Dual-function anti-PD1 / IL-7 molecule

Abstract

The present invention relates to bifunctional molecules containing IL-7 variants and having specific scaffold structures, and uses thereof.

Description

[Technical Field]

[0001] This invention relates to the field of immunotherapy. The invention provides a novel scaffold structure for a bifunctional molecule containing an IL-7 variant. [Background technology]

[0002] Interleukin-7 is an immunostimulant cytokine member of the IL-2 superfamily and plays a crucial role in the adaptive immune system by promoting immune responses. This cytokine activates immune function by promoting the survival and differentiation of T and B cells, the survival of lymphocytes, and the stimulation of natural killer (NK) cell activity. IL-7 also regulates lymph node development via lymphoid tissue inducer (LTi) cells and promotes the survival and division of naive T cells or memory T cells. Furthermore, IL-7 enhances the human immune response by promoting the secretion of IL-2 and interferon-γ. The IL-7 receptor is a heterodimer consisting of IL-7Rα (CD127) and a common γ chain (CD132). While the γ chain is expressed in all hematopoietic cell types, IL-7Rα is primarily expressed in lymphocytes, including B and T lymphocyte progenitor cells, naive T cells, and memory T cells. IL-7Rα expression in regulatory T cells has been observed to be lower compared to effector / naive T cells that express it at higher levels. Therefore, CD127 is used as a surface marker to distinguish these two populations. IL-7Rα is also expressed in innate lymphoid cells as NK cells and intestinal lymphoid tissue (GALT)-derived T cells. The IL-7Rα (CD127) chain is shared with TSLP (tumor stromal lymphopoietin), and the CD132 (γ chain) is shared with IL-2, IL-4, IL-9, IL-15, and interleukin-21. Two major signaling pathways: (1) the Janus kinase / STAT pathway (i.e., Jak-Stat-3 and 5) and (2) the phosphatidyl-inositol-3 kinase pathway (i.e., PI3K-Akt) are induced by CD127 / CD132. IL-7 administration is well tolerated by patients and leads to increased proliferation of CD8 and CD4 cells, as well as a relative decrease in CD4+ T regulatory cells. Recombinant naked IL-7 or IL-7 fused to the N-terminal domain of the Fc of an antibody is being tested clinically. The latter is based on the rationale that fusion with the Fc domain extends the half-life of IL-7, thereby enhancing the long-term effectiveness of the treatment.

[0003] Recombinant IL-7 cytokines have a poor pharmacokinetic profile, limiting their clinical use. After injection, recombinant IL-7 is rapidly distributed and eliminated, resulting in a poor half-life in humans (ranging from 6.8 to 9.5 hours) (Sportes et al., Clin Cancer Res. January 15, 2010; Vol. 16 (No. 2): pp. 727-735) or mice (2.5 hours) (Hyo Jung Nam et al., Eur. J. Immunol. 2010. Vol. 40: pp. 351-358). Fusion of IL-7 with an IgG Fc domain extends the half-life of IL-7. This is because IgG can bind to the neonatal Fc receptor (FcRn) and participate in molecular transcytosis and endosomal recycling. In the case of IL-7 Fc fusion molecules, an extension of the circulating half-life was observed in mice (t1 / 2 = 13 hours), and the IL-7 Fc fusion molecule remained at a detectable level (200 pg / mL) up to 8 days after administration (Hyo Jung Nam et al., Eur. J. Immunol. 2010, Vol. 40: pp. 351-358). Although the half-life of IL-7 cytokines fused to the Fc domain is increased, high-frequency in vivo injection of this molecule was necessary to obtain biological effects. In the context of immune cytokine molecules, cytokines are fused to antibodies that preferentially enrich the cytokines against cells expressing target antigens (e.g., targeting cancer antigens, immune checkpoint blockades, co-stimulatory molecules, etc.). However, the affinity of IL-7 cytokines to CD127 / CD132 receptors (in the nanomolar to picomolar range) may be higher than the affinity of antibodies to the target. In this case, cytokines drive the pharmacokinetics of the product, leading to rapid depletion of available drugs in vivo through the target-mediated pharmacokinetic (TMDD) mechanism. This rapid elimination has been described for immune cytokines such as IL-2 or IL-15, demonstrating that the pharmacokinetic properties of fusion proteins can directly affect drug performance (List et Neri Clin Pharmacol. 2013; Vol. 5 (Supplement 1): pp. 29-45). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2020 / 127377 [Patent Document 2] International Publication No. 2019144945A1 [Patent Document 3] International Publication No. 2006061219 [Patent Document 4] International Publication No. 2014 / 194302 [Patent Document 5] International Publication No. 2017 / 040790 [Patent Document 6] International Publication No. 2017 / 19846 [Patent Document 7] International Publication No. 2017 / 024465 [Patent Document 8] International Publication No. 2017 / 025016 [Patent Document 9] International Publication No. 2017 / 132825 [Patent Document 10] International Publication No. 2017 / 133540 [Patent Document 11] International Publication No. 2006 / 121168 [Patent Document 12] International Publication No. 2020 / 127366 [Patent Document 13] International Publication No. 96 / 34103 [Patent Document 14] International Publication No. 94 / 04678 [Patent Document 15] International Publication No. 2014 / 145806 [Patent Document 16] U.S. Patent No. 8,216,805 [Patent Document 17] U.S. Patent Application Publication No. 2012 / 0149876 [Patent Document 18] International Publication No. 01 / 58957 [Patent Document 19] International Publication No. 2018 / 053106, pp. 36-43 [Non-licensed literature]

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[0006] Therefore, there remains a significant need in the art for new and improved IL-7 variants that improve distribution and reduce the exclusion of IL-7 products, particularly bifunctional molecules containing IL-7. The inventors have made significant progress with the invention disclosed herein.

[0007] Bifunctional molecules are currently a development goal in the fields of immunology, particularly oncology. Indeed, they offer novel pharmacological properties through simultaneous engagement with two targets, can enhance the safety profile through targeted redistribution to tumors compared to combinations of two distinct molecules, and can potentially reduce the development and manufacturing costs associated with single formulations. However, while these molecules offer advantages, they can also present some drawbacks. The design of bifunctional molecules must encompass several key attributes, such as binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector function, compatibility with the binding of additional domains, and production yield and cost commensurate with clinical development. For example, a bifunctional molecule targeting PD-1 and possessing an IL-7 variant is described in International Publication No. 2020 / 127377.

[0008] However, there is still a strong need for improved scaffold structures for bifunctional molecules. [Means for solving the problem]

[0009] The inventors provide IL-7 mutations and optimized scaffold structures to enhance the biological effects in vivo by improving the distribution and exclusion of bifunctional molecules. The inventors observed that IL-7 mutations, when combined with optimized scaffold structures, enable better distribution and longer in vivo half-lives of bifunctional molecules.

[0010] In particular, the bifunctional molecules provided herein exhibit favorable pharmacokinetics and pharmacodynamics in vivo, especially compared to bifunctional molecules including IL-7 wild-type. Furthermore, as detailed below and in the examples, these novel molecules are associated with advantageous and unexpected properties.

[0011] The present invention relates to a bifunctional molecule having a specific scaffold structure and comprising an interleukin 7 (IL-7) variant (IL-7m) conjugated to a binding portion that binds to PD-1, wherein the scaffold structure essentially consists of a dimeric Fc domain, a single monovalent antigen-binding domain that binds to PD-1 at the N-terminus of one monomer of the Fc domain, and either i) a single IL-7m is linked at the C-terminus of the same monomer of the Fc domain, or ii) the single monovalent antigen-binding domain comprises a variable heavy chain and a variable light chain, and a single IL-7m is linked at the C-terminus of the light chain of the antigen-binding domain.

[0012] This particular scaffold structure is associated with an improvement in the pharmacokinetic profile. This improvement is surprising because, in the absence of IL-7m, no improvement is observed with respect to this scaffold structure.

[0013] This bifunctional molecule with a specific scaffold structure is advantageous for cis-targeting of two targets on the same cell, enabling selective delivery of IL-7m to target cells.

[0014] Furthermore, in the context of bifunctional molecules containing IL-7, these molecules can induce synergistic activation and good in vivo antitumor efficacy. Finally, surprisingly, bifunctional molecules with specific scaffold structures exhibit better productivity and avoid by-products due to mispairing of chains, which is a major advantage for industrial-scale production and safety.

[0015] Furthermore, in addition to improved pharmacokinetic profiles and better productivity, novel and advantageous biological efficiencies have been identified that enhance the activity of effector memory stem cell-like T cells, a subset of tumor-reactive intratumor T cells that are crucial for their immunological activity.

[0016] In certain aspects of this disclosure, the bifunctional molecule comprises one IL7 variant, in particular the W142H IL7 variant, and one antigen-binding domain specific to PD-1 having a particular scaffold structure, i.e., the molecule is called anti-PD-1*1 IL7 W142H*1. This bifunctional molecule is characterized by one monovalent antigen-binding domain having high affinity and antagonist activity to PD-1 on one side, and one IL7 variant having low affinity to the IL7 receptor (IL7R) on the other side.

[0017] Surprisingly, this bifunctional molecule (anti-PD-1*1 IL7 W142H*1) exhibits better preferential targeting of tumor-specific T cells (cis-targeting) (315 times compared to 58 times; see Figure 11C) than the same molecule with wild-type IL7. In addition, this molecule blocks the inhibitory activity of Tregs, yielding better results in neutralizing Treg-induced inhibition than the same molecule with wild-type IL7 (see Figure 19). While this bifunctional molecule (anti-PD-1*1 IL7 W142H*1) shows a dual effect affecting both Treg neutralization and strong T cell proliferation, it was unexpected that the selected IL7 variant in this molecule exhibited low affinity for the IL7 receptor.

[0018] As is well known to those skilled in the art, tumor cells may not be adequately eliminated by T cells due to a phenomenon called T cell exhaustion, which is observed in many cancers. For example, as described by Jiang, Y., Li, Y., and Zhu, B (Cell Death Dis 6, e1792 (2015)), exhausted T cells in the tumor microenvironment may be associated with overexpression of inhibitory receptors, decreased effector cytokine production and cytolytic activity, leading to failure to eliminate cancer and, in general, immune evasion of cancer. Therefore, the recovery of exhausted T cells is a clinical strategy conceived for cancer treatment.

[0019] Even when exhausted T cells have reduced IL7R expression and the molecule's IL7 variant has low affinity for IL7R, this bifunctional molecule (anti-PD-1*1 IL7 W142H*1) restores the proliferation of chronically stimulated T cells and maintains the survival of exhausted T cells at the same level as recombinant IL7. Surprisingly, this bifunctional molecule (anti-PD-1*1 IL7 W142H*1) can protect T lymphocytes from apoptosis. In the context of reduced IL7R expression and lower affinity for IL7R, these effects are remarkable and unpredictable.

[0020] Surprisingly, this bifunctional molecule (anti-PD-1*1 IL7 W142H*1) can induce proliferation of stem cell-like TCF1+ CD8 T cells (Ki67+ TCF1+ CD8+ T cells) (Figure 24) at the same level as rIL7 (data not shown).

[0021] Recently, Caushi et al. (2021, Nature, Vol. 596, pp. 126-132) examined mutation-associated neoantigen (MANA)-specific CD8 T cells in the context of patients unresponsive to anti-PD-1 treatment and observed low levels of IL7R in these tumor-specific TILs (tumor-infiltrating lymphocytes). Conveniently, despite the low affinity for IL7R and reduced IL7R expression in exhausted T cells, the bifunctional molecule (anti-PD-1*1 IL7 W142H*1) not only restores proliferation but also maintains the survival of chronically stimulated T cells.

[0022] Four different preclinical in vivo models are being used for further therapeutic validation: two different immunocompetent models (PD1-sensitive model (AK7 orthotopic); and PD1-resistant model (Hepa1.6 orthotopic) (Figures 20 and 21)); and two different humanized models (TNBC ectopic model and ectopic lung cancer). In these in vivo preclinical models, the bifunctional molecule (anti-PD-1*1 IL7 W142H*1) increases survival and reduces or completely inhibits tumor growth. Surprisingly, improved therapeutic efficacy has been observed compared to the same molecule with wild-type IL7 or an anti-PD-1 antibody.

[0023] In particular, the PD1-resistant Hepa1.6 model is especially important due to tumor T cell removal from the tumor (Gauttier et al., 2020, Clin Invest, Vol. 130, pp. 6109-6123). In this model, anti-PD1 was expected to have no efficacy, but the bifunctional molecule (anti-PD-1*1 IL7 W142H*1) achieved a complete tumor response in 60% of cases, clearly superior to the same molecule with wild-type IL7 (47%) (Figure 21). In addition, the molecule can promote the selective expansion and proliferation of stem cell-like memory CD8+ TILs (Figure 22). This is particularly important in the context of this model demonstrating tumor T cell removal. Despite T cell removal from the tumor in this resistance model, the CD8 TIL composition increased sharply after treatment with the bifunctional molecule (anti-PD-1*1 IL7 W142H*1), and the T cell subset was dramatically modified. A subset of stem cell-like memory T cells with very low Treg, high CD8+ T cell counts, and high proliferative activity was observed (Figures 23 and 24). Conversely, this molecule provides significantly lower levels of exhausted T cells compared to anti-PD-1 treatment. This strong advantage, particularly in TILS, is illustrated in detailed examples demonstrating intratumoral proliferation of a stem cell-like memory CD8 T cell subpopulation (TCF1+Tox- cells) by this bifunctional molecule. This proliferation allows for the acquisition of an active pool of stem cell-like memory T cells. These T stem cells represent an intermediate differentiation stage between i) naive T cells (which are PD1-negative) that are not tumor antigen-specific or are abundant against tumor cells in the presence of anti-PD1 drugs, and ii) mature exhausted T cells (in particular, already fully exhausted T cells) that are not abundant enough or are overstimulated and no longer respond to anti-PD1 treatment. These exhausted T cells are stressed cells and undergo apoptosis, as exemplified by their genetic profile, but a pool of stem cell-like memory T cells (TCF1+) can proliferate and differentiate into numerous T cells, enabling long-term persistence along with a higher antitumor response.

[0024] A broad memory antitumor response was obtained with the bifunctional molecule (anti-PD-1*1 IL7 W142H*1), as demonstrated by re-challenging mice cured of the same tumor type (three tumor models were tested: a PD1-sensitive model (AK7 orthotopic); a PD1 partially sensitive model (MC38 ectopic); and a PD1-resistant model (Hepa1.6 orthotopic)). These pre-treated animals did not develop any new tumors without any further treatment, establishing the ability of the bifunctional molecule (anti-PD-1*1 IL7 W142H*1) to confer long-term protection through the activation of memory T cell subsets (Figure 20B).

[0025] In a humanized mouse model of TNBC, the activity of a bifunctional molecule (anti-PD-1*1 IL7 W142H*1) was evaluated using humanized conditions with human peripheral blood mononuclear cells (PBMCs) from four different human donors. The effect of the bifunctional molecule (anti-PD-1*1 IL7 W142H*1) on tumor growth was clearly superior to that of an anti-PD-1 antibody, strongly reducing tumor growth (Figure 25). The same superior effect was observed in another humanized mouse model (lung cancer), particularly in a model with increased serum IFN-gamma secretion, indicating a direct effect of the induced response (Figure 26).

[0026] In summary, despite low affinity for IL7R on target cells and reduced IL7R expression, the bifunctional molecule (anti-PD-1*1 IL7 W142H*1) can revitalize the cancer immune cycle by synergistically affecting TCR signaling, inhibiting Tregs while promoting the expansion and proliferation of T effectors, facilitating mucosal T cell migration, and finally revitalizing exhausted T cells. Surprisingly, the effect was superior to or equivalent to that of the same molecule with recombinant IL7 or wild-type IL7.

[0027] The present invention is a bifunctional molecule comprising a single antigen-binding domain and a single IL-7 variant, - The bifunctional molecule comprises a first monomer containing an antigen-binding domain whose C-terminus is optionally covalently linked to the N-terminus of a first Fc chain via a peptide linker, and a second monomer containing a complementary second Fc chain lacking the antigen-binding domain and the IL-7 variant; - i) an IL-7 variant is optionally covalently linked to the C-terminus of the first Fc chain via a peptide linker; or ii) a single antigen-binding domain comprises a variable heavy chain and a variable light chain, with the IL-7 variant covalently linked to the C-terminus of the light chain; - The antigen-binding domain binds to PD-1; and - The IL-7 variant exhibits at least 75% identity with wild-type human IL-7 (wth-IL-7), which contains or consists of the amino acid sequence shown in SEQ ID NO: 1, and the IL-7 variant i) reduces the affinity of the IL-7 variant to the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 to IL-7R, and ii) improves the pharmacokinetics of the bifunctional molecule containing the IL-7 variant compared to the bifunctional molecule containing wth-IL-7. Regarding bifunctional molecules.

[0028] In particular, the IL-7 variant includes at least one amino acid mutation selected from the group consisting of (i) W142G, W142A, W142V, W142C, W142L, W142I, W142M, W142H, W142Y, and W142F, preferably 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, with the amino acid numbering as shown in Sequence ID No. 1.

[0029] Preferably, the IL-7 variant includes an amino acid substitution selected from the group consisting of W142H, W142F, and W142Y, with the amino acid numbering as shown in SEQ ID NO: 1. More preferably, the IL-7 variant includes the amino acid substitution W142H. Even more preferably, the IL-7 variant includes or consists of the amino acid sequences shown in SEQ ID NOs: 2 to 15. Most preferably, the IL-7 variant includes or consists of the amino acid sequence shown in SEQ ID NO: 5.

[0030] In certain embodiments, the IL-7 variant is preferably ligated at its N-terminus to the C-terminus of the first Fc chain.

[0031] In another embodiment, the bifunctional molecule 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;L234A / L235A;N297A+M252Y / S254T / T256E(YTE);N297A+N298A+ M252Y / S254T / T256E+ The heavy chain constant domain of human IgG1, preferably the Fc domain, is selected from the group consisting of K444A, K322A, K444A, K444E, K444D, K444G, K444S, P329GL234A / L235A / P329G, M428L, L309D, Q311H, N434S, M428L+N434S(LS), and L309D+Q311H+N434S(DHS), preferably N297A which is optionally combined with M252Y / S254T / T256E, and L234A / L235A or L234A / L235A / P329G. Preferably, the substitution or combination of substitutions is T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S Selected from the group consisting of 239D / I332E / G236A;N297A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A and K444A, preferably selected from the group consisting of N297A combined with M252Y / S254T / T256E by optional selection, and L234A / L235A.

[0032] Alternatively, the bifunctional molecule contains a heavy chain constant domain of human IgG4, preferably an Fc domain, having a substitution or combination of substitutions selected from the group consisting of S228P;L234A / L235A, S228P+M252Y / S254T / T256E+K444A, K444E, K444D, K444G, K444S, P329G, and L234A / L235A / P329G. Preferably, the substitution or combination of substitutions is selected from the group consisting of S228P;L234A / L235A, S228P+M252Y / S254T / T256E, and K444A.

[0033] In certain embodiments, the bifunctional molecule according to the present invention comprises an Fc domain derived from IgG1 or IgG4 containing mutant LALA (L234A / L352A) or LALA PG (L234A / L235A / P329G).

[0034] In one embodiment, the first Fc chain and the second Fc chain form a heterodimeric Fc domain, particularly a knob-into-hole heterodimeric Fc domain. Specifically, the first Fc chain is a hole chain or H chain and contains the substitutions T366S / L368A / Y407V / Y349C and optionally N297A, and the second Fc chain is a knob chain or K chain and contains the substitutions T366W / S354C and optionally N297A. Preferably, the first Fc chain is a hole chain or H chain and contains the substitutions T366S / L368A / Y407V / Y349C and N297A, and the second Fc chain is a knob chain or K chain and contains the substitutions T366W / S354C and N297A. More specifically, the second Fc chain contains or consists of SEQ ID NO: 75, and / or the first Fc chain contains or consists of SEQ ID NO: 77.

[0035] In a very specific embodiment, the bifunctional molecule according to the present invention comprises a first monomer having an antigen-binding domain whose C-terminus is optionally covalently linked to the N-terminus of a first heterodimer Fc chain via a peptide linker, wherein the first heterodimer Fc chain comprises a first monomer having its C-terminus optionally covalently linked to the N-terminus of an IL-7 variant via a peptide linker, and a second monomer having a complementary second heterodimer Fc chain lacking an antigen-binding domain.

[0036] In another embodiment, in the bifunctional molecules disclosed herein, 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 GGGGSGGGGSGGGGGS (SEQ ID NO: 70), and is fused to the antigen-binding domain or Fc domain by a peptide linker, preferably (GGGGS)3. Preferably, the IL-7 variant is fused to the antigen-binding domain or Fc domain by a peptide linker of SEQ ID NO: 70.

[0037] In one embodiment, the antigen-binding domain in the bifunctional molecule according to the present invention is a Fab domain, Fab', a single-chain variable fragment (scFV), or a single-domain antibody (sdAb). Preferably, the antigen-binding domain is a Fab domain or Fab'.

[0038] In particular, the antigen-binding domains are found in pembrolizumab, nivolumab, pizilizumab, semiprimab, camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317, spartalizumab, MK-3477, SCH-900475, PF-06801591, JNJ-63723283, genolimuzumab, LZM-009, BCD-100, SHR-1201, BAT- The antigen-binding domain is derived from an antibody selected from the group consisting of 1306, AK-103, MEDI-0680, MEDI0608, JS001, BI-754091, CBT-501, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, AGEN2034, MGA012, or IBI308, 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4. Preferably, the antigen-binding domain is derived from an antibody selected from the group consisting of pembrolizumab, nivolumab, pizilizumab, semiprimab, camrelizumab, spartalizumab, and genolimuzumab. More preferably, the antigen-binding domain is derived from pembrolizumab or nivolumab.

