to a bifunctional molecule comprising wth-IL-7. 2. The molecule of Claim 1, wherein said IL-7 variant comprises an amino acid substitution selected from the group consisting of W142H, W142F, and W142Y, wherein amino acid numbering is as set forth in SEQ ID NO:1. Said IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, and C47S-C92S and C34S-C129S, wherein the amino acid numbering is: 3. The molecule of claim 1 or 2, which is as set forth in SEQ ID NO:1. 4. The IL-7 variant of any one of claims 1-3, wherein said IL-7 variant comprises an amino acid substitution selected from the group consisting of D74E, D74Q, and D74N, wherein the amino acid numbering is as set forth in SEQ ID NO:1. Molecules described in . 5. The molecule of any one of claims 1-4, wherein said IL-7 variant comprises or consists of the amino acid sequences set forth in SEQ ID NOs:2-15. The binding moiety is optionally T250Q/M428L; M252Y/S254T/T256E+H433K/N434F; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A+M252Y/S254T/T256E; K322A; 6. A molecule according to any one of claims 1 to 5, comprising the heavy chain constant domain of human IgG1, preferably the Fc domain, with a substitution or combination of substitutions selected from the group consisting of: , and L234A/L235A. The binding portion optionally has a substitution or combination of substitutions selected from the group consisting of: S228P; L234A/L235A, S228P+M252Y/S254T/T256E.17, and K444A. 6. A molecule according to any one of claims 1 to 5, which preferably comprises an Fc domain. 8. A molecule according to any one of claims 1 to 7, wherein said immune cells are T cells, preferably exhausted T cells. The target is expressed by T cells and the binding moiety is PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, from the group consisting of Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8 9. The molecule of claim 8, which binds to a target of choice. 3. The target is expressed by T-exhausted cells and the binding moiety preferably binds to a target selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, LAG3 and TIM3. 8. The molecule according to 8. The binding portion is an antibody or an antigenic fragment thereof, and the N-terminus of the IL-7 variant is the C-terminus of the heavy chain constant domain or light chain constant domain of the antibody or antibody fragment thereof. 11. A molecule according to any one of claims 1 to 10, fused to the C-terminus of the chain constant domain, optionally via a peptide linker. Said IL-7 variant is selected from the group consisting of GGGGS (SEQ ID NO:68), GGGGSGGGS (SEQ ID NO:67), GGGGSGGGGS (SEQ ID NO:69) and GGGGSGGGGSGGGGS (SEQ ID NO:70), preferably (GGGGS) ₃ 12. The molecule of claim 11, fused to said binding moiety by a peptide linker. Said molecule comprises a first monomer and a second monomer, said first monomer C-terminally to the N-terminus of the first heterodimeric Fc chain, optionally a peptide linker wherein said first heterodimeric Fc chain is covalently linked at its C-terminus to the N-terminus of said IL-7 variant, optionally via a peptide linker 13. Any one of claims 1 to 12, wherein the second monomer comprises a complementary second heterodimeric Fc chain lacking an antigen binding domain, linked in a bond. molecule. In said second monomer, said complementary second heterodimeric Fc chain is covalently linked to said IL-7 variant, optionally via a peptide linker, preferably C 14. The molecule of claim 13, wherein the terminus is covalently linked to the N-terminus of said IL-7 variant, optionally via a peptide linker. Said molecule comprises a first monomer and a second monomer, said first monomer C-terminally to the N-terminus of the first heterodimeric Fc chain, optionally a peptide linker wherein said first heterodimeric Fc chain lacks an IL-7 variant and said second monomer lacks an antigen-binding domain comprising a complementary second heterodimeric Fc chain, said second heterodimeric Fc chain covalently linked to said IL-7 variant, optionally via a peptide linker, preferably C-terminal is linked to the N-terminus of said IL-7 variant, optionally via a peptide linker. Said molecule comprises a first monomer and a second monomer, said first monomer C-terminally to the N-terminus of the first heterodimeric Fc chain, optionally a peptide linker wherein said second monomer is to the N-terminus of a C-terminally complementary second heterodimeric Fc chain, optionally a peptide linker wherein only one of the heterodimeric Fc chains, preferably the first, is covalently linked at its C-terminus to the N-terminus of said IL-7 variant. 13. A molecule according to any one of claims 1 to 12, which is linked with. 17. A molecule according to any one of claims 13 to 16, wherein said antigen binding domain is a Fab domain, Fab', single chain variable fragment (scFV) or single domain antibody (sdAb). The antigen-binding domain comprises (i) a heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:55, 56, 57, 58, 59, 60, 61, or 62; and ( ii) comprising or consisting essentially of a light chain comprising CDR1 of SEQ ID NO: 64 or SEQ ID NO: 65, CDR2 of SEQ ID NO: 66 and CDR3 of SEQ ID NO: 16 molecule. The antigen-binding domain is
(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, or 25;
(b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28
18. A molecule according to any one of claims 13 to 17 which comprises or consists essentially of. 20. Any one of claims 13-19, wherein said antigen binding domain comprises or consists essentially of a heavy chain variable region (VH) of SEQ ID NO:24 and a light chain variable region (VL) of SEQ ID NO:28. Molecules described in . 21. An isolated nucleic acid sequence or group of isolated nucleic acid molecules encoding a bifunctional molecule according to any one of claims 1-20. 22. A host cell containing the isolated nucleic acid of claim 21. A medicament comprising a bifunctional molecule according to any one of claims 1 to 20, a nucleic acid according to claim 21, or a host cell according to claim 22, optionally together with a pharmaceutically acceptable carrier. Composition. A molecule according to any one of claims 1 to 20, a nucleic acid according to claim 21, or a nucleic acid according to claim 22, for use as a medicament, in particular for the treatment of cancer or an infectious disease. 24. The host cell of claim 23, or the pharmaceutical composition of claim 23.

Images