[0039] In a very specific embodiment, the antigen-binding domain of the bifunctional molecule according to the present invention comprises or essentially comprises (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, or 62; and (ii) a light chain containing CDR1 of SEQ ID NOs: 64, 65, 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16 or 90. Preferably, the antigen-binding domain comprises or essentially comprises (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 61; and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16.

[0040] More preferably, the antigen-binding domain comprises or essentially consists of (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; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 88, or SEQ ID NO: 99. Even more preferably, the antigen-binding domain comprises or essentially consists of the heavy chain variable region (VH) of SEQ ID NO: 24 and the light chain variable region (VL) of SEQ ID NO: 28.

[0041] In a very specific embodiment, the bifunctional molecule according to the present invention comprises an antigen-binding domain comprising or essentially the heavy chain variable region (VH) of SEQ ID NO: 24 and the light chain variable region (VL) of SEQ ID NO: 28, and an amino acid substitution W142H, the amino acid numbering as shown in SEQ ID NO: 1, and preferably an IL-7 variant comprising or essentially the SEQ ID NO: 5.

[0042] Preferably, in the bifunctional molecule according to the present invention: (i) The antigen-binding domain includes or is essentially derived from the heavy chain variable region (VH) of SEQ ID NO: 24 and the light chain variable region (VL) of SEQ ID NO: 28. (ii) The IL-7 variant contains or essentially consists of the sequence specified in SEQ ID NO: 5 (iii) The second Fc chain contains or essentially consists of SEQ ID NO: 75, and / or the first Fc chain contains or consists of SEQ ID NO: 77.

[0043] Preferably, such a bifunctional molecule further comprises the peptide linker of SEQ ID NO: 70.

[0044] In a very specific embodiment, the bifunctional molecule according to the present invention comprises a first monomer of SEQ ID NO: 83, a second monomer of SEQ ID NO: 75, and a third monomer of SEQ ID NO: 37, 38, or 80, preferably SEQ ID NO: 38 or 80. Preferably, the bifunctional molecule comprises a first monomer containing or consisting of SEQ ID NO: 83, a second monomer containing or consisting of SEQ ID NO: 75, and a third monomer containing or consisting of SEQ ID NO: 80.

[0045] The present invention also relates to a group of isolated nucleic acid sequences or isolated nucleic acid molecules that encode the bifunctional molecules according to the present disclosure.

[0046] The present invention also relates to a host cell comprising an isolated nucleic acid sequence or a group of isolated nucleic acid molecules encoding the bifunctional molecules according to this disclosure.

[0047] The present invention further relates to a pharmaceutical composition comprising a bifunctional molecule, nucleic acid, or host cell according to this disclosure, together with an optionally pharmaceutically acceptable carrier.

[0048] Finally, the present invention relates to a bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition according to the Disclosure for use as a pharmaceutical, particularly for use in the treatment of cancer or infectious diseases; the use of a bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition according to the Disclosure for use in the manufacture of a pharmaceutical, particularly for use in the treatment of cancer or infectious diseases; and a method for treating a disease in a subject, particularly cancer or infectious disease, comprising administering a therapeutically effective amount of a bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition according to the Disclosure.

[0049] Optionally, the present invention relates to a bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition according to the present disclosure for use in the treatment of cancer or viral infection by stimulating effector memory stem cell-like T cells; the use of a bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition according to the present disclosure for the manufacture of a pharmaceutical, particularly for use in the treatment of cancer or viral infection by stimulating effector memory stem cell-like T cells; and a method for treating cancer or viral infection in a subject, comprising administering a therapeutically effective amount of a bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition according to the present disclosure, thereby stimulating effector memory stem cell-like T cells.

[0050] In detail, cancer includes hematopoietic cancers, solid tumors, carcinomas, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, urethral cancer, environment-induced cancers and any combination thereof, metastatic or non-metastatic, melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric or gastroesophageal cancer, cervical cancer, hematopoietic neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndrome, acute myeloid leukemia, Kaposi's sarcoma; cervix, anus, penis The group consists of squamous cell carcinoma of the vulva and oropharyngeal cancer 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, classical Hodgkin lymphoma, and lymphoproliferative disorders associated with Epstein-Barr virus (EBV) and / or Kaposi's sarcoma herpesvirus; hepatocellular carcinoma associated with hepatitis B and / or C virus; Merkel cell carcinoma associated with Merkel cell polyomavirus (MPV); and cancers associated with human immunodeficiency virus infection (HIV).

[0051] More specifically, viral infections are caused by viruses selected from the group consisting of HIV, hepatitis viruses, e.g., hepatitis A, B, or C, herpesviruses, e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein-Barr virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

[0052] Finally, the present invention relates to a therapeutic agent or a bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition for use in combination with a therapeutic agent selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, anti-angiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes derived from tumor antigens, bone marrow checkpoint inhibitors, immunotherapy, and HDAC inhibitors. In particular, the therapeutic agent is an immune checkpoint blocker or adaptive immune cell (T and B lymphocyte) activator selected from the group consisting of anti-CTLA4, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonist, CD40-L, TLR agonist, anti-ICOS, ICOS-L, and B cell receptor agonist. Such combinations can be used, in particular, for the treatment of cancer or viral infections as disclosed herein. [Brief explanation of the drawing]

[0053] [Figure 1] This is a schematic diagram of the various molecules used in Examples 1 and 2. [Figure 2]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 antibodies were added at different concentrations. Chromogenic development was performed using peroxidase-bound anti-human Fc antibody. Colorimetric analysis was determined at 450 nm using TMB substrate. Anti-PD-1 with one (anti-PD-1*1 gray triangle, gray) or two anti-PD-1 arms (anti-PD-1*2, black diamond) was tested as a control. B. Bifunctional molecules containing IL7 variants (anti-PD-1*2 IL7 W142H*2, black circle, black), (anti-PD-1*2 IL7 W142H*1, black square, black), (anti-PD-1*1 IL7 W142H*2, gray circle, gray), and (anti-PD-1*1 IL7 W142H*1, gray inverted triangle, gray) were also tested. B. Antagonistic ability to block PD-1 / PD-L1 as measured by ELISA. PD-L1 was immobilized, and the complex antibody + biotinylated recombinant human PD-1 was added. This complex contains a constant concentration of PD1 (0.6 μg / mL) and different concentrations of anti-PD1*2 IL7 W142H*1 (black square, solid line), anti-PD1*2 IL7 W142H*2 (white circle, dashed line), anti-PD-1*1 (gray, gray triangle, gray dashed line), anti-PD1*1 IL7 W142H*2 (gray, gray circle, gray solid line), or anti-PD1*1 IL7 W142H*1 (gray, gray inverted triangle, gray solid line). All tested constructs contain the GGGGSGGGGSGGGGS linker between the Fc domain and the IL-7 domain. [Figure 3A]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. Recombinant PD-1 protein was immobilized, and then different concentrations of the bifunctional molecule and a fixed amount of recombinant CD127 protein (histidine-tagged, Sino reference number 10975-H08H) were added. Chromogenic development was performed with a mixture of biotin-bound anti-histidine antibody and peroxidase-bound streptavidin. Colorimetric analysis was determined at 450 nm using a TMB substrate. Anti-PD1*2 IL7 W142H1*1 (black square) or anti-PD1*2 IL7 W142H*2 (black circle, gray) were tested. [Figure 3B] Anti-PD-1 IL7 molecules constructed with monovalent or bivalent anti-PD-1 and one IL-7 W142H cytokine activate pSTAT5 with high efficacy. B. pSTAT5 signaling assay using an anti-PD-1*2 scaffold fused to the IL-7 W142*1 cytokine. Human PBMCs isolated from the peripheral blood of healthy volunteers were incubated with anti-PD1*2 IL7 WT*2 (black inverted triangle) or anti-PD1*2 IL7 W142H*1 (black square, dashed line) for 15 minutes. The 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+ cell percentage relative to the CD3+ population. [Figure 3C] Anti-PD-1 IL7 molecules constructed with monovalent or bivalent anti-PD-1 and one IL-7 W142H cytokine activate pSTAT5 with high efficacy. C. pSTAT5 signaling assay after treatment with anti-PD-1*1 IL7 W142H*1 (black circle), anti-PD-1*2 IL7WT*2 (black square), or anti-PD1*2 IL7 W142H*1 (black triangle). All tested W142H constructs contained the GGGGSGGGGSGGGGS linker between the IgG1m and Fc domains and the IL-7 domain. [Figure 4]Anti-PD-1 IL-7 molecules, constructed in monovalent or bivalent form, significantly promote T cell proliferation in vivo. Mice were intraperitoneally injected with a single 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 an isotype control. 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 the 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 and n=2-8 mice per group in two independent experiments. [Figure 5] Anti-PD-1*2 IL7*1, anti-PD-1*1 IL7*1, and anti-PD-1*1 IL7*2 synergistically activate TCR signaling. Promega PD-1 / PD-L1 Bioassay: (1) Effector T cells (Jarcut cells that stably express PD-1 and NFAT-induced luciferase) and (2) Activation Target Cells (CHO K1 cells that stably express PDL1 and surface proteins designed to activate cognitive TCRs in an antigen-independent manner) were co-cultured. After adding BioGlo® luciferin, luminescence was quantified to represent T cell activation. A. Anti-PD1*2 (black circle, black) and anti-PD-1*2 IL7 W142H*1 (white circle, white) were added at serial concentrations. Isotype antibodies were used as negative controls for activation (black squares). B. Combination of anti-PD1*1 + isotype IL7 W142H*2 control (white circle, white dashed line), anti-PD-1*1 IL7 W142H*2 (black circle, gray), anti-PD-1*1 IL7 W142H*1 (white circles, gray) was added at sequential concentrations. All tested W142H constructs contained IgG1m and the GGGGSGGGGSGGGGS linker between the Fc domain and the IL-7 domain. [Figure 6]The anti-PD-1*2 IL7*1, anti-PD-1*1 IL7*1, and anti-PD-1*1 IL7*2 W142H mutations preferentially bind to PD-1+ CD127+ cells rather than PD-1- CD127+ cells, activating pSTAT5 signaling. U937 cells expressing CD127+ or cells co-expressing CD127+ and PD-1+ were stained with cell proliferation dye (CPDe450 or CPDe670), co-cultured in a 1:1 ratio, and then incubated with different concentrations of the anti-PD-1 IL-7 bifunctional molecule. After incubation, the cells were stained 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 a bifunctional molecule, 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: U937 PD-1+ CD127+ (white histogram) and U937 PD-1- CD127+ (black histogram). N=2 independent experiments. This assay tested anti-PD-1*2 IL7 W142*1, anti-PD-1*1 IL7 W142*1, and anti-PD-1*1 IL7 W142*2. These include the IgG1m isotype and the GGGGSGGGGSGGGGS linker between the Fc domain and the IL-7 domain. [Figure 7] Pharmacokinetics of anti-PD-1*2 IL7*1, anti-PD-1*1 IL7*1, and anti-PD-1*1 IL7*2 W142H mutant molecules after intraperitoneal injection. Humanized PD1 mice were intraperitoneally injected with a single dose (34 nM / kg) of anti-PD-1*2 IL7 IL7*2 IgG4m (white triangle), anti-PD-1*2 IL7 W142H*1 IgG1m (black inverted triangle), anti-PD-1*1 IL7 W142H*1 IgG1m (black circle, gray), or anti-PD-1*1 IL7 W142H*2 IgG1m (white circle, gray). Serum drug concentrations were evaluated by ELISA up to 72 hours after injection. [Figure 8]Productivity of anti-PD-1 / IL7 bifunctional antibodies in mammalian cells. CHO-S cells were transiently transfected with DNA encoding anti-PD-1*2 / IL7*1 or anti-PD-1*1 / IL7*1 molecules in a ratio of (1:3:3; strand A: strand B: VL). The supernatant containing the antibodies was purified using protein A chromatography. The amount of bifunctional antibodies obtained after purification was quantified using UV spectroscopy (DO 280 nm) and normalized to the production volume. Productivity of raw data for construction. [Figure 9A] Size exclusion chromatography of anti-PD-1*1 / IL-7wt*1. Purified antibodies were separated by size using gel filtration chromatography with SuperDex 200 (10 / 300GL). Peaks corresponding to aggregates, heterodimer antibodies, and Fc homodimers are graphically represented along with the calculated percentage of each compound. [Figure 9B] Size exclusion chromatography of anti-PD-1*1 / IL-7v*1. Purified antibodies were separated by size using gel filtration chromatography with a SuperDex 200 (10 / 300GL). Peaks corresponding to aggregates, heterodimer antibodies, and Fc homodimers are graphically represented along with the calculated percentage of each compound. [Figure 10]The anti-PD-1* / IL7*1 molecule activates pSTAT5 with high efficiency. Figure 10A. pSTAT5 signaling assay of human primary T cells treated with anti-PD-1*1 / IL-21*1, anti-PD-1*1 / IL-15*1, anti-PD-1*1 / IL-7wt*1, and anti-PD-1*1 / IL-7v*1. Human PBMCs isolated from peripheral blood of healthy volunteers were incubated with the molecules for 15 minutes. The cells were then fixed, permeabilized, and stained with anti-CD3-BV421 and anti-pSTAT5 AF647 (clone 47 / Stat5(pY694)). Data correspond to %pSTAT5+ cells relative to the CD3+ population. Figure 10B. pSTAT5 signaling in human CD127+ CD132+ U937 cell lines after treatment with anti-PD-1*2 IL7v*2 (black inverted triangle) or anti-PD-1*1 IL7v1*1 (white triangle) molecules. The graph on the left corresponds to the percentage of pSTAT5+ cells, and the graph on the right corresponds to the EC50 (nM) calculated based on the concentration required to reach 50% pSTAT5 activation. Data represent the mean + / - SD of three independent experiments. [Figure 11A] The anti-PD-1*1 / IL-7*1 molecule preferentially binds to PD-1+ cells with higher efficiency than PD-1- cells. Figure 11A. Binding of the anti-PD-1*1 / IL-7wt*1 molecule in PD-1+ CD127+ U937 cells (solid line) versus PD-1- CD127+ U937 cell line (dashed line). [Figure 11B] The anti-PD-1*1 / IL-7*1 molecule preferentially binds to PD-1+ cells with higher efficiency than PD-1- cells. Figure 11B. Binding of the anti-PD-1*1 / IL-7v*1 molecule in PD-1+ CD127+ U937 cells (solid line) versus PD-1- CD127+ U937 cell line (dashed line). [Figure 11C]The anti-PD-1*1 / IL7*1 molecule preferentially binds to PD-1+ cells with higher efficiency than PD-1- cells. Figure 11C. Comparison of pSTAT5 activation in PD-1+ CD127+ cells versus PD-1- CD127+ cells. The EC50nM ratio of pSTAT5 activation in PD-1+ CD127+ cells (white histogram) versus PD-1- CD127+ cells (black histogram) was calculated and reported graphically. N=3 independent experiments. [Figure 12] Anti-PD-1*1 IL7*1 synergistically activates TCR signaling. Promega PD-1 / PD-L1 bioassay: (1) Effector T cells (Jarcut cells stably expressing PD-1 and NFAT-induced luciferase) and (2) Activation target cells (CHO K1 cells stably expressing PD-L1 and surface proteins designed to activate cognitive TCRs in an antigen-independent manner) were co-cultured. After adding BioGlo® luciferin, luminescence was quantified, representing T cell activation. Figure 12A. Anti-PD1*1 (black square, black), anti-PD-1*1 + isotype*1 IL-7wt*1 as separate compounds (white inverted triangle), and anti-PD-1*1 IL7wt*1 (black triangle, black) were added at serial concentrations. Graph on the right shows the calculated EC50 (nM) for each construct. Figure 12B. Anti-PD1*1 (black square, black), anti-PD-1*1 + isotype*1 IL-7v*1 as separate compounds (white inverted triangle), and anti-PD-1*1 IL7v*1 (black triangle, black) were added in series concentrations. The graph on the right shows the EC50 (nM) calculated for each construct. The data represents at least three independent experiments. [Figure 13]Anti-PD-1*1 / IL7v*1 demonstrated higher pharmacokinetics in vivo than anti-PD-1*2 / IL7v*1 or anti-PD-1*2 / IL7v*2 molecules. C57BL / 6 mice were injected intravenously (Figure 13A) or intraperitoneally (Figure 13B) with a single dose (34 nmol / kg) of the bifunctional anti-PD-1 / IL-7v molecule. Serum antibody concentrations were quantified at multiple time points using anti-human Fc-specific ELISA. The graph on the left shows data in nanomolar concentrations. The graph on the right calculates the area under the curve for each construct, defining in vivo drug exposure. Data are mean + / - SEM values ​​for 2-4 mice / group. [Figure 14] Pharmacokinetic studies of individual anti-PD1*2 / IL7*1 molecules. Data represent the pharmacokinetics and AUC of individual constructs of anti-PD-1 / IL-7 molecules constructed with an anti-PD-1*1 or anti-PD-1*2 skeleton and one or two fused IL7 molecules. Data are mean + / - SEM, including 1-4 mice / group from independent experiments. [Figure 15] Pharmacokinetic studies in mice after single injection of anti-PD-1 monotherapy (anti-PD-1*1 and anti-PD-1*2). C57 BL6 mice were injected with one dose (34 nmol / kg) of anti-PD-1 antibody constructed with either an anti-PD-1 titer of 1 (anti-PD-1*1, black square, dashed line) or an anti-PD-1 titer of 2 (anti-PD-1*2, black circle, solid line) (Figure 15A). (Figure 15A) Intravenous injection and (Figure 15B) Intraperitoneal injection. Serum antibody concentrations were quantified at multiple time points using anti-human Fc-specific ELISA. Data are expressed in nM concentration. [Figure 16A]The anti-PD-1*1 IL7v*1 molecule significantly promotes T cell proliferation in vivo. Mice were intraperitoneally injected with one dose (34 nM / kg) of the anti-PD-1 IL-7v molecule (anti-PD-1*2 IL7v*1, anti-PD-1*1 IL7v*1, anti-PD-1*1 IL7v*2, anti-PD-1*2 IL7wt*1, anti-PD-1*1, or anti-PD-*2). On day 4, tumors and blood were collected, and T cells in different T cell subpopulations were stained with the ki67 proliferation marker. The percentage of KI67 in the CD3 CD4+ and CD3 CD8+ populations in the blood was quantified. Statistical significance (*p<0.05) was calculated using one-way ANOVA for multiple comparisons with control mice in groups of n=3-8 mice. [Figure 16B] The anti-PD-1*1 IL7v*1 molecule significantly promotes T cell proliferation in vivo. Mice were intraperitoneally injected with one dose (34 nM / kg) of the anti-PD-1 IL-7v molecule (anti-PD-1*2 IL7v*1, anti-PD-1*1 IL7v*1, anti-PD-1*1 IL7v*2, anti-PD-1*2 IL7wt*1, anti-PD-1*1, or anti-PD-*2). On day 4, tumors and blood were collected, and T cells in different T cell subpopulations were stained with the ki67 proliferation marker. The percentage of KI67 in the CD3 CD4+ and CD3 CD8+ populations of intratumoral TCF1+ stem cell-like CD8 T cells (CD45 / CD3 / CD8 / CD44 / TCF1+ / TOX-) was quantified. Statistical significance (*p<0.05) was calculated using one-way ANOVA for multiple comparisons with control mice in groups of n=3 to 8 mice. [Figure 17]The anti-PD-1*1 IL7*1 molecule demonstrated significant efficacy in an orthotopic model of anti-PD-1 resistant hepatocellular carcinoma. Humanized PD-1 KI immunocompetent mice were used for the experiment. Hepa1.6 hepatocellular carcinoma cells were orthotopically injected via the portal vein. On day 4, the mice were treated with three doses: PBS (negative control), anti-PD-1*2, and anti-PD-1*1 IL7*1. Two independent experiments were performed. Figure 17A. Survival of mice treated with anti-PD-1*1 IL7v*1 versus anti-PD-1*2 and PBS. Figure 17B. Survival of mice treated with anti-PD-1*1 IL7wt*1 versus anti-PD-1*2 and PBS. [Figure 18] The anti-PD-1*1 IL7v*1 molecule demonstrates high efficacy in a mesothelioma orthotopic model. Humanized PD-1 KI immunocompetent mice were used for the experiment. AK7 mesothelioma cells were injected intraperitoneally. On day 4, mice were treated with three doses: PBS (negative control), anti-PD-1*2, and anti-PD-1*1 IL7v*1. Figure 18A. Tumor volume measured by bioluminescence. AK7 cells stably express luciferase, enabling in vivo quantification of bioluminescence. Figure 18B. Survival of treated mice. [Figure 19]The anti-PD-1*1 IL7v*1 molecule inactivates the inhibitory function of Tregs to a greater extent than IL-7 cytokine alone and anti-PD-1*1 IL7wt*1. CD8+ effector T cells and autologous CD4+ CD25high CD127low Tregs were isolated from the peripheral blood of healthy donors and stained with cell proliferation dye (CPDe670 for CD8+ T cells). Next, Treg / CD8+ Tregs were co-cultured for 5 days in a 1:1 ratio in the presence or absence of rIL-7, anti-PD-1*2, recombinant IL-7 cytokine, anti-PD-1*1 IL7WT*1, or anti-PD-1*1 IL7v*1 (anti-PD-1*1 IL7W142H*) (0.12nM) on OKT3 coated plates (2 μg / mL) for 5 days. Effector T cell proliferation was analyzed by cytofluorometry based on the loss of CPD markers. The data represent the % inhibitory activity of Treg, analyzed using the formula 100 - ((% of Teff co-cultured with Treg / %) of Teff growth alone) * 100). Data from n=4 donors in independent experiments + / - SEM. Statistical significance was determined by one-way ANOVA using Dunnett's test for multiple comparisons. [Figure 20] Significant long-term monotherapy efficacy of the anti-PD-1*1 IL7v*1 molecule in the AK7 orthotopic model, an anti-PD-1 sensitive orthotopic model. hPD-1KI mice were intraperitoneally injected with AK7 mesothelioma cells and treated with PBS (n=8 mice), anti-PD-1*2 (n=8 mice), anti-PD-1*1 IL7v*1 (W142H*1) (n=14), and anti-PD-1*1 IL7WT*1. (A) Overall survival after treatment. Statistical significance was calculated by log-rank test (*p<0.05). (B) Anti-PD-1*1 IL7v*1 induces a long-term memory response after tumor rechallenge. Mice cured by anti-PD-1*1 IL7v*1 treatment (n=7) were rechallenged with AK7 mesothelioma cells via intraperitoneal injection (3e6 cells). As a control, naive mice (n=3) were also injected, and tumor volume and growth were observed. The graph shows the growth of luciferase-transduced AK7 tumors quantified by bioluminescence after intraperitoneal injection of D-luciferin (150 mg / kg and analysis using bioimager) (mean + / - SEM). [Figure 21] Preclinical efficacy of the anti-PD-1*1 IL7v*1 molecule in orthotopic models of hepatocellular carcinoma. After Hepa 1.6 tumor inoculation, mice were treated with PBS, anti-PD-1, anti-PD-1*1 IL7v*1 (anti-PD-1*1 IL7W142H*1), or anti-PD-1*1 IL7wt*1 on days 4 / 6 and 8. Overall survival was obtained for three independent experiments and combined for illustrative purposes: PBS (n=23); anti-PD-1*2 (n=26), isotype-IL-7 (n=14), anti-PD-1*1 IL7v*1 (n=20), and anti-PD-1*1 IL7wt*1 (n=19). Statistical significance (p<0.05) was calculated by log-rank test. [Figure 22A] Genetic signatures after in vivo treatment with the anti-PD-1*1 IL7v*1 molecule indicate an increase in stem cell-like memory CD8 T cell subsets in the tumor microenvironment. After inoculation with Hepa 1.6 tumors, mice were treated on days 4 / 6 and 8 with PBS, anti-PD-1 (34.3 nmol / kg), anti-PD-1*1 IL7v*1, or anti-PD-1*1 IL7wt*1 (34.3 nmol / kg) (n=4 per group). On day 10, tumors were collected and gene expression was analyzed using a Nanostring Pancancer immunotherapy panel. (A) Heatmap of differentially expressed genes (DEGs) between the PBS, anti-PD-1, and anti-PD-1*1 IL7v*1 groups, based on STRING protein-protein network analysis of genes commonly upregulated between anti-PD-1 and BICKI IL7v treatment. [Figure 22B]Genetic signatures after in vivo treatment with the anti-PD-1*1 IL7v*1 molecule indicate an increase in stem cell-like memory CD8 T cell subsets in the tumor microenvironment. After inoculation with Hepa 1.6 tumors, mice were treated on days 4, 6, and 8 with PBS, anti-PD-1 (34.3 nmol / kg), anti-PD-1*1 IL7v*1, or anti-PD-1*1 IL7wt*1 (34.3 nmol / kg) (n=4 per group). On day 10, tumors were collected and gene expression was analyzed using a Nanostring Pancancer immunotherapy panel. Genetic signature enrichment was observed between early-activated T cells and exhausted T cells. The gene signatures of exhausted T cells and naive-like / stem cell-like memory T cells were matched using the naive-like / stem cell-like memory T cells (TCF7, CCR7, SELL, IL7R) and exhausted CD8 T cell scores (LAG3, PRF1, CD8A, HAVRC2, GZMB, CD8B1, KLRD1, TNFRSF9, TIGIT, CTSW, CCL4, CD63, IFNG, CXCR6, FASL, CSF1) from Andreatta et al. (Nature comm 2021, vol. 12, p. 2965). [Figure 22C]Genetic signatures after in vivo treatment with the anti-PD-1*1 IL7v*1 molecule indicate an increase in stem cell-like memory CD8 T cell subsets in the tumor microenvironment. After inoculation with Hepa 1.6 tumors, mice were treated on days 4, 6, and 8 with PBS, anti-PD-1 (34.3 nmol / kg), anti-PD-1*1 IL7v*1, or anti-PD-1*1 IL7wt*1 (34.3 nmol / kg) (n=4 per group). On day 10, tumors were collected and gene expression was analyzed using a Nanostring Pancancer immunotherapy panel. Genetic signature enrichment was observed between early-activated T cells and exhausted T cells. The gene signatures of exhausted T cells and naive-like / stem cell-like memory T cells were matched using the naive-like / stem cell-like memory T cells (TCF7, CCR7, SELL, IL7R) and exhausted CD8 T cell scores (LAG3, PRF1, CD8A, HAVRC2, GZMB, CD8B1, KLRD1, TNFRSF9, TIGIT, CTSW, CCL4, CD63, IFNG, CXCR6, FASL, CSF1) from Andreatta et al. (Nature comm 2021, vol. 12, p. 2965). [Figure 23]Anti-PD-1*1 IL7v*1 molecule-induced proliferation of stem cell-like memory CD8 T cell (TCF1+) subsets in the tumor microenvironment. (A, B, and C) After inoculation with Hepa 1.6 tumors, mice were treated with PBS, anti-PD-1 (34.3 nmol / kg), or anti-PD-1*1 IL7v*1 or anti-PD-1*1 IL7wt*1 (34.3 nmol / kg) (n=4 per group) on days 4, 6, and 8. On day 10, tumors were collected and T cells were stained for flow cytometry analysis for the expression of CD3 / CD8 / CD44 markers and TCF1 / TOX factors. (A) Percentage of CD4, CD8, and Treg subpopulations in the tumor microenvironment. (B) Percentage of CD44+ CD8+ activated T cells expressing TCF1+ / -TOX markers. (C) Proliferation of CD44+ CD8+ activated T cells expressing the TCF1+ / -TOX marker, measured by the percentage of the KI67 marker in the Hepa1.6 model (D) and the MC38 subcutaneous model. The proliferation of CD44+ CD8+ activated T cells expressing the TCF1+ / -TOX marker was measured by the percentage of the KI67 marker after treatment (anti-PD-1*2 anti-PD-1*1 IL7v*1 (W142H*1)). [Figure 24] Anti-PD-1*1 IL7v*1 maintained the survival of chronically stimulated T cells (A) and induced in vitro proliferation of stem cell-like T cells (B). Human PBMCs isolated from the peripheral blood of two healthy volunteers were chronically stimulated every 3 days with anti-CD3 and anti-CD28 agonist antibodies. T cells were treated with anti-PD-1, human IL-7, and anti-PD-1*1 IL7v*1 between each stimulation. One day after 5 stimulations, T cells were stained with viability dye, anti-CD3, anti-CD8, anti-TCF1, and anti-ki67 proliferation markers. (A) Viability was quantified in all cells. (B) KI67 percentage was quantified in the CD3+ CD8+ TCF1- / + population. Data are mean + / - SD for N=2 donors per group. [Figure 25]Efficacy of monotherapy with anti-PD-1*1 IL7v*1 molecule in a humanized mouse model of breast cancer cells. NXG immunodeficient mice were subcutaneously injected with MDA-MB231 breast cancer cells (3e6 cells), and on day 8, they were humanized intraperitoneally with human PBMCs (3e6 cells). They were then treated with PBS, anti-PD-1*2, or anti-PD-1*1 IL7v*1 on days 12, 15, and 18 after tumor inoculation. Data are mean + / - SEM values ​​per group and per donor (N=3-4 mice). [Figure 26] Efficacy of monotherapy with anti-PD-1*1 IL7v*1 molecule in a humanized mouse model of lung cancer A549. NXG immunodeficient mice were subcutaneously injected with A549 lung cancer cells and humanized intraperitoneally with human PBMCs (10e6 cells) on day 21. They were then treated with PBS, anti-PD-1*2, or anti-PD-1*1 IL7v*1 inoculation on days 25, 28, 31, and 34 after tumor inoculation. (A) A549 tumor growth, mean + / - SEM for n=5 mice per group. Statistical significance**p<0.05 was calculated using Dunnett's multiple comparison test by comparing anti-PD-1*1 IL7v*1 versus anti-PD-1*2. (B) Human IFNg secretion quantified by ELISA in mouse serum collected on day 35 (n=2-5 mice per group). [Figure 27] Anti-PD-1*1 IL7v*1 demonstrated a favorable pharmacokinetic profile compared to the anti-PD-1*1 IL7wt*1 molecule and induced CD8 T cell proliferation in vivo. Animals were intravenously injected with one dose of either anti-PD-1*1 IL-7v*1 (anti-PD-1*1 IL7 W142H*1) or anti-PD-1*1 IL7wt*1 at doses of 0.8 mg / kg (n=1 cynomolgus monkey), 4.01 mg / kg (n=1 cynomolgus monkey), and 25 mg / kg (n=1 cynomolgus monkey). (A) Drug concentrations in animal serum were quantified by MSD immunoassay. (B) CD8 T cell proliferation was measured by flow cytometry in peripheral blood T cells after injection of different doses of anti-PD-1*1 IL7v*1 antibody (n=1 cynomolgus monkey per dose). [Figure 28]Anti-PD-1*1 IL7v*1 constructed with the IgG1 N297A isotype or the IgG1 N297A LALA PG isotype demonstrated similar efficacy in activating pSTAT5. Stimulated human PBMCs were treated with different doses of anti-PD-1*1 IL7v*1 constructed with the IgG1 N297A isotype or the IgG1 LALA P329G mutation. IL-7R signaling activation was measured by intracellular pSTAT5 staining in CD4 and CD8 T cells and analyzed by flow cytometry. [Figure 29] The anti-PD-1 IL7 W142H variant demonstrates high binding efficiency to PD-1. PD-1 binding ELISA assay was performed. Human recombinant PD-1 (rPD1) protein was immobilized, and antibodies were added at different concentrations. Chromogenic development was performed using peroxidase-bound anti-human Fc antibody. Colorimetric analysis was determined at 450 nm using TMB substrate. A bifunctional molecule (gray rhombic) containing the IL7 W142H N297A variant with one anti-PD-1 arm was tested as a control. We also tested bifunctional molecules containing IL7 variants with one anti-PD-1 arm (IL7 W142H N297A DHS gray square), (IL7 W142H N297A LS black triangle), (IL7 W142H N297A YTE X), (IL7 W142H N297A VL CNDOwt Ж), and (IL7 W142H N297A VL vAv3-11 gray circle). [Figure 30]The anti-PD-1 IL7 molecule, constructed with monovalent anti-PD-1 and one IL-7 W142H mutant cytokine, activates pSTAT5 with high efficiency. A pSTAT5 signaling assay was performed using an anti-PD-1*1 scaffold fused with the IL-7 W142H*1 cytokine mutant. Human T lymphocytes isolated from the peripheral blood of healthy volunteers were incubated with the bifunctional molecule for 15 minutes. The 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 in the CD3+ population. The bifunctional molecule, containing the IL7 W142H N297A variant with one anti-PD-1 arm (black, black rhombus) and wild-type IL7 (inverted white triangle), and the anti-PD-1 molecule constructed with monovalent anti-PD-1 (white rhombus) were tested as controls. We also tested bifunctional molecules including IL7 variants with one anti-PD-1 arm (IL7 W142H N297A DHS (gray, gray square)), (IL7 W142H N297A LS (black, black triangle)), (IL7 W142H N297A YTE (black X)), (IL7 W142H N297A VL CNDOwt black*), and (IL7 W142H N297A VL vAv3-11 gray, gray circle). [Modes for carrying out the invention]

[0054] Introduction The present invention relates to a bifunctional molecule comprising a single monovalent antigen-binding domain that has a specific scaffold structure and binds to a target specifically expressed on the surface of immune cells, particularly PD-1, and a single immunostimulatory cytokine, particularly IL-7. This scaffold structure essentially consists of a dimeric Fc domain, a single monovalent anti-PD-1 antigen-binding domain linked at the N-terminus of one monomer of the Fc domain, and, if the antigen-binding domain includes a variable heavy chain and a variable light chain, a single immunostimulatory cytokine, particularly IL-7, linked at the C-terminus of the monomer or light chain of the Fc domain. These novel bifunctional molecules exhibit an improved pharmacokinetic profile and better productivity, among other advantages.

[0055] The inventors have surprisingly shown that constructs containing a single IL-7 variant exhibit improved properties in terms of both activity and pharmacokinetics compared to constructs containing two IL-7 variants.

[0056] definition To facilitate understanding of the present invention, certain terms are defined below in this specification. Additional definitions are provided throughout the detailed description.

[0057] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have meanings that are generally understood by those skilled in the art. In some cases, terms that have generally understood meanings are also defined herein for clarity and / or for easy reference. Where such definitions are included herein, they should not necessarily be construed as meanings that are different from those generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and are widely used by those skilled in the art using conventional methodologies.

[0058] As used herein, the terms “wild-type interleukin-7,” “wt-IL-7,” and “wt-IL7” refer to mammalian endogenous secreted glycoproteins, particularly IL-7 polypeptides, derivatives, and analogs having substantial amino acid sequence identity with wild-type functional mammalian IL-7 and substantially equivalent biological activity in, for example, a standard bioassay or assay of IL-7 receptor binding affinity. For example, wt-IL-7 refers to i) a natural or naturally occurring IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, iii) a biologically active polypeptide analog of an IL-7 polypeptide, or iv) the 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 or may not contain its peptide signal. Alternative names for this molecule are “pre-B cell growth factor” and “lympopoietin-1.” Preferably, the term “wt-IL-7” refers to human IL-7 (wt-IL7). For example, the amino acid sequence of human wt-IL-7 is approximately 152 amino acids (in the absence of the signal peptide), its Genbank acceptance number is NP_000871.1, and the gene is located on chromosome 8q12-13. Human IL-7 is described, for example, in UniProtKB - P13232. As used herein, the terms “programmed death 1”, “programmed cell death 1”, “PD1”, “PD-1”, “PDCD1”, “PD-1 antigen”, “human PD-1”, “hPD-1”, and “hPD1” are used interchangeably and refer to the programmed death-1 receptor, also known as CD279, including variants and isoforms of human PD-1, as well as analogs having at least one common epitope with PD-1. PD-1 is an important regulator of the threshold of immune response and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands, PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. For example, the amino acid sequence of human PD-1 is disclosed under GenBank acceptance number NP_005009.PD1 has four splice variants expressed in human peripheral blood mononuclear cells (PBMCs). Therefore, the PD-1 protein includes full-length PD-1, as well as alternative splice variants of PD-1, such as PD-1Aex2, PD-1Aex3, PD-1Aex2,3, and PD-1Aex2,3,4. Unless otherwise specified, the term includes any variant and isoform of human PD-1 expressed naturally by PBMCs or by cells transfected with the PD-1 gene.

[0059] As used herein, the term “antibody” is used in its broadest sense to describe a type of immunoglobulin molecule. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. The heavy chain constant domains corresponding to different classes of immunoglobulin are called alpha, delta, epsilon, gamma, and mu, respectively. Unless otherwise specifically indicated, the term “antibody” includes intact immunoglobulins, as well as any other modified configurations of immunoglobulin molecules containing antigen recognition sites of the required specificity, including “antibody fragments” or “antigen-binding fragments” (Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), their variants, molecules containing the antibody portion, diabodies, linear antibodies, single-chain antibodies, and glycosylated variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term “antibody” refers to humanized antibodies.

[0060] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the antibody three-dimensional structure. The CDRs of the antibody heavy chain are typically referred to as "HCDR1," "HCDR2," and "HCDR3." The framework regions of the antibody heavy chain are typically referred to as "HFR1," "HFR2," "HFR3," and "HFR4."

[0061] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the antibody three-dimensional structure. κ-light chain and λ-light chain refer to the two main antibody light chain isotypes. The CDRs of the antibody light chain are typically referred to as "LCDR1," "LCDR2," and "LCDR3." The framework regions of the antibody light chain are typically referred to as "LFR1," "LFR2," "LFR3," and "LFR4."

[0062] As used herein, the “antigen-binding fragment” or “antigen-binding domain” of an antibody means a molecule corresponding to a part of the antibody, i.e., a part of the antibody structure of the present invention that exhibits antigen-binding ability to a particular antigen, possibly in its natural form. In particular, such a fragment exhibits the same or substantially the same antigen-binding specificity to that antigen compared to the antigen-binding specificity of the corresponding quadruple-chain antibody. Advantageously, the antigen-binding fragment has a similar binding affinity to the corresponding quadruple-chain antibody. However, antigen-binding fragments having a reduced antigen-binding affinity compared to the corresponding quadruple-chain antibody are also included in the present invention. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. Such antigen-binding fragments may also be referred to as “functional fragments” of an antibody. Antigen-binding fragments of an antibody are fragments containing a hypervariable domain or a portion thereof, referred to as a CDR (complementarity-determining region).

[0063] As used herein, the term "humanized antibody" is intended to refer to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been transplanted into a human framework sequence (e.g., a chimeric antibody containing a minimal sequence derived from a non-human antibody). Furthermore, the "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which one or more residues derived from CDRs are replaced by residues derived from at least one CDR of a non-human antibody (donor antibody), while maintaining the desired specificity, affinity, and volume of the original antibody. Additional framework region modifications may be present within the human framework sequence. Preferably, the humanized antibody has a T20 humanity score greater than 80%, 85%, or 90%. "Humanity" can be measured using a T20 score analyzer to quantify the humanity of the variable region of an antibody, as described, for example, Gao SH, Huang K, Tu H, Adler A S., BMC Biotechnology. 2013: Vol. 13: p. 55, or a web-based tool to calculate the T20 score of an antibody sequence using T20 Cutoff Human Databases: http: / / abAnalyzer.lakepharma.com.

[0064] A "chimeric antibody" refers to an antibody produced by combining genetic material derived from a non-human source, preferably such as a mouse, with genetic material derived from a human. Such antibodies are derived from both a human antibody and a non-human antibody linked by a chimeric region. Chimeric antibodies generally contain a constant domain derived from a human and a variable domain derived from another mammalian species, and when used in therapeutic treatment, they reduce the risk of reaction to exogenous antibodies derived from non-human animals.

[0065] As used herein, the terms “crystallizable fragment region,” “Fc region,” or “Fc domain” are synonymous and refer to the tail region of an antibody that interacts with a cell surface receptor called the Fc receptor. The Fc region or domain typically consists of two optionally identical domains derived from the second and third constant domains (i.e., the CH2 domain and the CH3 domain) of the two heavy chains of the antibody. The portion of the Fc domain refers to either the CH2 domain or the CH3 domain. Optionally, the Fc region or domain may optionally include all or part of the hinge region between CH1 and CH2. Thus, the Fc domain may include the hinge, the CH2 domain, and the CH3 domain. Optionally, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4 and optionally has an IgG1 hinge-CH2-CH3 and an IgG4 hinge-CH2-CH3.

[0066] In the context of IgG antibodies, each IgG isotype has three CH regions. Therefore, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-215 on the EU index, as in the case of Kabat; "Hinge" refers to positions 216-230 on the EU index, as in the case of Kabat; "CH2" refers to positions 231-340 on the EU index, as in the case of Kabat; and "CH3" refers to positions 341-447 on the EU index, as in the case of Kabat.

[0067] "Amino acid change" or "amino acid modification" as used herein means a change in the amino acid sequence of a polypeptide. "Amino acid modifications" include substitutions, insertions, and / or deletions in the polypeptide sequence. "Amino acid substitution" or "substitution" as used herein means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid. "Amino acid insertion" or "insertion" means adding an amino acid at a specific position in the parent polypeptide sequence. "Amino acid deletion" or "deletion" means removing an amino acid at a specific position in the parent polypeptide sequence. Amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") having similar chemical properties (e.g., charge, bulk, and / or hydrophobicity). As used herein, "amino acid position" or "amino acid position number" is used synonymously to refer to the position of a particular amino acid in an amino acid sequence, generally designated by a single-letter code for the amino acid. The first amino acid in an amino acid sequence (i.e., starting from the N-terminus) should be considered to be position 1.

[0068] A conservative substitution is the replacement of 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). Generally, conservative amino acid substitutions will not substantially alter the functional properties of a protein. Conservative substitutions and their corresponding rules are well documented in the state of the art. For example, conservative substitutions can be defined by substitutions within the group of amino acids reflected in the table below.

[0069] [Table 1]

[0070] [Table 2]

[0071] [Table 3]

[0072] As used herein, “sequence identity” between two sequences is described by the parameters “sequence identity,” “sequence similarity,” or “sequence homology.” For the purposes of this invention, the “identity percentage” between two sequences (A) and (B) is determined by comparing the two sequences aligned in the most optimal manner across a comparison window. Sequence alignment can be performed by methods well known in the art, for example, using the Needleman-Bunsch global alignment algorithm. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. Once the overall alignment is obtained, the identity percentage can be obtained by dividing the total number of aligned identical amino acid residues by the total number of residues in the longest sequence between sequences (A) and (B). Sequence identity is typically determined using sequence analysis software. To compare two amino acid sequences, you can use the Emboss needle, a pairwise sequence alignment tool for proteins, which is provided by EMBL-EBI or available at www.ebi.ac.uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein, for example, with the following initial settings: (I) Matrix: BLOSUM62, (ii) Gap Open: 10, (iii) Gap Extension: 0.5, (iv) Output Format: Pair, (v) End Gap Penalty: False, (vi) End Gap Open: 10, (vii) End Gap Extension: 0.5.

[0073] Alternatively, sequence identity can typically be determined using Clustal Omega, a sequence analysis software that uses the HHalign algorithm and its initial settings as its core alignment engine. This algorithm, along with its initial settings, is described in Soding, J. (2005), "Protein homology detection by HMM-HMM comparison," Bioinformatics, Vol. 21, pp. 951-960.

[0074] The terms “derive from” and “derived from,” as used herein, refer to a compound having a structure derived from the structure of a parent compound or parent protein, whose structure is sufficiently similar to that disclosed herein, and which, based on its similarity, would be expected by those skilled in the art to exhibit the same or similar properties, activities, and utility as the claimed compound.

[0075] Where used herein, “pharmaceutical composition” refers to one or more preparations of an activating agent, including those comprising the bifunctional molecule according to the present invention together with other optional chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of an activating agent to a living organism. The compositions of the present invention may be in any conventional route of administration or in a form suitable for use. In one embodiment, “composition” is typically intended to be a combination of an activating agent, such as a compound or composition, and a carrier, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, or inert (e.g., a detectable activating agent or label) or active carrier, whether naturally occurring or not, including a pharmaceutically acceptable carrier. Where used herein, “acceptable vehicle” or “acceptable carrier” is any known compound or combination of compounds that is known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0076] "Effective dose" or "therapeutic effective dose," as used herein, refers to the amount of an activator required to impart a therapeutic effect to a subject, either alone or in combination with one or more other activators, for example, the amount of activator required to treat a target disease or disorder or to produce a desired effect. "Effective dose" will vary depending on the activator, the disease and its severity, the characteristics of the subject to be treated, including age, physical condition, size, sex, and weight, the duration of treatment, the nature of the combination therapy (if applicable), the specific route of administration, and similar factors within the scope of the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art and can be addressed through routine experimental work. Generally, it is preferable to use the maximum dose of each individual component or combination thereof, i.e., the highest safe dose based on sound medical judgment.

[0077] As used herein, the term “pharmaceutical” refers to any substance or composition having therapeutic or preventive properties for a disorder or disease.

[0078] The term “treatment” refers to any action aimed at improving a patient’s health condition, such as the therapy, prevention, avoidance, and mitigation of a disease or its symptoms. This term encompasses both curative and / or preventative treatments of a disease. Curative treatment is defined as treatment that results in a cure or treatment that reduces, improves, and / or eliminates, decreases, and / or stabilizes the disease or its symptoms or the suffering it directly or indirectly causes. Preventative treatment includes both treatments that result in the prevention of disease, and treatments that reduce and / or delay the progression and / or onset of disease or the risk of its occurrence. In certain embodiments, such term refers to the improvement or eradication of a disease, disorder, infection, or associated symptoms. In other embodiments, this term refers to minimizing the spread or worsening of cancer. Treatments according to the present invention do not necessarily imply 100% or complete cure. Rather, there are varying degrees of treatment that a person skilled in the art would recognize as having potential benefits or therapeutic effects. Preferably, the term “treatment” refers to applying or administering a composition comprising one or more active agents to a subject with a disorder / disease.

[0079] As used herein, the terms “disorder” or “disease” refer to a failure of an organ, part, structure, or system of the body to function properly due to a genetic or developmental error, infection, toxin, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, these terms refer to a health disorder or disease, such as a disease that interferes with normal physical or mental functioning. More preferably, the term “disorder” refers to an immune disorder and / or inflammatory disease affecting animals and / or humans, such as cancer.

[0080] As used herein, “immune cells” refers to cells involved in innate and adaptive immunity, such as leukocytes (white blood cells), lymphocytes (T cells, B cells, natural killer (NK) cells, and natural killer T cells (NKT)), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, and dendritic cells), derived from hematopoietic stem cells (HSCs) produced in the bone marrow. In particular, immune cells can be selected from a non-exclusive list including B cells, T cells, especially CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells, and monocytes. As used herein, “T cells” include, for example, CD4+ T cells, CD8+ T cells, T helper 1 T cells, T helper 2 T cells, T helper 17 T cells, and inhibitory T cells.

[0081] As used herein, the terms “T effector cells,” “Teff,” or “effector cells” describe a group of immune cells, including several T cell types, that respond to stimuli such as co-stimulation. This term includes, in particular, T cells that function to eliminate antigens (e.g., by producing cytokines that modulate the activation of other cells, or by cytotoxic activity). This term includes, in particular, CD4+ cells, CD8+ cells, cytotoxic T cells, and helper T cells (Th1 and Th2).

[0082] As used herein, the terms “regulatory T cells,” “Treg cells,” or “Treg” refer to a subgroup of T cells that modulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases. 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 originate from the same lineage as naive CD4 cells.

[0083] The term "exhausted T cells" refers to a population of T cells in a dysfunctional (i.e., "exhausted") state. T cell exhaustion is characterized by progressive loss of function, altered transcriptional profiles, and sustained expression of inhibitory receptors. Exhausted T cells lose their ability to produce cytokines, their high proliferative capacity, and their cytotoxic capacity, which ultimately leads to their own elimination. Exhausted T cells typically exhibit lower expression of CD62L and CD127, along with higher levels of CD43, CD69, and inhibitory receptors.

[0084] The term "effector memory stem cell-like T cells" refers to a subset of tumor-responsive intratumor T cells that exhibit characteristics of exhausted cells and central memory cells, including the expression of the checkpoint protein PD-1 and the transcription factor Tcf1. These cells are characterized by Tcf1 + PD-1 + CD8 + These cells are called T cells. They reside in the tumor microenvironment and are important for the immune regulation of cancer, which is facilitated by immunotherapy. They are important for maintaining the T cell response in chronic viral infections and cancer, and provide the explosive proliferation seen after PD-1 immunotherapy. These cells have slow self-renewal and also give rise to more terminally differentiated, exhausted CD8 T cells. These cells and their characteristics are further defined in the following papers, whose disclosure is incorporated herein by reference: Siddiqui et al., 2019, Immunity, Vol. 50, pp. 195-211; and Jadhav et al., 2019, PNAS, Vol. 116, pp. 14113-14118).

[0085] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, resulting in selective damage, destruction, or elimination from the human body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0086] As used herein, the term "antagonist" refers to a substance that inhibits or reduces the activity or functionality of another substance. In particular, this term refers to an antibody that binds to a cell receptor (e.g., PD-1) as a reference substance (e.g., PD-L1 and / or PD-L2) and prevents it from producing all or part of its normal biological effect (e.g., the creation of an immunosuppressive microenvironment). The antagonist activity of the humanized antibody according to the present invention can be evaluated by competitive ELISA.

[0087] As used herein, the term "agonist" refers to a substance that activates the function of an activating 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 a biologically native ligand (e.g., induces the receptor's activating effect).

[0088] Pharmacokinetics (PK) refers to the movement of a drug within the body, while pharmacodynamics (PD) refers to the body's biological response to a drug. PK describes drug exposure by characterizing absorption, distribution, bioavailability, metabolism, and excretion as functions of time. PD describes drug responses in terms of biochemical or molecular interactions. PK and PD analyses are used to characterize drug exposure, predict and evaluate changes in dosage, estimate elimination and absorption rates, assess the relative bioavailability / bioequivalence of formulations, characterize within- and between-subject variability, understand concentration-response relationships, and establish safety margins and efficacy characteristics. "Improved PK" means an improvement in one of the above characteristics, such as an increase in the molecular half-life, particularly when the molecule exhibits a longer serum half-life after injection into the subject.

[0089] As used herein, the terms “pharmacokinetics” and “PK” are interchangeable and refer to the fate of a compound, substance, or drug administered to a living organism. Pharmacokinetics, in more detail, includes ADME or LADME schemes representing release (i.e., the release of the substance from a composition), absorption (i.e., the entry of the substance into the bloodstream), distribution (i.e., the dispersion or diffusion of the substance throughout the body), metabolism (i.e., the transformation or breakdown of the substance), and excretion (i.e., the removal or clearance of the substance from the organism). The two stages of metabolism and excretion can also be grouped together under the heading of elimination. Those skilled in the art can monitor various pharmacokinetic parameters, including, among others, elimination half-life, elimination constant rate, clearance (i.e., the volume of plasma from which the drug is cleared per unit time), Cmax (maximum serum concentration), and drug exposure (determined by the area under the curve; see Scheff et al., Pharm Res. May 2011; Vol. 28 (No. 5): pp. 1081-1089).

[0090] As used herein, the term “isolated” means that the substance described (e.g., antibodies, polypeptides, nucleic acids, etc.) has been substantially separated from or concentrated compared to other substances that exist with it in nature. In particular, an “isolated” antibody is one that has been identified, separated, and / or recovered from components of its natural environment.

[0091] When used herein, the term "and / or" should be interpreted as a specific disclosure of each of the two designated features or components, with or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were presented individually.

[0092] The terms "a" or "an" can refer to one or more of the elements they modify, unless the context makes it clear that either one or more of the elements are being referred to (for example, "reagent" can mean one or more reagents).

[0093] When used herein in relation to any value (including the lower and upper limits of a numerical range), the term "approximately" means any value that has an acceptable deviation of 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%). The use of the term "approximately" at the beginning of a string of values ​​qualifies each of the values ​​(i.e., "approximately 1, 2, and 3" refers to approximately 1, approximately 2, and approximately 3). Furthermore, if a list of values ​​is provided herein (for example, about 50%, 60%, 70%, 80%, 85%, or 86%), the list shall include all of its intermediate and decimal values ​​(for example, 54%, 85.4%).

[0094] When used herein in relation to any given biological sequence, the term “essentially” means that the biological sequence varies by up to 10% in biological sequence length from the reference sequence contained in the sequence listing. In particular, “essentially consisting of” means that the biological sequence consists of that sequence, but is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, additions, deletions, or mixtures thereof, preferably 1, 2, 3, 4, or 5 substitutions, additions, deletions, or mixtures thereof, provided that the biological sequence varies by up to 10% in biological sequence length from the reference sequence contained in the sequence listing.

[0095] Bifunctional molecules This invention relates to a bifunctional molecule having a scaffold structure associated with improved properties.

[0096] More specifically, the present invention relates to a bifunctional molecule having a specific scaffold structure and comprising a single monovalent antigen-binding domain that binds to PD-1 and a single IL-7m. This scaffold structure essentially consists of a dimeric Fc domain, a single monovalent antigen-binding domain that binds to PD-1 linked at the N-terminus of one monomer of the Fc domain, and either i) a single IL-7m is linked to the C-terminus of the same monomer of the Fc domain and an optional peptide linker, or ii) the single monovalent antigen-binding domain comprises a variable heavy chain and a variable light chain, and the single IL-7m is linked to the C-terminus of the light chain of the antigen-binding domain.

[0097] In a particular embodiment, the bifunctional molecule comprises a first monomer comprising an anti-PD-1 antigen-binding domain optionally covalently linked to a first Fc chain via a peptide linker, wherein the first Fc chain is optionally covalently linked to IL-7m via a peptide linker, and a second monomer comprising a complementary second Fc chain lacking an antigen-binding domain and an IL-7 variant, wherein the first and second Fc chains form a dimeric Fc domain.

[0098] In an alternative embodiment, the bifunctional molecule comprises a first monomer containing an anti-PD-1 antigen-binding domain optionally covalently linked to a first Fc chain via a peptide linker, and a second monomer containing a complementary second Fc chain lacking an antigen-binding domain and IL-7m, wherein the first and second Fc chains form a dimeric Fc domain, a single monovalent antigen-binding domain comprising a variable heavy chain and a variable light chain, and a single IL-7m is linked to the C-terminus of the light chain of the antigen-binding domain.

[0099] Therefore, each of the two monomers contains one Fc chain capable of forming a dimeric Fc domain. In one embodiment, the dimeric Fc fusion protein is a homodimeric Fc domain. In another embodiment, the dimeric Fc fusion protein is a heterodimeric Fc domain.

[0100] More specifically, if the dimeric Fc domain is a heterodimeric Fc domain, the bifunctional molecule comprises an antigen-binding domain optionally covalently linked to the N-terminus of a first heterodimeric Fc chain via a peptide linker, wherein the first heterodimeric Fc chain comprises a first monomer having IL-7m optionally covalently linked to its C-terminus via a peptide linker, and a second monomer comprising a complementary second heterodimeric Fc chain lacking the antigen-binding domain and IL-7m. Optionally, the second monomer comprising the complementary second heterodimeric Fc chain lacks any other functional moieties, in particular another antigen-binding domain, or any cytokine. More specifically, the bifunctional molecule comprises a first monomer having an antigen-binding domain whose C-terminus is optionally covalently linked to the N-terminus of a first heterodimer Fc chain via a peptide linker, wherein the first heterodimer Fc chain comprises a first monomer having its C-terminus optionally covalently linked to the N-terminus of IL-7m via a peptide linker, and a second monomer having a complementary second heterodimer Fc chain lacking the antigen-binding domain and IL-7m, preferably lacking any other functional moiety, particularly another antigen-binding domain, or any cytokine. Such a bifunctional molecule is illustrated in Figure 1, for example, as “Construction 3,” along with IL-7 W142H as an example of IL-7m.

[0101] Optionally, a single antigen-binding domain is selected from the group consisting of Fab, Fab', scFV, and sdAb.

[0102] Therefore, in one embodiment, the bifunctional molecule according to the present invention is (a) an anti-PD1 antigen-binding domain comprising (i) a heavy chain having a first Fc chain, and (ii) a light chain, (b) IL-7m, and (c) Complementary second Fc chain, The molecule contains or consists of IL-7m, preferably with its N-terminus covalently linked to the C-terminus of the first Fc chain via a peptide linker. The first and second Fc chains together form a dimeric Fc domain.

[0103] In certain embodiments, a bifunctional molecule is - A single antibody heavy chain containing VH-CH1-hinge-CH2-CH3 linked to IL-7m at its C-terminus. - A single antibody light chain comprising a VL-CL (constant light chain), a VH-CH1 moiety, and a VL-CL moiety, which together form an antigen-binding domain that binds to PD-1 expressed on the surface of immune cells, and - CH2-CH3, optionally including hinge-CH2-CH3, one Fc chain that forms a dimeric Fc domain together with the CH2-CH3 of the antibody heavy chain. Includes.

[0104] In an alternative embodiment, if the dimeric Fc domain is a heterodimeric Fc domain, the bifunctional molecule comprises a first monomer containing an antigen-binding domain optionally covalently linked to the N-terminus of a first heterodimeric Fc chain via a peptide linker, and a second monomer containing a complementary second heterodimeric Fc chain lacking an antigen-binding domain and IL-7m, wherein the antigen-binding domain comprises a variable heavy chain and a variable light chain, and IL-7m optionally linked to the C-terminus of the light chain of the antigen-binding domain via a peptide linker. Optionally, the N-terminus of IL-7m optionally linked to the C-terminus of the light chain of the antigen-binding domain via a peptide linker.

[0105] Therefore, in this embodiment, the bifunctional molecule according to the present invention is (a) a heavy chain having a first Fc chain, and (ii) an anti-PD1 antigen-binding domain comprising a light chain, (b) IL-7m, and (c) Complementary second Fc chain The system contains or consists of IL-7m, preferably with its N-terminus covalently linked to the C-terminus of the light chain via a peptide linker (optionally). The first and second Fc chains together form a dimeric Fc domain.

[0106] In certain embodiments, a bifunctional molecule is - A single antibody heavy chain containing VH-CH1-hinge-CH2-CH3, - A single antibody light chain comprising a VL-CL (constant light chain) linked to IL-7m at its C-terminus, a VH-CH1 moiety, and a VL-CL moiety, which together form an anti-PD-1 antigen-binding domain that binds to PD-1 expressed on the surface of immune cells, and - CH2-CH3, optionally including hinge-CH2-CH3, one Fc chain that forms a dimeric Fc domain together with the CH2-CH3 of the antibody heavy chain. Includes.

[0107] In any embodiment, the IL-7m, anti-PD1 antigen-binding domain, Fc domain, and optionally a linker are further defined below.

[0108] IL-7 and IL-7 variants IL-7m can stimulate or activate immune cells. These immune cells can be selected from a non-exclusive list including B cells, T cells, particularly CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells, and monocytes. In a preferred embodiment, the immune cells are T cells, more specifically CD8+ T cells, effector T cells, or exhausted T cells. In a particularly preferred embodiment, the immune cells are effector memory stem cell-like T cells.

[0109] In detail, IL-7m has a size ranging from 10 kDa to 50 kDa. Preferably, IL-7m is a peptide, polypeptide, or protein. In one embodiment, IL-7m is a non-antibody entity or portion.

[0110] IL-7m may be mutated or modified so as to alter its biological activity, for example, to increase, decrease, or completely inhibit its biological activity.

[0111] In a very specific embodiment, IL-7m is either IL-7 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the wild-type cytokine, or having 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions, and combinations thereof.

[0112] In certain embodiments, an IL-7 variant or its variant comprises or comprises a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 1, or a sequence having 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions, and combinations thereof with respect to the sequence of SEQ ID NO: 1.

[0113] The terms “interleukin-7 variant,” “mutant IL-7,” “IL-7 variant,” “IL-7 variant,” “IL-7m,” or “IL-7v” are used synonymously herein. A “variant” or “mutant” of the IL-7 protein is defined as an amino acid sequence in which one or more amino acids are changed. A variant may have “conservative” or “non-conservative” modifications. Such modifications include amino acid substitutions, deletions, and / or insertions. Preferably, the modification is a substitution, particularly a conservative substitution. The variant IL-7 proteins included in the present invention relate in particular to IL-7 proteins that do not retain substantially equivalent biological properties (e.g., activity, binding ability, and / or structure) compared to wild-type IL-7. An IL-7 protein variant or mutant contains at least one mutation. In particular, at least one mutation reduces the affinity of IL-7m for IL-7R, but does not result in a loss of IL-7R recognition. Therefore, IL-7 variants or mutants retain the ability to activate IL-7R, as measured, for example, by the pStat5 signal, as disclosed in Bitar et al., Front. Immunol., 2019, Vol. 10, etc. The biological activity of the IL-7 protein can be measured using an in vitro cell proliferation assay or by measuring P-Stat5 of T cells by ELISA or FACS. Preferably, the IL-7 variant according to the present invention reduces the biological properties (e.g., activity, binding ability, and / or structure) by at least 1 / 2, 1 / 5, 1 / 10, 1 / 25, 1 / 50, 1 / 25, 1 / 750, 1 / 1000, 1 / 250, 1 / 500, or 1 / 8000 compared to wild-type IL-7, preferably with-IL7. More preferably, the IL-7 variant exhibits reduced binding to the IL-7 receptor but retains the ability to activate IL-7R.For example, binding to the IL-7 receptor may be reduced by at least 10%, 20%, 30%, 40%, 50%, or 60% compared to wild-type IL-7, while retaining at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the ability to activate IL-7R compared to wild-type IL-7. Preferably, IL-7m is a variant of human wild-type IL-7, such as the one described in SEQ ID NO: 1.

[0114] In one embodiment, the IL-7 variant according to the present invention maintains at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, and 60% of the biological activity compared to wild-type human IL-7, preferably at least 80%, 90%, 95%, and more preferably 99% compared to wild-type IL-7.

[0115] In one embodiment, an IL-7 variant or mutant has at least one amino acid mutation that differs from wt-IL-7, i) reducing the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wt-IL-7 for IL-7R, and ii) improving the pharmacokinetics of the IL-7 variant compared to wt-IL-7. More specifically, the IL-7 variant or mutant further retains the ability to activate IL-7R, particularly via pStat5 signaling.

[0116] In another embodiment, a bifunctional molecule containing an IL-7 variant or mutant has at least one amino acid mutation that differs from wt-IL-7, i) reducing the affinity of the bifunctional molecule to the IL-7 receptor (IL-7R) compared to the affinity of a bifunctional molecule containing wt-IL-7 to IL-7R, and ii) improving the pharmacokinetics of the bifunctional molecule containing the IL-7 variant or mutant compared to a bifunctional molecule containing wt-IL-7. More specifically, the bifunctional molecule containing the IL-7 variant or mutant further retains the ability to activate IL-7R, particularly via pStat5 signaling. For example, the binding of a bifunctional molecule containing an IL-7 variant or mutant to the IL-7 receptor may be reduced by at least 10%, 20%, 30%, 40%, 50%, or 60% compared to a bifunctional molecule containing wild-type IL-7, while retaining at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the ability to activate IL-7R compared to a bifunctional molecule containing wild-type IL-7.

[0117] According to the present invention, IL-7m exhibits reduced affinity for the IL-7 receptor (IL-7R) compared to the affinity of with-IL-7 for IL-7R. In particular, IL-7m exhibits reduced affinity for CD127 and / or CD132 compared to the affinity of with-IL-7 for CD127 and / or CD132, respectively. Preferably, IL-7m exhibits reduced affinity for CD127 compared to the affinity of with-IL-7 for CD127.

[0118] Preferably, at least one amino acid mutation reduces the affinity of IL-7m to IL-7R, particularly to CD132 or CD127, to at least 1 / 10, 1 / 10

[0119] Preferably, at least one amino acid mutation reduces the affinity of IL-7m to IL-7R, but does not reduce the biological activity of IL-7m compared to IL-7 wt, particularly as measured by the pStat5 signaling pathway.

[0120] Alternatively, at least one amino acid mutation reduces the affinity of IL-7m to IL-7R, but does not significantly reduce the biological activity of IL-7m compared to IL-7 wt, particularly as measured by the pStat5 signaling pathway.

[0121] In addition to or instead of this, IL-7m improves the pharmacokinetics of IL-7 variants or mutants or bifunctional molecules containing IL-7, respectively, compared to wild-type IL-7 or bifunctional molecules containing wild-type IL-7. In particular, the IL-7m according to the present invention improves the pharmacokinetics of IL-7 variants by at least 10-fold, 100-fold, or 1000-fold compared to with-IL-7. Specifically, the IL-7m according to the present invention improves the pharmacokinetics of bifunctional molecules containing IL-7 variants or mutants by at least 10-fold, 100-fold, or 1000-fold compared to bifunctional molecules containing with-IL-7. Pharmacokinetic profile comparisons can be performed by any method known to those skilled in the art, such as injecting the drug in vivo and performing dose ELISA of the serum drug at multiple time points, as shown in Example 2.

[0122] As used herein, the terms “pharmacokinetics” and “PK” are used synonymously and refer to the fate of a compound, substance, or drug administered to a living organism. Pharmacokinetics includes, in particular, ADME or LADME schemes representing release (i.e., release of the substance from a composition), absorption (i.e., entry of the substance into the bloodstream), distribution (i.e., dispersion or diffusion of the substance throughout the body), metabolism (i.e., transformation or breakdown of the substance), and excretion (i.e., removal or clearance of the substance from the organism). The two stages of metabolism and excretion can also be grouped together under the heading of elimination. Those skilled in the art can monitor various pharmacokinetic parameters, including, among others, elimination half-life, elimination constant rate, clearance (i.e., volume of plasma from which the drug is cleared per unit time), Cmax (maximum serum concentration), and drug exposure (determined by the area under the curve; see Scheff et al., Pharm Res. May 2011; Vol. 28 (No. 5): pp. 1081-1089).

[0123] Therefore, the improvement in pharmacokinetics due to the use of IL-7m, particularly in bifunctional molecules, refers to an improvement in at least one of the parameters mentioned above. Preferably, the improvement refers to an improvement in the elimination half-life of the bifunctional molecule, i.e., an increase in half-life duration or Cmax.

[0124] In certain embodiments, at least one mutation in IL-7m improves the elimination half-life of a bifunctional molecule containing IL-7m compared to a bifunctional molecule containing IL-7 wt.

[0125] In one embodiment, 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 the 152-amino acid wild-type human IL-7 (wth-IL-7) protein disclosed in SEQ ID NO: 1. Preferably, 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.

[0126] In particular, at least one mutation occurs at amino acid positions 74 and / or 142 of IL-7. In addition or instead, 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 Sequence ID No. 1.

[0127] In particular, at least one mutation is an amino acid substitution or group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, Q11E, Y12F, M17L, Q22E, K81R, D74E, D74Q, and D74N, or any combination thereof. Such mutations refer to the amino acid positions shown in SEQ ID NO: 1. For example, the mutation W142H represents the substitution of tryptophan in wth-IL7 with histidine to obtain IL-7m having histidine at amino acid position 142. Such a variant is described, for example, in SEQ ID NO: 5.

[0128] In certain embodiments, the IL-7 variant includes a substitution of any amino acid other than P at amino acid position 142 of SEQ ID NO: 1. Such substitutions have been tested in International Publication No. 2019144945A1, the disclosure of which is incorporated herein by reference. For example, W at position 142 can be substituted with G, A, V, C, L, I, M, H, Y, or F. Optionally, the substitution may be selected from the group consisting of W142G, W142A, W142V, W142C, W142L, W142I, W142M, W142H, W142Y, and W142F. In preferred embodiments, the substitution is selected from the group consisting of W142H, W142Y, and W142F, more specifically W142H.

[0129] In one embodiment, IL-7m includes a set of substitutions to break disulfide bonds between C2 and C141, between C47 and C92, and between C34-C129. In particular, IL-7m includes two sets of substitutions to break 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. For example, cysteine ​​residues may be substituted with serine to prevent disulfide bond formation. Therefore, amino acid substitutions can be selected from the group consisting of 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"). These 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: 2-4 (SS1, SS2, and SS3, respectively). Preferably, IL-7m contains the amino acid substitutions C2S-C141S and C47S-C92S. Even more preferably, IL-7m represents the sequence shown in SEQ ID NO: 3.

[0130] In another embodiment, IL-7m comprises at least one mutation selected from the group consisting of W142H, W142F, and W142Y. Such IL-7m are specifically described in the sequences shown in SEQ ID NOs. 5 to 7, respectively. Preferably, IL-7m comprises the mutation W142H. More preferably, IL-7m represents the sequence shown in SEQ ID NO. 5.

[0131] 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 are specifically described in the sequences shown in SEQ ID NOs. 12-14, respectively. Preferably, IL-7m comprises the D74E mutation. More preferably, IL-7m represents the sequence shown in SEQ ID NO. 12.

[0132] In another embodiment, IL-7m includes at least one mutation selected from the group consisting of Q11E, Y12F, M17L, Q22E, and / or K81R. Such mutations point 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.

[0133] In one embodiment, IL-7m includes i) W142H, W142F, or W142Y, and / or ii) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or iii) at least one mutation consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0134] In one embodiment, IL-7m comprises a W142H substitution and i) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or ii) at least one mutation consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0135] In one embodiment, IL-7m comprises a D74E substitution and i) W142H, W142F, or W142Y, and / or ii) at least one mutation consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0136] In one embodiment, IL-7m comprises mutations C2S-C141S and C47S-C92S, and at least one substitution consisting of i) W142H, W142F, or W142Y, and / or ii) D74E, D74Q, or D74N, preferably D74E or D74Q.

[0137] In one embodiment, IL-7m includes i) D74E and W142H substitutions, and ii) mutations C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0138] The IL-7m protein may or may not contain its peptide signal. Furthermore, variants of IL-7 may contain modified polypeptide sequences of IL-7 (e.g., oxidized, reduced, deaminated, or truncated forms).

[0139] In one embodiment, 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 obtained or derived from a recombinant expression system, i.e., from a culture of host cells (e.g., microorganisms, insects, plants, or mammals), or from a transgenic plant or animal genetically engineered to contain a nucleic acid molecule encoding the IL-7m polypeptide. Preferably, the recombinant IL-7 is human recombinant IL-7m (e.g., human IL-7m produced in a recombinant expression system).

[0140] In one embodiment, IL-7m represents 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 present invention comprises an IL-7 variant containing or comprising the amino acid sequence shown in SEQ ID NOs: 2 to 15. More preferably, the bifunctional molecule according to the present invention comprises an IL-7 variant containing or comprising the amino acid sequence shown in SEQ ID NO: 2.

[0141] More preferably, the bifunctional molecule according to the present invention comprises an IL-7 variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, 5, or 12.

[0142] In one embodiment, the present invention provides a bifunctional molecule comprising an IL-7 variant that exhibits reduced immunogenicity compared to wild-type IL-7 protein, particularly by removing a T cell epitope within IL-7 that may stimulate an immune response. An example of such IL-7 is described in International Publication No. 2006061219.

[0143] Furthermore, the present invention relates to any fusion protein comprising an IL-7 variant or mutant as disclosed herein. The IL-7 variant or mutant may be fused at its N-terminus or C-terminus.

[0144] The present invention provides a bifunctional molecule comprising, in particular, IL-7m, an anti-PD-1 binding domain, and an Fc fragment, and optionally a peptide linker.

[0145] In particular, the Fc domain is preferably covalently conjugated to IL-7 by a peptide linker as disclosed above herein (e.g., by gene fusion or chemical coupling).

[0146] In particular, the conjugation of IL-7m and the Fc domain can be direct or indirect (i.e., via a linker) by covalent bonding, and / or chemical, enzymatic, or genetic means. Conjugation can be carried out by any acceptable binding means known in the art. In this regard, coupling can be carried out by one or more covalent bonds, ionic bonds, hydrogen bonds, hydrophobic bonds, or van der Waals bonds that are cleavable or non-cleavable in a physiological medium or within a cell.

[0147] In particular, chemical conjugation can be carried out by the attachment of an exposed sulfhydryl group (Cys), affinity tag (e.g., 6-histidine, Flag tag, Strep tag, SpyCatcher, etc.) to either an Fc domain or IL7-m, or by the incorporation of unnatural amino acids or compounds for click chemistry conjugation.

[0148] In preferred embodiments, the conjugation is obtained by gene fusion (i.e., by expressing a nucleic acid construct as a gene fusion product encoding a binding portion and IL-7 in a suitable system). In one aspect, the present invention is characterized by a fusion protein comprising a first portion containing an immunoglobulin (Ig) chain, particularly an Fc domain, and a second portion containing interleukin-7 (IL-7).

[0149] The availability of IL-7 variants is described in detail in International Publication No. 2020 / 12377, which is incorporated herein by reference.

[0150] Antigen-binding domain that targets PD-1 on immune cells According to the present invention, the antigen-binding domain specifically binds to PD-1 expressed on the surface of immune cells. In particular, the antigen-binding domain does not target targets expressed on tumor cells.

[0151] As used herein, the term “antigen-binding domain” refers to any portion of a peptide, polypeptide, protein, fusion protein, or antibody that has the ability to bind PD-1. Examples of such terms include antibodies or their antigen-binding fragments, as well as antibody mimes or antibody mimetics.

[0152] In one embodiment, the "antigen-binding domain" is selected from the group consisting of antibodies or fragments thereof, and antibody mimetics or antibody mimetics. Those skilled in the field of biochemistry will be familiar with antibody mimetics or antibody mimetics as discussed in Gebauer and Skerra, 2009, Curr Opin Chem Biol, Vol. 13 (No. 3): pp. 245-255. Examples of antibody mimetic compounds include: afibody (also known as trinectin; Nygren, 2008, FEBS J, Vol. 275, pp. 2668-2676); CTLD (also known as tetranectin; Innovations Pharmac. Technol. (2006), pp. 27-30); adnectin (also known as monobody; Meth, Mol. Biol., Vol. 352 (2007), pp. 95-109); antikalin (Drug Discovery Today (2005), Vol. 10, pp. 23-33); DARPins (ankyrin; Nat. Biotechnol. (2004), Vol. 22, pp. 575-582); avimer (Nat. Biotechnol. (2005), Vol. 23, pp. 1556-1561); microbody (FEBS J, (2007), Vol. 274, pp. 86-95); Aptamer (Expert. Opin. Biol. Ther. (2005), Vol. 5, pp. 783-797); Knitz domain (J. Pharmacol. Exp. Ther. (2006), Vol. 318, pp. 803-809); Affilin (Trends. Biotechnol. (2005), Vol. 23, pp. 514-522); Afitin (Krehenbrink et al., 2008, J. Mol. Biol. Vol. 383 (No. 5): pp. 1058-1068), Alpha body (Desmet, J. et al., 2014, Nature Communications. Vol. 5: p. 5237), Fynomer (Grabulovski D et al., 2007, J Biol Chem. Vol. 282 (No. 5): pp. 3196-3204, and Avacta Life Sciences (Wetherby, UK).

[0153] Preferably, the antigen-binding domain is an antibody fragment. More preferably, the antigen-binding domain is a human, humanized, or chimeric antigen-binding fragment.

[0154] With regard to the “binding ability” of the antigen-binding domain, the term “binding” or “conjugate” refers to antibodies containing antibody fragments and derivatives that recognize and contact another peptide, polypeptide, protein, or molecule, particularly PD-1. With regard to a specific target, the terms “specific binding,” “specifically binding,” “specific to,” “selectively binding,” and “selective to” mean that the antigen-binding domain recognizes and binds to a specific target, but substantially does not recognize or bind to other molecules in the sample. For example, an antibody that specifically (or preferentially) binds to an antigen is an antibody or antigen-binding domain that binds to this antigen more easily and / or for a longer period of time, for example, with higher affinity, binding strength, than to other molecules. Preferably, the term “specific binding” is used. -7 This means contact with an antibody or antigen-binding domain having a binding affinity equal to or lower than M. In certain embodiments, the antibody or antigen-binding domain is 10 -8 M, 10 -9 M, or 10 -10 It binds with an affinity equal to or lower than that of M.

[0155] Optionally, the antigen-binding domain may be a Fab domain, Fab', a single-chain variable fragment (scFV), or a single-domain antibody (sdAb). The antigen-binding domain preferably includes a heavy-chain variable region (VH) and a light-chain variable region (VL).

[0156] When the antigen-binding domain is Fab or Fab', the bifunctional molecule contains one heavy chain constant domain and one light chain constant domain (i.e., CH and CL), with the heavy chain linked to IL-7m at its C-terminus.

[0157] In one aspect, PD-1 is specifically expressed by immune cells of healthy subjects or subjects suffering from a disease, particularly a disease such as cancer. This means that PD-1 shows a higher expression level in immune cells than in other cells, or the ratio of immune cells expressing PD-1 to all immune cells is higher than the ratio of other cells expressing PD-1 to all other cells. Preferably, the expression level or ratio is 2, 5, 10, 20, 50, or 100 times higher. More specifically, the expression level or ratio can be determined for a specific type of immune cell, such as a T cell, more specifically a CD8+ T cell, an effector T cell, or an exhausted T cell, or in a specific context, for example, for a subject suffering from a disease such as cancer or an infection.

[0158] As used herein, "immune cell" refers to cells involved in innate and adaptive immunity, such as leukocytes (white blood cells) derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells, and natural killer T cells (NKTs)), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells), etc. In particular, immune cells include B cells, T cells, particularly CD4 + T cells and CD8 + T cells, NK cells, NKT cells, APC cells, macrophages, dendritic cells, and monocytes can be selected in a non-exhaustive list.

[0159] Preferably, the antigen-binding domain specifically binds to PD-1 expressed by immune cells selected from the group consisting of B cells, T cells, natural killer cells, dendritic cells, monocytes, and innate lymphoid cells (ILCs).

[0160] Even more preferably, the immune cell is a T cell. As used herein, "T cell" or "T lymphocyte" includes, for example, CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T regulatory cells, T helper type 17 T cells, and inhibitory T cells. In a very specific aspect, the immune cell is an exhausted T cell.

[0161] In certain aspects, immune cells are effector memory stem cell-like T cells.

[0162] In certain embodiments, the immune cells are exhausted T cells or effector memory stem cell-like T cells, and PD-1 is expressed on the surface of these exhausted T cells or effector memory stem cell-like T cells. T cell exhaustion is a condition in which T cells progressively lose their function, proliferative capacity, and cytotoxic capacity, ultimately leading to their own elimination. T cell exhaustion can be induced by persistent antigen exposure or several factors, including inhibitory receptors, including PD-1.

[0163] In a preferred embodiment, the antigen-binding domain has antagonist activity against PD-1.

[0164] Numerous antibodies against PD-1 have already been described in the art. Those skilled in the art are familiar with methods for constructing a single antigen-binding domain from known antibodies based on their sequences.

[0165] Several anti-PD-1 antibodies have already been clinically approved, while others are still in clinical development. For example, anti-PD-1 antibodies can be selected from the following group: pembrolizumab (also known as keytruda lambrolizumab, MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pizilizumab (CT-011), semiprimab (Libtayo), camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (tisreizumab (Ti Sleizumab, 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 (Si-Yang Liu et al., J. Hematol.) See Oncol. Vol. 10: p. 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 International Publication No. 2014 / 194302), AGEN2034 (see International Publication No. 201 Monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 as described in International Publication No. 7 / 040790, MGA012 (see International Publication No. 2017 / 19846), or IBI308 (see International Publication Nos. 2017 / 024465, 2017 / 025016, 2017 / 132825, and 2017 / 133540), International Publication No. 2006 / 121168. Other bifunctional or bispecific molecules that target PD-1 include RG7769 (Roche), XmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda), and XmAb23104 (Xencor).In particular, the antigen-binding domain targeting PD-1 comprises six CDRs or VH and VLs of the anti-PD1 antibody 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 comprises six CDRs or VH and VLs of the anti-PD1 antibody selected from pembrolizumab (also known as keytruda lambrolizumab, MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), which may be, for example, Fab or scFc domains.

[0166] In certain embodiments, the anti-PD1 antibody from which the antigen-binding domain is derived may be pembrolizumab (also known as keytruda lambrolizumab, MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538).

[0167] In particular, the target is PD-1, and the antigen-binding domain of the bifunctional molecule is an antibody, a fragment or derivative thereof, or an antibody mimetic that is specific to PD-1. Next, in certain embodiments, the antigen-binding domain contained in the bifunctional molecule according to the present invention is derived from an anti-PD1 antibody or its antigen-binding fragment, preferably a human, humanized, or chimeric anti-PD1 antibody or its antigen-binding fragment. Preferably, the antigen-binding domain is a PD-1 antagonist. Thus, the bifunctional molecule can have a synergistic effect on the activation of T cells, particularly exhausted T cells, and more specifically, on TCR signaling, by combining the effect of an IL-7 variant or mutant on the IL-7 receptor with the blockade of the inhibitory effect of PD-1. Preferably, the antigen-binding domain is a PD-1 antagonist.

[0168] In a very specific aspect of this disclosure, the antigen-binding domain is derived from an antibody that targets PD-1, which is disclosed in International Publication No. 2020 / 127366, the disclosure of which is incorporated herein by reference.

[0169] Therefore, the antigen-binding domain is (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 Includes, - Heavy chain CDR1 (HCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 51, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 51. - Heavy chain CDR2 (HCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 53, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 53. - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 54, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 54. - Light chain CDR1 (LCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 63, where X is G or T, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 63. - Light chain CDR2 (LCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 66 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. - Light chain CDR3 (LCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 16 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16.

[0170] In one embodiment, the antigen-binding domain is (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 Includes, - Heavy chain CDR1 (HCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 51, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 51. - Heavy chain CDR2 (HCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 53, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 53. - Heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 54, wherein X1 is D and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, E, or X1 is E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, E, and S, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 54. - Light chain CDR1 (LCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 63, where X is G or T, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 63. - Light chain CDR2 (LCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 66 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. - Light chain CDR3 (LCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 16 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16.

[0171] In another embodiment, the antigen-binding domain comprises or essentially comprises (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, or 62; and (ii) a light chain containing CDR1 of SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16, or SEQ ID NO: 90.

[0172] In another embodiment, the antigen-binding domain comprises (ii) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, or 62; and (ii) a light chain containing CDR1 of SEQ ID NO: 64 or SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16.

[0173] In another embodiment, the antigen-binding domain is (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 55, and (ii) a light chain containing CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 56, and (ii) a light chain containing CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 57, and (ii) a light chain containing CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 58, and (ii) a light chain containing CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 59, and (ii) a light chain containing CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 60, and (ii) a light chain containing CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 61, and (ii) a light chain containing CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 62, and (ii) a light chain containing CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 55, and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 56, and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 57, and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 58, and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 59, and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 60, and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 61, and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 62, and (ii) a light chain containing CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16. It includes or essentially consists of.

[0174] In another embodiment, the antigen-binding domain is (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 55; and (ii) a light chain containing CDR1 of SEQ ID NO: 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 90; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 56; and (ii) a light chain containing CDR1 of SEQ ID NO: 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 90; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 57; and (ii) a light chain containing CDR1 of SEQ ID NO: 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 90; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 58; and (ii) a light chain containing CDR1 of SEQ ID NO: 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 90; or (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 59; and (ii) a light chain containing CDR1 of SEQ ID NO: 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 90; or (i) Heavy chains containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NO: 60; and (ii) Light chains containing CDR1 of SEQ ID NO: 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 90. It includes or essentially consists of.

[0175] In one embodiment, the anti-PD1 antigen-binding fragment according to the present invention includes framework regions, particularly heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3, and HFR4, and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3, and LFR4.

[0176] Preferably, the anti-PD1 antigen-binding fragment according to the present invention includes a human framework region or a humanized framework region. For the purposes of this specification, "human acceptor framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework, as defined below. The human acceptor framework derived from a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence. The "Human Consensus Framework" is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences.

[0177] In particular, the anti-PD1 antigen-binding fragments each include heavy chain variable region framework (HFR) HFR1, HFR2, HFR3, and HFR4, which comprise the amino acid sequences of SEQ ID NOs. 41, 42, 43, and 44, and optionally have one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 27, 29, and 32 of SEQ ID NO. 43, specifically 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.

[0178] In certain embodiments, the anti-PD1 antigen-binding fragment comprises light chain variable region framework (LFR) LFR1, LFR2, LFR3, and LFR4, each containing the amino acid sequence of SEQ ID NOs: i) 45, 46, 47, and 48, ii) 91, 92, 93, and 94, or iii) 95, 96, 97, and 98, and having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the humanized anti-PD1 antigen-binding fragment comprises LFR1 of SEQ ID NOs: 45, 91, or 95, LFR2 of SEQ ID NOs: 46, 92, or 96, LFR3 of SEQ ID NOs: 47, 93, or 97, and LFR4 of SEQ ID NOs: 48, 94, or 98.

[0179] Alternatively, the anti-PD1 antigen-binding fragment comprises light chain variable region framework (LFR) LFR1, LFR2, LFR3, and LFR4, each containing the amino acid sequences of SEQ ID NOs. 45, 46, 47, and 48, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the humanized anti-PD1 antigen-binding fragment comprises LFR1 of SEQ ID NOs. 45, LFR2 of SEQ ID NOs. 46, LFR3 of SEQ ID NOs. 47, and LFR4 of SEQ ID NOs. 48.

[0180] Alternatively, the anti-PD1 antigen-binding fragment comprises light chain variable region framework (LFR) LFR1, LFR2, LFR3, and LFR4, each containing the amino acid sequences of SEQ ID NOs: 91, 92, 93, and 94, and having one, two, or three modifications selected optionally from substitutions, additions, deletions, and any combination thereof. Preferably, the humanized anti-PD1 antigen-binding fragment comprises LF1 of SEQ ID NOs: 91, LFR2 of SEQ ID NOs: 92, LFR3 of SEQ ID NOs: 93, and LFR4 of SEQ ID NOs: 94. Preferably, such frameworks are associated with CDR1 of SEQ ID NOs: 89, CDR2 of SEQ ID NOs: 66, and CDR3 of SEQ ID NOs: 90, and have one, two, or three modifications selected optionally from substitutions, additions, deletions, and any combination thereof.

[0181] Alternatively, the anti-PD1 antigen-binding fragment includes light chain variable region framework (LFR) LFR1, LFR2, LFR3, and LFR4, each containing the amino acid sequences of SEQ ID NOs. 95, 96, 97, and 98, and having one, two, or three modifications selected optionally from substitutions, additions, deletions, and any combination thereof. Preferably, the humanized antigen-binding fragment includes LFR1 of SEQ ID NOs. 95, LFR2 of SEQ ID NOs. 96, LFR3 of SEQ ID NOs. 97, and LFR4 of SEQ ID NOs. 98, and has one, two, or three modifications selected optionally from substitutions, additions, deletions, and any combination thereof.

[0182] The VL domain and VH domain of the antigen-binding domain contained in the bifunctional molecule according to the present invention may preferably include four framework regions separated by three complementarity-determining regions, which are operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the amino terminus to the carboxyl terminus).

[0183] In one embodiment, the antigen-binding domain includes or is essentially composed of: (a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17; (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Includes.

[0184] In another embodiment, 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, each having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112, respectively; (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, which optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27 or SEQ ID NO: 28. It includes or essentially consists of.

[0185] In another embodiment, the antigen-binding domain is (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 It includes or essentially consists of.

[0186] In one aspect, the bifunctional molecule comprises framework regions, particularly the heavy chain variable region framework regions (HFRs) HFR1, HFR2, HFR3, and HFR4 and the light chain variable region framework regions (LFRs) LFR1, LFR2, LFR3, and LFR4, particularly the amino acid sequences of SEQ ID NOs: 41, 42, 43, and 44, respectively, and optionally HER3, i.e., HFR1, HFR2, HFR3, and HFR4 having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position except positions 27, 29, and 32 of SEQ ID NO: 43. Preferably, the bifunctional molecule 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. In addition thereto, the bifunctional molecule may comprise light chain variable region framework regions (LFRs) LFR1, LFR2, LFR3, and LFR4 having the amino acid sequences of SEQ ID NOs: 45, 46, 47, and 48, respectively, and optionally one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the bifunctional molecule 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. Alternatively, the bifunctional molecule may comprise light chain variable region framework regions (LFRs) LFR1, LFR2, LFR3, and LFR4 having the amino acid sequences of SEQ ID NOs: 91, 92, 93, and 94, respectively, and optionally one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the bifunctional molecule comprises LFR1 of SEQ ID NO: 91, LFR2 of SEQ ID NO: 92, LFR3 of SEQ ID NO: 93, and LFR4 of SEQ ID NO: 94. Alternatively, the bifunctional molecule may comprise light chain variable region framework regions (LFRs) LFR1, LFR2, LFR3, and LFR4 having the amino acid sequences of SEQ ID NOs: 95, 96, 97, and 98, respectively, and optionally one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the bifunctional molecule comprises LFR1 of SEQ ID NO: 95, LFR2 of SEQ ID NO: 96, LFR3 of SEQ ID NO: 97, and LFR4 of SEQ ID NO: 98.

[0187] In another aspect, the antigen-binding domain comprises or consists essentially of any combination of VH and VL described in Table D (Table 4) and E (Table 5).

[0188] In another aspect, the antigen-binding domain comprises or consists essentially of any of the following combinations of a heavy chain variable region (VH) and a light chain variable region (VL).

[0189] [Table 4]

[0190] In a very specific aspect, 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.

[0191] In another aspect, the antigen-binding domain comprises or consists essentially of any of the following combinations of a heavy chain variable region (VH) and a light chain variable region (VL):

[0192] [Table 5]

[0193] Peptide linker In certain aspects, the bifunctional molecule according to the invention further comprises a peptide linker for connecting the antigen-binding domain and IL-7m to the Fc chain. The peptide linker typically has a length and flexibility sufficient to ensure that the IL-7m and the antigen-binding domain, which are linked by the linker therebetween, have sufficient spatial freedom to perform their functions.

[0194] In one aspect of the disclosure, IL-7m is preferably linked to the Fc chain through a peptide linker. In one aspect of the disclosure, the antigen-binding domain can be linked to the Fc chain by a hinge naturally found in the heavy chain for connecting the VH domain, particularly the CH1 domain, to the CH2 domain of the Fc chain.

[0195] As used herein, the term “linker” refers to a sequence of at least one amino acid. Such linkers may be useful in preventing steric hindrance. Linkers are typically 3 to 44 amino acid residues in length. Preferably, linkers have 3 to 30 amino acid residues. In some embodiments, linkers have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues.

[0196] The linker sequence may be a naturally occurring sequence or a sequence that does not exist in nature. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to which the bifunctional molecule is administered. One useful group of linker sequences is those derived from the hinge region of heavy chain antibodies, as described in International Publications 96 / 34103 and 94 / 04678. Another example is polyalanine linker sequences. A more preferred example of linker sequences is Gly / Ser linkers of various lengths, including (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2)3, particularly (Gly4Ser)3. Preferably, the linker is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, and (Gly3Ser2)3. More preferably, the linker is (GGGGS)3.

[0197] In one embodiment, the linker contained in the bifunctional molecule is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2)3, and is preferably (Gly4Ser)3. Preferably, the linker is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, and (Gly3Ser2)3.

[0198] FC Domain The Fc domain of a bifunctional molecule is part of the antigen-binding domain, particularly the heavy chain of IgG immunoglobulin. Indeed, if the antigen-binding domain is Fab, the bifunctional molecule may contain a single heavy chain including a variable heavy chain (VH), CH1, hinge, CH2, and CH3 domains. However, the bifunctional molecule may also have other structures, such as scFv, or diabodies. For example, this may include an Fc domain linked to the antigen-binding domain.

[0199] The Fc domain may be derived from a human immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4, or other classes of heavy chain constant domains. Preferably, the bifunctional molecule contains an IgG1 or IgG4 heavy chain constant domain.

[0200] Preferably, the Fc domain includes the CH2 and CH3 domains. Optionally, this may include all or part of the hinge region, the CH2 domain, and / or the CH3 domain. In some embodiments, the CH2 and / or CH3 domains are derived from a human IgG4 or IgG1 heavy chain. Preferably, the Fc domain includes all or part of the hinge region. The hinge region may be derived from an immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, or IgG4. More preferably, the hinge region is derived from a human or humanized IgG1 or IgG4 heavy chain.

[0201] The IgG1 hinge region has three cysteine ​​molecules, two of which are involved in the disulfide bond between the two heavy chains of the immunoglobulin. These same cysteine ​​molecules enable efficient and consistent disulfide bond formation between the Fc moieties. Therefore, the preferred hinge region of the present invention is derived from IgG1, more preferably human IgG1. In some embodiments, the first cysteine ​​in the human IgG1 hinge region is mutated to another amino acid, preferably serine.

[0202] The hinge region of IgG4 is known to be inefficient in the formation of interchain disulfide bonds. However, the preferred hinge region of the present invention may be derived from the 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., Vol. 30: pp. 105-108). More preferably, the hinge region is derived from the human IgG4 heavy chain.

[0203] A bifunctional molecule contains a dimeric Fc domain. Therefore, each of the two monomers contains one Fc chain, and these Fc chains can form a dimeric Fc domain. A dimeric Fc domain can be a homodimer, where each Fc monomer is identical or essentially identical. Alternatively, a dimeric Fc domain can be a heterodimer, where each Fc monomer is different but complementary to facilitate the formation of the heterodimeric Fc domain.

[0204] More specifically, the Fc domain is a heterodimer Fc domain. The heterodimer Fc domain is constructed by altering the amino acid sequence of each monomer. The heterodimer Fc domain relies on the amino acid variants in the constant region being different in each chain, which promotes heterodimer formation and / or facilitates the purification of the heterodimer more easily than the homodimer. There are a number of mechanisms that can be used to generate the heterodimers of the present invention. In addition, as those skilled in the art will understand, these mechanisms can be combined to ensure a high degree of heterodimerization. Therefore, amino acid variants that lead to the production of heterodimers are called "heterodimerizing variants." Examples of heterodimerizing variants include steric variants (e.g., the "knobs and holes" variant or "skew" variant described below, and the "charge pair" variant described below), as well as "pi variants" that enable the separation and purification of homodimers from heterodimers. International Publication No. 2014 / 145806, which is incorporated herein by reference in its entirety, discloses useful mechanisms for heterodimerization, including “knob-and-hole” variants, “electrostatic steering” variants, or “charge pair” variants, bi variants, and general additional Fc variants. See also Ridgway et al., Protein Engineering, Vol. 9 (No. 7): p. 617 (1996); Atwell et al., J. Mol. Biol., Vol. 270: p. 26 (1997); U.S. Patent No. 8,216,805; Merchant et al., Nature Biotech, Vol. 16: p. 677 (1998). All of these documents are incorporated herein by reference in their entirety. For “electrostatic steering,” see Gunasekaran et al., J. Biol. Chem., Vol. 285 (No. 25): p. 19637 (2010). This document is incorporated herein by reference in its entirety. For the variant, see U.S. Patent Application Publication No. 2012 / 0149876.This document is hereby incorporated by reference in its entirety into this specification.

[0205] 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" chain or K chain, which means that the first Fc chain contains substitutions that characterize the knob chain, and the second Fc chain is the "hole chain" or H chain, which means that the second Fc chain contains substitutions that characterize the hole chain. The reverse is also true, where the first Fc chain is the "hole" chain or H chain, which means that the first Fc chain contains substitutions that characterize the hole chain, and the second Fc chain is the "knob chain" or K chain, which means that the second Fc chain contains substitutions that characterize the knob chain. In a preferred embodiment, the first Fc chain is the "hole" chain or H chain, and the second Fc chain is the "knob" chain or K chain.

[0206] Optionally, the heterodimeric Fc domain can include one heterodimeric Fc chain containing the substitutions shown in Table F (Table 6) below, and the other heterodimeric Fc chain containing the substitutions shown in Table F (Table 6) below.

[0207]

Table 6

[0208] In a preferred embodiment, the first Fc chain is the "hole" chain or H chain and contains the substitutions T366S / L368A / Y407V / Y349C, and the second Fc chain is the "knob" chain or K chain and contains the substitutions T366W / S354C.

[0209] Optionally, the Fc chain may further contain additional substitutions.

[0210] In particular, with respect to bifunctional molecules that target cell surface molecules, especially those on immune cells, it may be necessary to disable their effector function. Manipulating the Fc region may also be desirable to reduce or increase the effector function of bifunctional molecules.

[0211] In certain embodiments, amino acid modifications may be introduced into the Fc region to create Fc region variants. In certain embodiments, the Fc region variants possess some, but not all, of the effector functions. Such bifunctional molecules may be useful in applications where, for example, the in vivo half-life of an antibody is important, but certain effector functions are unnecessary or even detrimental. Numerous substitutions, or substitutions or deletions that alter effector functions, are known in the art.

[0212] In one embodiment, the constant region of the Fc domain contains a mutation that reduces affinity for the Fc receptor or reduces Fc effector function. For example, the constant region may contain a mutation that eliminates a glycosylation site within the constant region of the IgG heavy chain. Preferably, the CH2 domain contains a mutation that eliminates a glycosylation site within the CH2 domain.

[0213] In certain embodiments, the Fc domain is modified to increase binding to FcRn, thereby increasing the half-life of the bifunctional molecule. In other embodiments or additional embodiments, the Fc domain is modified to decrease binding to FcγR, thereby reducing ADCC or CDC, or to increase binding to FcγR, thereby increasing ADCC or CDC.

[0214] Modifications to the amino acids near the junction of the Fc and non-Fc portions can dramatically increase the serum half-life of the Fc fusion protein, as shown in International Publication No. 01 / 58957. Therefore, the junction region of the protein or polypeptide of the present invention may preferably contain modifications within about 10 amino acids of the junction compared to the naturally occurring sequences of the immunoglobulin heavy chain and erythropoietin. Such amino acid changes may result in increased hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which the C-terminal lysine residue is 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 the serum half-life.

[0215] In one embodiment, the constant region of the Fc domain has one of the mutations listed in Table G (Table 7) below, or any combination thereof.

[0216] [Table 7A]

[0217] [Table 7B]

[0218] In certain embodiments, the bifunctional molecules are optionally selected as 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;L234A / L23 The human IgG1 heavy chain constant domain or IgG1 Fc domain comprises a substitution or combination of substitutions selected from the group consisting of 5A;P329G;N297A+M252Y / S254T / T256E;K322A and K444A, preferably a substitution or combination of substitutions selected from the group consisting of N297A in combination with M252Y / S254T / T256E and L234A / L235A in combination with P329G.

[0219] In certain embodiments, the bifunctional molecule is 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;L234A / L235A;P329G;N297A+M252Y / S254T / T256E; The human IgG1 heavy chain constant domain or IgG1 Fc domain has a substitution or combination of substitutions selected from the group consisting of K322A, K444A, K444E, K444D, K444G, K444S, M428L, L309D, Q311H, N434S, M428L+N434S and L309D+Q311H+N434S, preferably N297A in combination with M252Y / S254T / T256E and optionally L234A / L235A in combination with P329G.

[0220] A bifunctional molecule comprising a human IgG1 heavy chain constant domain or IgG1 Fc domain having the substitution L234A / L235A / P329G combination significantly reduces or completely suppresses ADCC, ADCP, and / or CDC induced by the bifunctional molecule, thereby reducing nonspecific cytotoxicity.

[0221] In another embodiment, the bifunctional molecule comprises a human IgG4 heavy chain constant domain or a human IgG4 Fc domain having a substitution or combination of substitutions selected from the group consisting of S228P;L234A / L235A;P329G, S228P+M252Y / S254T / T256E, and K444A. More preferably, the bifunctional molecule, preferably the bifunctional molecule according to the present invention, comprises an IgG4 Fc region having S228P that stabilizes IgG4.

[0222] In another embodiment, the bifunctional molecule comprises a human IgG4 heavy chain constant domain or a human IgG4 Fc domain having a substitution or combination of substitutions selected from the group consisting of S228P;L234A / L235A;L234A / L235A / P329G, P329G, S228P+M252Y / S254T / T256E, K444A K444E, K444D, K444G and K444S. More preferably, the bifunctional molecule, preferably the bifunctional molecule according to the present invention, comprises an IgG4 Fc region having S228P that stabilizes IgG4.

[0223] As used herein, " / " and "+" refer to cumulative mutations. Thus, the mutation S228P+M252Y / S254T / T256E means the following mutations: S228P, M252Y, S254T, and T256E.

[0224] A bifunctional molecule containing a human IgG4 heavy chain constant domain or IgG4 Fc domain having a substituted P329G reduces ADCC and / or CDC induced by the bifunctional molecule, thereby reducing nonspecific cytotoxicity.

[0225] All subclasses of human IgG possess a C-terminal lysine residue (K444) in the antibody heavy chain that is easily cleaved in circulation. This cleavage in the blood can impair or reduce the biological activity of the bifunctional molecule by releasing linked IL-7m to the bifunctional molecule.

[0226] To circumvent this problem, the K444 amino acid in the IgG domain can be replaced with another amino acid, which is a mutation commonly used in antibodies, to reduce proteolytic cleavage. In one embodiment, the bifunctional molecule comprises at least one further amino acid substitution consisting of K444A, K444E, K444D, K444G, or K444S, preferably K444A.

[0227] More specifically, the K444 amino acid in the IgG domain can be replaced with alanine, a mutation commonly used in antibodies, to reduce proteolytic cleavage. In one embodiment, the bifunctional molecule includes at least one further amino acid substitution consisting of K444A.

[0228] Optionally, the bifunctional molecule may include an additional cysteine ​​residue in the C-terminal domain of the Fc domain, creating an additional disulfide bond and potentially limiting the flexibility of the bifunctional molecule.

[0229] In one embodiment, the bifunctional molecule comprises, in particular as disclosed in Table H (Table 8) below, one heavy chain constant domain of SEQ ID NO: 39 or 52, and / or one light chain constant domain of SEQ ID NO: 40, in particular one heavy chain constant domain or Fc domain of SEQ ID NO: 39 or 52, and one light chain constant domain of SEQ ID NO: 40.

[0230] [Table 8]

[0231] In one particular embodiment, the bifunctional molecule according to the present invention comprises a heterodimer of an Fc domain containing a “knob-into-hole” modification as described herein. Preferably, such an Fc domain is an IgG1 or IgG4 Fc domain as described herein, and more preferably an IgG1 Fc domain containing the mutant N297A as disclosed above.

[0232] For example, the first Fc chain is a "hole" chain or H chain containing substitutions T366S / L368A / Y407V / Y349C and optionally N297A, and the second Fc chain is a "knob" chain or K chain containing substitutions T366W / S354C and optionally N297A. Preferably, the first Fc chain is a "hole" chain or H chain containing substitutions T366S / L368A / Y407V / Y349C and N297A, and the second Fc chain is a "knob" chain or K chain containing substitutions T366W / S354C and N297A. More specifically, the second Fc chain may contain or consist of SEQ ID NO: 75, and / or the first Fc chain may contain or consist of SEQ ID NO: 77.

[0233] More specifically, the IL-7m according to the present invention is linked to the knob and / or hole chain of the heterodimer Fc domain. Thus, the bifunctional molecule according to the present invention may contain a single IL-7m linked to either the hole chain or the knob chain of the Fc domain. Preferably, the bifunctional molecule according to the present invention contains a single IL-7m linked to the hole chain of the Fc domain.

[0234] In the first embodiment, the bifunctional molecule comprises IL-7m ligated to the C-terminus of a knob chain of an Fc domain, such a knob chain of an Fc domain ligated to an antigen-binding domain.

[0235] In a second embodiment, the bifunctional molecule comprises IL-7m ligated to the C-terminus of a hole chain of an Fc domain, the hole chain of such an Fc domain being ligated to an antigen-binding domain at its N-terminus.

[0236] Optionally, the bifunctional molecule may contain a single IL-7m ligated to the C-terminus of the hole chain of the Fc domain, or it may contain only a single antigen-binding domain ligated to the N-terminus of the hole chain of the Fc domain. In such embodiments, the knob chain domain lacks the IL-7m antigen-binding domain.

[0237] Optionally, the bifunctional molecule may contain a single IL-7m ligated to the C-terminus of the knob chain of the Fc domain, or it may contain only a single antigen-binding domain ligated to the N-terminus of the knob chain of the Fc domain. In such embodiments, the whole-chain domain lacks both IL-7m and the antigen-binding domain.

[0238] Therefore, the object of the present invention relates to a polypeptide comprising an antigen-binding domain (or at least a portion thereof corresponding to a heavy chain), an Fc chain (knob Fc chain or hole Fc chain), preferably a hole chain of the Fc domain, and IL-7m, from the N-terminus to the C-terminus. The complementary chain comprises IL-7m and a complementary Fc chain lacking the antigen-binding domain, preferably a knob chain of the Fc domain.

[0239] In very specific embodiments, the bifunctional molecule targets PD-1 and includes: (a) A heavy chain 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, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36, respectively, wherein the substitutions correspond to a hole chain or a knob chain, preferably a hole chain, and more specifically, Table As disclosed in F (Table 6), particularly in SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36, either T363S / L365A / Y4047V / Y346C or T363W / S351C, preferably T363S / L365A / Y4047V / Y346C, and optionally N294A in any of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36, heavy chain; (b) A light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37 or SEQ ID NO: 38. Includes.

[0240] In another embodiment, the bifunctional molecule comprises or consists of any of the following combinations of a heavy chain (CH) and a light chain (CL):

[0241] [Table 9]

[0242] The heavy chain corresponds to a hole chain or a knob chain, preferably a hole chain, and more specifically as disclosed in Table F (Table 6), and is either T366S / L368A / Y407V / Y349C or T366W / S354C, preferably T366S / L368A / Y407V / Y349C, particularly in SEQ ID NOs. 29, 30, 31, 32, 33, 34, 35, or 36, and optionally includes the substitution N297A in any of SEQ ID NOs. 36, where the position of the substitution is specified according to EU numbering.

[0243] In a very specific embodiment, the bifunctional molecule targets PD-1 and comprises or consists of SEQ ID NO: 37 or 38.

[0244] Therefore, the bifunctional molecule may contain one heavy chain including any of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, and 36, and the Fc chain is optionally modified to promote heterodimerization of the Fc chain to form a heterodimeric Fc domain. More specifically, the heavy chain corresponds to a hole chain or a knob chain, preferably a hole chain, and more specifically as disclosed in Table F (Table 6), in particular including a substitution of either T366S / L368A / Y407V / Y349C or T366W / S354C, preferably T366S / L368A / Y407V / Y349C, and optionally N297A, in any of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36, where the position of the substitution is defined according to EU numbering. The heavy chain is optionally linked to IL-7m at its C-terminus via a linker.

[0245] Preferred structures In certain embodiments, the molecule comprises a first monomer comprising an antigen-binding domain optionally covalently linked to a first Fc chain via a peptide linker, wherein the first Fc chain optionally covalently linked to an IL-7 variant via a peptide linker, and a second monomer preferably comprising a complementary second Fc chain lacking the antigen-binding domain and the 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 specifically, the molecule comprises a first monomer comprising an antigen-binding domain optionally covalently linked via a peptide linker to the N-terminus of a first heterodimer Fc chain, wherein the first heterodimer Fc chain has its C-terminus optionally covalently linked via a peptide linker, and a second monomer comprising a complementary second heterodimer Fc chain lacking the antigen-binding domain and the IL-7 variant, and preferably lacking any other molecule. More specifically, the molecule comprises a first monomer and a second monomer, the first monomer comprising an antigen-binding domain whose C-terminus is optionally covalently linked to the N-terminus of the first heterodimer Fc chain via a peptide linker, the first heterodimer Fc chain comprising an antigen-binding domain whose C-terminus is optionally covalently linked to the N-terminus of an IL-7 variant via a peptide linker, and the second monomer comprising a complementary second heterodimer Fc chain lacking the antigen-binding domain and the IL-7 variant, preferably lacking any other molecules. Such a molecule is shown, for example, as “Construction 3” in Figure 1.

[0246] In particular, the bifunctional molecule according to the present invention comprises a single anti-PD-1 antigen-binding domain and a single IL-7 W142H variant (this construct is also called anti-PD-1*1 IL-7 W142H*1). In particular, such a construct 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. The 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.

[0247] In another embodiment, the bifunctional molecule according to the present invention comprises a single anti-PD-1 antigen-binding domain and a single IL-7 W142H variant (this construct is also called anti-PD-1*1 IL-7 W142H*1). In particular, such a construct 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: 24 and SEQ ID NO: 88 or 90.

[0248] In one embodiment, the present invention relates to a bifunctional molecule comprising a single antigen-binding domain and a single IL-7 variant, - The bifunctional molecule comprises a first monomer containing an antigen-binding domain whose C-terminus is optionally covalently linked to the N-terminus of a first Fc chain via a peptide linker, and a second monomer containing a complementary second Fc chain lacking the antigen-binding domain and the IL-7 variant; - i) an IL-7 variant is optionally covalently linked to the C-terminus of the first Fc chain via a peptide linker; or ii) a single antigen-binding domain comprises a variable heavy chain and a variable light chain, with the IL-7 variant covalently linked to the C-terminus of the light chain; - The antigen-binding domain binds to PD-1; and - The IL-7 variant exhibits at least 75% identity with wild-type human IL-7 (wth-IL-7), which contains or consists of the amino acid sequence shown in SEQ ID NO: 1, and 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 the IL-7R, and ii) improves the pharmacokinetics of the bifunctional molecule containing the IL-7 variant compared to the bifunctional molecule containing wth-IL-7. Regarding bifunctional molecules.

[0249] In particular, the IL-7 variants are (i) W142G, W142A, W142V, W142C, W142L, W142I, W142M, W142H, W142Y and W142F, preferably 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, comprising at least one amino acid mutation selected from the group, the amino acid numbering being as shown in SEQ ID NO: 1, preferably comprising the amino acid substitution W142H, and more preferably the amino acid substitution as defined in SEQ ID NO: 5, for example.

[0250] In another embodiment, the present invention relates to a bifunctional molecule comprising a single antigen-binding domain and a single IL-7 variant, - The bifunctional molecule comprises a first monomer containing an antigen-binding domain whose C-terminus is optionally covalently linked to the N-terminus of a first Fc chain via a peptide linker, and a second monomer containing a complementary second Fc chain lacking the antigen-binding domain and the IL-7 variant; - i) the IL-7 variant is optionally covalently linked to the C-terminus of the first Fc chain via a peptide linker; or ii) a single antigen-binding domain comprises a variable heavy chain and a variable light chain, with the IL-7 variant covalently linked to the C-terminus of the light chain; - The antigen-binding domain is as described in the paragraph above, "Antigen-binding domain targeting PD-1 on immune cells"; - IL7 is as described in the above paragraph, “IL7 and IL-7 Variants”; - The Fc domain is as described in the paragraph titled "Fc Domain" above; - The peptide linker is as described in the paragraph titled "Peptide Linker" above. Regarding bifunctional molecules.

[0251] Preferably, the peptide linker is (GGGGS)3, or GGGGSGGGGSGGGGS, or as specified in Sequence ID No. 70.

[0252] Preferably, the Fc domain is derived from human IgG1 or IgG4. The first Fc chain is a hole chain or H chain and contains substitutions T366S / L368A / Y407V / Y349C and optionally N297A, and the second Fc chain is a knob chain or K chain and contains substitutions T366W / S354C and optionally N297A. Preferably, the Fc chain is a hole chain or H chain and contains substitutions T366S / L368A / Y407V / Y349C and N297A, and the second Fc chain is a knob chain or K chain and contains substitutions T366W / S354C and N297A. More preferably, the second Fc chain contains or consists of SEQ ID NO: 75, and / or the first Fc chain contains or consists of SEQ ID NO: 77.

[0253] Preferably, IL-7 contains the amino acid substitution W142H, with the amino acid numbering as shown in SEQ ID NO: 1, and in particular, the substitution as specified in SEQ ID NO: 5, for example.

[0254] Preferably, the antigen-binding domain is pembrolizumab, nivolumab, pizilizumab, semiprimab, camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317, spartalizumab, MK-3477, SCH-900475, PF-06801591, JNJ-63723283, genolimuzumab, LZM-009, BCD-100, SHR-1201, BA The antigen-binding domain is derived from an antibody selected from the group consisting of T-1306, AK-103, MEDI-0680, MEDI0608, JS001, BI-754091, CBT-501, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, AGEN2034, MGA012, or IBI308, 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4. More preferably, the antigen-binding domain comprises or essentially consists of (i) a heavy chain containing CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 53, and CDR3 of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, or 62; and (ii) a light chain containing CDR1 of SEQ ID NO: 64 or 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16. More preferably, the antigen-binding domain comprises or essentially comprises (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) a light chain comprising CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16. Preferably, the antigen-binding domain comprises or essentially comprises (a) a heavy chain variable region (VH) comprising or essentially comprising the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25, preferably SEQ ID NO: 24; and (b) a light chain variable region (VL) comprising or comprising the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 88, or SEQ ID NO: 90, preferably SEQ ID NO: 28. Preferably, the antigen-binding domain essentially comprises (a) a heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25, preferably SEQ ID NO: 24; and (b) a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28, preferably SEQ ID NO: 28.

[0255] In certain embodiments, the bifunctional molecule according to the present invention comprises an anti-PD-1 antigen-binding domain comprising or essentially comprising the heavy chain variable region (VH) of SEQ ID NO: 24 and the light chain variable region (VL) of SEQ ID NO: 28, and an IL-7 variant comprising the amino acid substitution W142H, preferably the amino acid substitution as defined in SEQ ID NO: 5, with the amino acid numbering as shown in SEQ ID NO: 1.

[0256] More specifically, the bifunctional molecule according to the present invention (i) the anti-PD-1 antigen-binding domain comprises or is essentially derived from the heavy chain variable region (VH) of SEQ ID NO: 24 and the light chain variable region (VL) of SEQ ID NO: 28, 88, or 99. (ii) The IL-7 variant contains or is essentially derived from the sequence specified in SEQ ID NO: 5 (iii) The second Fc chain contains or consists of SEQ ID NO: 75, and / or the first Fc chain contains or consists of SEQ ID NO: 77, (iv) The peptide linker optionally contains or consists of SEQ ID NO: 70. Includes.

[0257] More specifically, the bifunctional molecule according to the present invention (i) the anti-PD-1 antigen-binding domain comprises or is essentially derived from the heavy chain variable region (VH) of SEQ ID NO: 24 and the light chain variable region (VL) of SEQ ID NO: 28. (ii) The IL-7 variant contains or is essentially derived from the sequence specified in SEQ ID NO: 5 (iii) The second Fc chain contains or consists of SEQ ID NO: 75, and / or the first Fc chain contains or consists of SEQ ID NO: 77, (iv) The peptide linker optionally contains or is essentially derived from SEQ ID NO: 70 Includes.

[0258] In a very specific embodiment, the bifunctional molecule comprises a light chain containing or consisting of SEQ ID NO: 38, and a heavy chain containing SEQ ID NO: 35, wherein the Fc chain is optionally modified to promote heterodimerization of the Fc chain to form a heterodimeric Fc domain. In one embodiment, the heavy chain is optionally linked to IL-7m at its C-terminus via a linker. In an alternative embodiment, the light chain is optionally linked to IL-7m at its C-terminus via a linker.

[0259] In a very specific embodiment, the bifunctional molecule may comprise a second monomer of SEQ ID NO: 75 and a first monomer comprising an Fc chain of SEQ ID NO: 77, optionally linked via a linker to an antigen-binding domain (e.g., SEQ ID NO: 79) at its N-terminus. More preferably, the bifunctional molecule may comprise a second monomer of SEQ ID NO: 75 and a first monomer comprising an Fc chain of SEQ ID NO: 77, optionally linked via a linker to an antigen-binding domain (e.g., SEQ ID NO: 79) at its N-terminus and optionally linked via a linker to any IL-7m disclosed herein at its C-terminus.

[0260] If IL-7m is optionally an IL-7 variant, the bifunctional molecule may include a second monomer of SEQ ID NO: 75, a first monomer of SEQ ID NO: 83, and a third monomer of SEQ ID NO: 37, 38, or 80, preferably SEQ ID NO: 38 or 80.

[0261] If IL-7m is optionally an IL-7 variant, the bifunctional molecule may include a second monomer of SEQ ID NO: 75, a first monomer of SEQ ID NO: 84, and a third monomer of SEQ ID NO: 37, 38, or 80, preferably SEQ ID NO: 38 or 80, which is optionally linked at its terminus by a linker to an IL-7 variant, particularly one of SEQ ID NOs from 2 to 15, particularly the variant of SEQ ID NO: 5.

[0262] In another very specific embodiment, the bifunctional molecule may comprise a second monomer of SEQ ID NO: 77 and a first monomer comprising the Fc chain of SEQ ID NO: 75, optionally linked via a linker at the N-terminus to an antigen-binding domain (e.g., SEQ ID NO: 79) and optionally linked via a linker at the C-terminus to any IL-7m disclosed herein.

[0263] In another very specific embodiment, the bifunctional molecule may comprise a second monomer of SEQ ID NO: 77, a first monomer comprising an Fc chain of SEQ ID NO: 75 optionally linked via a linker to an antigen-binding domain (e.g., SEQ ID NO: 79) at its N-terminus, and a third monomer of SEQ ID NO: 37, 38, or 80, preferably SEQ ID NO: 38 or 80, optionally linked via a linker to any IL-7m disclosed herein at its terminus.

[0264] In particular, IL-7m includes or is essentially composed of SEQ ID NO: 1, or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity therewith, or having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications selected from the group consisting of additions, deletions, substitutions, and combinations thereof with respect to the wild-type protein.

[0265] Optionally, the bifunctional molecule may include a second monomer of SEQ ID NO: 77, a first monomer of SEQ ID NO: 82, and a third monomer of SEQ ID NO: 37, 38, or 80, preferably SEQ ID NO: 38 or 80.

[0266] In a very specific embodiment, the bifunctional molecule according to the present invention comprises a first monomer of SEQ ID NO: 75, a second monomer of SEQ ID NO: 83, and a third monomer of SEQ ID NO: 80, 100, or 101. Preferably, the bifunctional molecule according to the present invention comprises a first monomer of SEQ ID NO: 75, a second monomer of SEQ ID NO: 83, and a third monomer of SEQ ID NO: 80.

[0267] Preparation of bifunctional molecules - Nucleic acid molecules encoding the bifunctional molecules of the present invention, recombinant expression vectors containing those nucleic acid molecules, and host cells To produce the bifunctional molecules according to the present invention, particularly in mammalian cells, a nucleic acid sequence or group of nucleic acid sequences encoding the bifunctional molecules is subcloned into one or more expression vectors. Such vectors are commonly used for transfecting 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 contents of this document are incorporated herein by reference), and in WH Freeman and Company's "Antibody engineering: a practical guide" (1992), where commentaries related to molecule production are scattered throughout the corresponding text.

[0268] Generally, such methods are (1) A step of transfecting or transforming a suitable host cell with a polynucleotide encoding the recombinant bifunctional molecule of the present invention, or a vector containing the polynucleotide. (2) A step of culturing host cells in an appropriate medium, and (3) Optionally, a step of isolating or purifying a bifunctional molecule from a culture medium or host cells. Includes.

[0269] The present invention further relates to nucleic acids encoding the bifunctional molecules as disclosed above, vectors comprising the nucleic acids of the present invention, preferably expression vectors, genetically modified host cells directly transformed with the vectors of the present invention or with sequences encoding recombinant bifunctional molecules, and methods for producing the bifunctional molecules of the present invention by recombinant techniques.

[0270] Nucleic acids, vectors, and host cells are described in more detail below in this specification.

[0271] nucleic acid sequence Furthermore, the present invention relates to nucleic acid molecules encoding a bifunctional molecule as defined above, or to a group of nucleic acid molecules encoding a bifunctional molecule as defined above. The nucleic acids encoding the bifunctional molecules 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.

[0272] In detail, nucleic acid molecules encoding a bifunctional molecule as defined herein are: - A first nucleic acid molecule encoding a first monomer comprising an antigen-binding domain optionally covalently linked to a first Fc chain via a peptide linker, wherein the first Fc chain is optionally covalently linked to IL7m via a peptide linker, and - A second nucleic acid molecule encoding a second monomer containing a complementary second Fc chain, - A third nucleic acid molecule encoding the light chain of the antigen-binding domain. Includes.

[0273] In another embodiment, a nucleic acid molecule encoding a bifunctional molecule as defined herein is: - A first nucleic acid molecule encoding a first monomer containing an antigen-binding domain optionally covalently linked to the first Fc chain via a peptide linker, - A second nucleic acid molecule encoding a second monomer containing a complementary second Fc chain, and - A third nucleic acid molecule encoding a light chain of an antigen-binding domain, wherein the light chain is optionally covalently linked to an IL7 variant according to the present invention via a peptide linker. Includes.

[0274] In one embodiment, the nucleic acid molecule is an isolated, particularly non-natural, nucleic acid molecule.

[0275] More specifically, the nucleic acid molecule encodes IL-7m, which has the amino acid sequence shown in SEQ ID NOs: 2-15.

[0276] In an alternative embodiment, a nucleic acid molecule encoding a bifunctional molecule as defined herein comprises a variable heavy chain domain having the sequence shown in SEQ ID NO: 73 and / or a variable light chain domain having the sequence shown in SEQ ID NO: 74.

[0277] vector In another embodiment, the present invention relates to a vector comprising a nucleic acid molecule or a group of nucleic acid molecules as defined above.

[0278] As used herein, “vector” refers to a nucleic acid molecule used as a vehicle for transferring genetic material into cells. The term “vector” encompasses plasmids, viruses, cosmids, and artificial chromosomes. Generally, genetically engineered vectors include an origin of replication, multiple cloning sites, and selectable markers. A vector is generally a nucleotide sequence, typically a DNA sequence, containing an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Modern vectors may also include other additional features of the transgene insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, target-directed sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically designed to express a transgene in target cells and generally contain regulatory sequences.

[0279] Those skilled in the art can clone bifunctional molecules and nucleic acid molecules encoding fusion proteins into vectors and then transform them into host cells. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombinant techniques. Using methods known to those skilled in the art, expression vectors containing the nucleic acid sequences and appropriate regulatory components for transcription / translation of the bifunctional molecules and variants described herein can be constructed.

[0280] Accordingly, the present invention also provides recombinant vectors comprising nucleic acid molecules encoding the bifunctional molecules according to the present 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 comprise a ribosome binding site and a transcription terminator for initiating translation, etc.

[0281] A suitable expression vector typically contains (1) a prokaryotic DNA element encoding a bacterial origin of replication and an antibiotic resistance marker to provide growth and selection of the expression vector in the bacterial host; (2) a eukaryotic DNA element controlling transcription initiation, such as a promoter; and (3) a DNA element controlling transcript processing, such as a transcription termination / polyadenylation sequence.

[0282] Expression vectors can be introduced into host cells using various techniques, including calcium phosphate transfection, liposome-mediated transfection, and electroporation. Preferably, transfected cells in which the expression vector is stably integrated into the host cell genome to produce stable transformants are selected and proliferated.

[0283] host cell In another embodiment, the present invention relates, for example, to a host cell comprising a vector or nucleic acid molecule or group of nucleic acid molecules as defined above, for the purpose of producing a bifunctional molecule.

[0284] As used herein, the term “host cell” is intended to include any individual cell or cell culture that may or is a recipient of the vectors, exogenous nucleic acid molecules, and polynucleotides encoding the bifunctional molecules according to the present invention. The term “host cell” is also intended to include the offspring or potential offspring of a single cell. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, as well as bacterial, yeast, fungal, plant cells, and insect and mammalian cells, such as animal cells of mouse, rat, rabbit, macaque, or human.

[0285] Suitable host cells are, in particular, eukaryotic hosts that provide suitable post-translational modifications such as glycosylation. Preferably, such suitable eukaryotic host cells may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe; insect cells such as Mythimna separate; plant cells such as tobacco; and mammalian cells such as BHK cells, 293 cells, CHO cells, NSO cells, and COS cells.

[0286] Preferably, the host cells of the present invention are selected from the group consisting of CHO cells, COS cells, NSO cells, and HEK cells.

[0287] Therefore, host cells stably or transiently express the bifunctional component according to the present invention. Such methods of expression are known to those skilled in the art.

[0288] Furthermore, methods for producing bifunctional molecules are provided herein. These methods include culturing host cells containing nucleic acids encoding the bifunctional molecule as provided above under conditions suitable for its expression, and optionally recovering the bifunctional component from the host cells (or host cell culture medium). In particular, in the case of recombinant production of bifunctional molecules, for example, a nucleic acid molecule encoding the bifunctional molecule as described above is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The bifunctional molecule is then isolated and / or purified by any method known in the art. Such methods include, but are not limited to, conventional regeneration treatments, treatment with protein precipitants (e.g., salt precipitation), centrifugation, osmotic cell lysis, sonication, ultracentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and combinations thereof. For example, as described by Coligan, bifunctional molecule isolation techniques include affinity chromatography with protein A Sepharose, size exclusion chromatography, and ion exchange chromatography. Protein A is preferably used for the isolation of the bifunctional molecule of the present invention.

[0289] Pharmaceutical composition and method of administration thereof The present invention also relates to a pharmaceutical composition comprising, preferably as an active ingredient or compound, any of the bifunctional molecules described herein, nucleic acid molecules as described above herein, a group of nucleic acid molecules, vectors, and / or host cells. The formulation can be sterilized and, if desired, can be mixed with pharmaceutically acceptable carriers, excipients, salts, antioxidants, and / or stabilizers, etc., which do not interact adversely with the bifunctional molecules, nucleic acids, vectors, and / or host cells of the present invention and do not impart any undesirable toxicological effects. Optionally, the pharmaceutical composition may further contain additional therapeutic agents.

[0290] In particular, the pharmaceutical compositions according to the present invention can be formulated for any conventional route of administration, including topical administration, enteral administration, oral administration, parenteral administration, intranasal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intraocular administration. To facilitate administration, the bifunctional molecules as described herein can be prepared into pharmaceutical compositions for in vivo administration. Means for preparing such compositions are described in the Art (see, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005)).

[0291] Pharmaceutical compositions can be prepared in the form of lyophilized formulations or aqueous solutions by mixing a bifunctional molecule of desired purity with an optional pharmaceutically acceptable carrier, excipient, antioxidant, and / or stabilizer. Such suitable carriers, excipients, antioxidants, and / or stabilizers are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

[0292] To facilitate delivery, either the bifunctional molecule or its encoding nucleic acid may be conjugated with the chaperone. The chaperone may be a naturally occurring substance such as a protein (e.g., human serum albumin, low-density lipoprotein, or globulin), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or a lipid. Alternatively, the chaperone may be a recombinant or synthetic molecule such as a synthetic polymer, for example, a synthetic polypeptide.

[0293] The pharmaceutical composition according to the present invention may be formulated to release the active ingredient (e.g., the bifunctional molecule of the present invention) substantially immediately after administration, or at any predetermined time or period after administration. In some embodiments, the pharmaceutical composition may be equipped with timed-release, delayed-release, and sustained-release delivery systems so that the delivery of the composition occurs before and with sufficient time to induce sensitization of the site to be treated. By means known in the art, the release and absorption of the composition can be prevented or minimized until it reaches the target tissue or organ, or timed release of the composition can be ensured. Such systems can avoid repeated administration of the composition, thereby improving convenience for both the subject and the physician.

[0294] Those skilled in the art will understand that the formulations of the present invention may be isotonic with human blood, that is, they have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by a vapor pressure type or ice-freezing type osmometer.

[0295] Pharmaceutical compositions typically must be sterile and stable under manufacturing and storage conditions. Prevention of microbial presence can be ensured by both sterilization procedures (e.g., by microfiltration) and / or the inclusion of various antibacterial and antifungal agents.

[0296] The amount of active ingredient that can be combined with a carrier to produce a single dosage form will vary depending on the target being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier to produce a single dosage form will be the amount of the composition that produces the therapeutic effect.

[0297] Subjects, regimens, and administration The present invention relates to a bifunctional molecule, nucleic acid or vector encoding it, host cell, or pharmaceutical composition, as disclosed herein, for use as a pharmaceutical, for use in the treatment of a disease, or for administration to a subject, or for use as a pharmaceutical. The present invention also relates to a method for treating a disease or disorder in a subject, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition or bifunctional molecule to the subject.

[0298] The subjects of treatment may be humans, particularly prenatal humans, newborns, children, infants, adolescents, or adults, especially adults aged at least 30 or 40 years, preferably at least 50 years, more preferably at least 60 years, and even more preferably at least 70 years.

[0299] In certain embodiments, the subject may be in an immunosuppressed or immunodeficient state.

[0300] Using conventional methods known to those skilled in the medical field, the bifunctional molecules or pharmaceutical compositions disclosed herein can be administered to the target area depending on the type or site of the disease to be treated, for example, orally, parenterally, enterally, by inhalation spray, topically, transrectally, transnasally, buccally, transvaginally, or via an implanted reservoir. Preferably, the bifunctional molecules or pharmaceutical compositions are administered by subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-bursal, intratumoral, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0301] The form of the pharmaceutical composition, the route of administration, and the dosage of the pharmaceutical composition or bifunctional molecule according to the present invention can be adjusted according to the type and severity of the infection, the patient, and especially the patient's age, weight, size, sex, and / or overall health condition, as can be seen by those skilled in the art. The compositions of the present invention can be administered in several forms, depending on whether topical or systemic treatment is desired.

[0302] Use in disease treatment The bifunctional molecules, nucleic acids, vectors, host cells, compositions, and methods of the present invention have numerous in vitro and in vivo uses and applications. In particular, any of the bifunctional molecules, nucleic acid molecules, groups of nucleic acid molecules, vectors, host cells, or pharmaceutical compositions provided herein may be used in therapeutic methods and / or for therapeutic purposes.

[0303] The present invention also relates to a bifunctional molecule, a nucleic acid or vector encoding it, or a pharmaceutical composition containing the same, for use in the treatment of disorders and / or diseases in a subject, and / or for use as a pharmaceutical or vaccine. The present invention also relates to the use of the bifunctional molecules described herein; nucleic acids or vectors encoding them, or pharmaceutical compositions containing the same, for the treatment of diseases and / or disorders in a subject. Finally, the present invention relates to a method for treating a disease or disorder in a subject, comprising the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition, or a bifunctional molecule, or nucleic acid or vector encoding it.

[0304] In one embodiment, the present invention relates to a method for treating a disease and / or disorder selected from the group consisting of cancer, infectious diseases, and chronic viral infections in a subject requiring such treatment, the method comprising the step of administering to the subject an effective amount of a bifunctional molecule or pharmaceutical composition as defined above. Examples of such diseases are described more specifically below in this specification.

[0305] In one embodiment, the treatment method includes (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of a bifunctional molecule, nucleic acid, vector, or pharmaceutical composition described herein.

[0306] Those requiring treatment may be individuals who have, are at risk of having, or are suspected of having the disease. Such patients can be identified through routine health checkups.

[0307] In another embodiment, the bifunctional molecules disclosed herein can be administered to a subject, for example, in vivo, to enhance immunity, preferably to treat disorders and / or diseases. Thus, in one embodiment, the present invention provides a method for modifying an immune response in a subject, comprising the step of administering to a subject a bifunctional molecule, nucleic acid, vector, or pharmaceutical composition of the present invention so as to modify an immune response in the subject. Preferably, the immune response is enhanced, increased, stimulated, or upregulated. The bifunctional molecules or pharmaceutical compositions can be used to enhance an immune response, such as T cell activation, in a subject requiring treatment. In certain embodiments, the bifunctional molecules or pharmaceutical compositions can be used to reduce T cell exhaustion or to reactivate exhausted T cells.

[0308] The present invention provides, in particular, a method for enhancing the immune response in a subject, comprising the step of administering to the subject a therapeutically effective amount of a bifunctional molecule, nucleic acid, vector, or pharmaceutical composition comprising the same described herein, such that the immune response in the subject is enhanced. In certain embodiments, the bifunctional molecule or pharmaceutical composition can be used to reduce T cell exhaustion or to reactivate exhausted T cells.

[0309] The bifunctional molecules containing the IL-7 variant according to the present invention target CD127+ immune cells, particularly CD127+ T cells. Such cells may be found in the following specific target areas: resident lymphoid cells in lymph nodes (mainly in the paracortical region, including occasional cells in the follicles), tonsils (interfollicular region), spleen (mainly in the periarteriole lymphoid sheath (PALS) of the white medulla and some scattered cells in the red medulla), thymus (mainly in the medulla; or cortex), bone marrow (scattered distribution), GALT (gut-associated lymphoid tissue, mainly in the interfollicular region and lamina propria) in the gastrointestinal tract (stomach, duodenum, jejunum, ileum, cecum, rectum), and MALT (mucosa-associated lymphoid tissue) of the gallbladder. Therefore, the bifunctional molecules of the present invention are of particular interest in treating these areas, especially diseases located in or involving cancer.

[0310] In certain embodiments, the bifunctional molecule comprises an IL-7 variant, particularly W142H, and an antigen-binding domain that binds to and antagonizes PD-1.

[0311] Such bifunctional molecules and pharmaceutical compositions containing them may be used in patients, particularly patients with cancer, to increase tumor-infiltrating lymphocytes (TILs), to protect T lymphocytes from apoptosis, to induce / improve the T memory response, to suppress T-regs and / or neutralize the inhibitory activity of Tregs, and to restore and / or maintain the proliferation of completely exhausted T cells, particularly exhausted tumor-infiltrating lymphocytes.

[0312] Such bifunctional molecules and pharmaceutical compositions containing them can be used to manufacture pharmaceuticals for increasing tumor-infiltrating lymphocytes (TILs) in patients, particularly patients with cancer; for protecting T lymphocytes from apoptosis; for inducing / improving the T memory response; for inhibiting T-regs and / or negating Treg inhibitory activity; and for restoring and / or maintaining the proliferation of completely exhausted T cells, particularly exhausted tumor-infiltrating lymphocytes.

[0313] The present invention also relates to a method for restoring and / or maintaining the proliferation of completely exhausted T cells, particularly exhausted tumor-infiltrating lymphocytes, to increase tumor-infiltrating lymphocytes (TILs) in a patient, particularly a patient with cancer, to protect T lymphocytes from cell death, to induce / improve the T memory response, to suppress T-regs and / or to neutralize the inhibitory activity of Tregs, the method comprising administering to the patient a therapeutically effective amount of a bifunctional molecule comprising an antigen-binding domain that binds to and antagonizes either of the following specific molecules, particularly IL-7 variants, particularly W142H, and PD-1.

[0314] cancer In another aspect, the present invention provides the use of a bifunctional molecule or pharmaceutical composition disclosed herein in the manufacture of a pharmacopoeia for treating cancer, for example, for inhibiting the growth of tumor cells in a subject.

[0315] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or to other parts of the body via the bloodstream and lymphatic system.

[0316] Accordingly, in one aspect, the present invention provides a method for treating cancer in a subject, for example, a method for inhibiting the growth of tumor cells, comprising the step of administering to the subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition according to the present invention. In particular, the present invention relates to the treatment of a subject using a bifunctional molecule so as to inhibit the growth of cancer cells.

[0317] In one aspect of this disclosure, the cancer to be treated is associated with exhausted T cells.

[0318] Any suitable cancers that can be treated with the present invention may be hematopoietic cancers or solid tumors. Such cancers include carcinomas, cervical cancers, colorectal cancers, esophageal cancers, gastric cancers, gastrointestinal cancers, head and neck cancers, kidney cancers, liver cancers, lung cancers, lymphomas, gliomas, mesotheliomas, melanomas, gastric cancers, urethral cancers, environmentally induced cancers, and any combination of such cancers. In addition, the present invention includes refractory or recurrent malignancies. Preferably, the cancers to be treated or prevented are selected from the group consisting of metastatic or non-metastatic cancers, melanomas, malignant mesotheliomas, non-small cell lung cancers, renal cell carcinomas, Hodgkin lymphomas, head and neck cancers, urothelial carcinomas, colorectal cancers, hepatocellular carcinomas, small cell lung cancers, metastatic Merkel cell carcinomas, gastric or gastroesophageal cancers, and cervical cancers.

[0319] In certain aspects, cancer is a hematological malignancy or a solid tumor. Such cancers may be selected from the group consisting of hematological and lymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndromes, and acute myeloid leukemia.

[0320] In certain aspects, cancer is a cancer induced by a virus or associated with immunodeficiency. Such cancers may be selected from the group consisting of Kaposi's sarcoma (e.g., associated with Kaposi's sarcoma herpesvirus); squamous cell carcinoma of the cervix, anus, penis and vulva and oropharyngeal cancer (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, classical Hodgkin lymphoma, 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 viruses); Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)); and cancers associated with human immunodeficiency virus infection (HIV).

[0321] Cancers favored for treatment typically include those responsive to immunotherapy, or conversely, cancers that are not responsive to immunotherapy.

[0322] infectious disease The bifunctional molecules, nucleic acids, groups of nucleic acids, vectors, host cells, or pharmaceutical compositions of the present invention can be used to treat patients exposed to specific toxins or pathogens. Accordingly, aspects of the present invention preferably provide a method for treating an infectious disease in a subject, comprising the step of administering to a subject a bifunctional molecule or a pharmaceutical composition containing the same according to the present invention, such that the subject is treated for an infectious disease.

[0323] 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.

[0324] Some examples of pathogenic viruses that cause infectious diseases treatable by the methods of the present invention include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial virus (RSV), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

[0325] Some examples of pathogenic bacteria that cause infectious diseases treatable by the methods of the present invention include Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, Salmonella, Bacillus, cholera, Tetanus, botulinum, anthrax, plague, leptospirosis, and Lyme disease bacteria.

[0326] Some examples of pathogenic fungi that cause infectious diseases treatable by the method of the present invention include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucor (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Coccidioides imitis. This includes *immitis* and *Histoplasma capsulatum*.

[0327] Some examples of pathogenic parasites that cause infectious diseases treatable by the method of the present invention include Entamoeba histolytica, Balantium coli, Naegleria fowleri, Acanthamoeba species, Giardia lambia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, and Toxoplasma gondii. This includes *Gondi* and *Nippostrongylus brasiliensis*.

[0328] Combination therapy The bifunctional molecule according to the present invention can be combined with several other possible strategies for overcoming immune evasion mechanisms using agents that are in clinical development or already on the market (see Table 1 (Table 10) in Antonia et al., Immuno-oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258-6268, 2014). Such combinations with the bifunctional molecule according to the present invention are 1. Reversing the inhibition of adaptive immunity (blocking the T cell checkpoint pathway); 2. Switching adaptive immunity (using agonist molecules, particularly antibodies, to promote T cell co-stimulation receptor signaling), 3. To improve the function of innate immune cells; 4. For example, activating the immune system through vaccine-based strategies (enhancing immune-cell effector function), It could be clearly useful.

[0329] Accordingly, combination therapies for treating diseases or disorders using any of the bifunctional molecules or pharmaceutical compositions containing them described herein and a suitable second agonist are also provided herein. In some embodiments, the bifunctional molecules and the second agonist may be present in the specific pharmaceutical compositions described above. Alternatively, as used herein, the terms “combination therapy” or “combination therapy” encompass the administration of these two agonists (e.g., the bifunctional molecules described herein and an additional or second suitable therapeutic agent) in a sequential manner, i.e., each therapeutic agent is administered at different times, and the administration of these therapeutic agents, or at least two of the agonists, is substantially simultaneous. The sequential or substantially simultaneous administration of each agonist may be influenced by any suitable route. The agonists may be administered by the same route or by different routes. For example, the first agonist (e.g., the bifunctional molecule) may be administered orally, and the additional therapeutic agent (e.g., an anticancer agent, an anti-infective agent; or an immunomodulator) may be administered intravenously. Alternatively, the selected combination of agents may be administered by intravenous injection, while the other agents in the combination may be administered orally.

[0330] In some embodiments, additional therapeutic agents include alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, mitotic inhibitors, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), activators of the cell death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell induction) antibodies, antibody-drug conjugates, bioreaction modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunological agents, apoptosis protein inhibitors (IAP) inhibitors, intercalating antibiotics, kinase inhibitors, kinesin inhibitors, and J The following can be selected from a non-inclusive list, including ak2 inhibitors, mammalian targets of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, polyvalent binding proteins, nonsteroidal 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, retinoid / deltoid plant alkaloids, small molecule inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, etc., epitopes or neoepitopes derived from tumor antigens, and combinations of one or more of these agonists.

[0331] For example, additional therapeutic agents can be selected from a group consisting of chemotherapy, radiotherapy, targeted therapy, anti-angiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes derived from tumor antigens, bone marrow checkpoint inhibitors, other immunotherapies, and HDAC inhibitors.

[0332] In one embodiment, the present invention relates to combination therapy as defined above, wherein the second therapeutic agent is more specifically selected from the group consisting of therapeutic vaccines, particularly immune checkpoint blockers or activators of adaptive immune cells (T and B lymphocytes), and antibody-drug conjugates. Preferably, any of the bifunctional molecules or activators suitable for co-use with the pharmaceutical composition according to the present invention include co-stimulatory receptors (e.g., OX40, CD40, ICOS, CD27, HVEM, or GITR), immunological cell death-inducing activators (e.g., chemotherapeutic agents, radiotherapy agents, anti-angiogenic agents, or activators for targeted therapy), activators that inhibit checkpoint molecules (e.g., CTLA4, LAG3, TIM3, B7H3, B7H4, BTLA, or TIGIT), cancer vaccines, activators that modify immunosuppressive enzymes (e.g., IDO1 or iNOS), activators that target Treg cells, activators for adoptive cell therapy, or activators that modulate myeloid cells.

[0333] In one embodiment, the present invention relates to combination therapy as defined above, wherein the second therapeutic agent is a checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes), selected from the group consisting of anti-CTLA4, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonist, CD40-L, TLR agonist, anti-ICOS, ICOS-L, and B cell receptor agonist.

[0334] The present invention also relates to a method for treating a disease in a subject, comprising the step of administering to the subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition described herein and an additional therapeutically effective amount of a second therapeutic agent.

[0335] Specific examples of additional or second therapeutic agents are provided in International Publication No. 2018 / 053106, pp. 36-43.

[0336] In preferred embodiments, the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiotherapy agents, immunotherapy agents, cell therapy agents (e.g., CAR-T cells), antibiotics, and probiotics.

[0337] Combination therapy can also rely on a combination of surgical intervention and the administration of bifunctional molecules.

[0338] More specifically, the bifunctional molecule according to the present invention is intended for use in combination with a second bifunctional molecule comprising a target specifically expressed on the surface of immune cells and at least one antigen-binding domain that binds to at least one immunostimulatory cytokine.

[0339] More specifically, in such a second bifunctional molecule, the immunostimulatory cytokine is selected from the group comprising IL-2, IL-4, IL-5, IL-6, IL-12A, IL-12B, IL-13; IL-15, IL-18, IL-21, IL-23, IL-24; IFNα, IFNβ, BAFF, LTα, and LTβ, or their variants. In a preferred embodiment, the immunostimulatory cytokine is IL-2 or a variant thereof.

[0340] More specifically, in such a second bifunctional molecule, the target specifically expressed on the surface of immune cells is selected from the group consisting of PD-1, CD28, 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, CD3, PDL2, and PDL1.

[0341] Preferably, the bifunctional molecule according to the present invention, comprising a single antigen-binding domain for PD-1 and a single IL-7 variant, is intended for use in combination with a bifunctional molecule comprising i) an antigen-binding domain for PD-1 and ii) IL-2 or an IL-2 variant.

[0342] The bifunctional molecule of the present invention and the second bifunctional molecule are administered simultaneously or sequentially. In the example of sequential administration, the bifunctional molecule of the present invention is administered before the administration of the second bifunctional molecule. In another example, the bifunctional molecule of the present invention is administered after the administration of the second bifunctional molecule.

[0343] In preferred embodiments, the present invention relates to a bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition for use in combination with a second bifunctional molecule comprising at least one antigen-binding domain that binds to a target specifically expressed on the surface of an immune cell, and at least one IL2, which is administered after the administration of the second bifunctional molecule.

[0344] kit Any of the bifunctional molecules or compositions described herein may be included in the kits provided by the present invention. This disclosure provides kits for use in enhancing immune responses and / or treating diseases or disorders (e.g., cancer and / or infections).

[0345] In the context of the present invention, the term “kit” means two or more components packaged in a container, recipient, or vice versa (one of which corresponds to a bifunctional molecule, nucleic acid molecule, vector, or cell of the present invention). A kit can be described herein as a set of products and / or tools sufficient to achieve a particular objective, which can be marketed as a single unit. The kits of the present invention are in appropriate packaging.

[0346] Specifically, the kit according to the present invention is - The bifunctional molecule defined above, - A nucleic acid molecule or group of nucleic acid molecules that encode the bifunctional molecule, - A vector containing the nucleic acid molecule or group of nucleic acid molecules, and / or - Cells containing the vector, nucleic acid molecule, or group of nucleic acid molecules. It may include.

[0347] Accordingly, the kit may contain, in a suitable container means, the pharmaceutical composition of the present invention, a fusion protein, or a bifunctional molecule, and / or a host cell, and / or a vector encoding the nucleic acid molecule of the present invention, and / or the nucleic acid molecule or related reagent of the present invention. In some embodiments, means for collecting a sample from an individual and / or means for assaying the sample may be provided. The compositions included in the kit according to the present invention may, in particular, be formulated into compositions compatible with syringes.

[0348] In some embodiments, the kit further comprises additional activators for treating cancer or infectious diseases, the additional activators may be in combination with the pharmaceutical compositions, fusion proteins, or bifunctional molecules of the present invention, host cells, and / or vectors encoding nucleic acid molecules of the present invention, and / or nucleic acid molecules of the present invention, or other components of the kit of the present invention, or may be provided individually in the kit. In particular, the kits described herein may comprise one or more additional therapeutic agents, such as those described in the “combination therapies” described above herein. The kits may be tailored to specific cancers for individuals and may comprise each of the second cancer therapies for individuals described above herein.

[0349] Instructions for the use of the bifunctional molecules or pharmaceutical compositions described herein generally include information regarding dosage, administration schedule, route of administration of the intended treatment, means for reconstituting the bifunctional molecules and / or means for diluting the bifunctional molecules of the present invention. Instructions provided in the kit of the present invention are typically instructions written on a label or accompanying document (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual). [Examples]

[0350] The availability of IL-7 variants is described in detail in International Publication No. 2020 / 12377, which is incorporated herein by reference.

[0351] (Example 1. Anti-PD-1 IL-7 molecules containing one IL-7 W142H cytokine and one or two anti-PD-1 arms demonstrated high efficacy in promoting cis-activity to PD-1+ IL-7R+ cells, stimulating IL-7R T cell proliferation in vivo, and exhibiting synergistic ability to reactivate TCR signaling.) The inventors designed and compared the biological activity of multiple structures of bifunctional molecules containing one or two anti-PD-1 binding domains and one or two IL7 W142H mutations, as shown in Figure 1.

[0352] 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 is also called BICKI-IL-7 W142H. In the examples, the control molecule called BICKI-IL-7 WT corresponds to construct 1 but has wild-type IL-7.

[0353] 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).

[0354] 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). The control construct, called anti-PD-1*1, is similar to construct 3 but lacks the IL-7 variant.

[0355] 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).

[0356] Constructs 2, 3, and 4 were genetically engineered to possess the IgG1 N298A isotype, and the amino acid sequences of the Fc region were mutated to create knobs in CH2 and CH3 of heavy chain A and holes in CH2 and CH3 of heavy chain B.

[0357] All anti-PD-1 IL-7 constructs exhibited high affinity for the PD-1 receptor, as demonstrated by ELISA assays (Figure 2A and Table 1 (Table 10)). Anti-PD-1 IL-7 molecules with two anti-PD-1 arms (anti-PD-1*2) had the same binding efficacy (equal EC50) compared to anti-PD-1*2 without IL-7. 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) showed the same binding efficacy compared to anti-PD-1*1 without IL-7, with the EC50s of anti-PD-1 IL7 being equal to 0.086 and 0.111 nM, respectively, compared to the anti-PD-1 EC50 of 0.238 nM. These data indicate that IL-7 fusion does not interfere with PD-1 binding, regardless of the construct tested.

[0358] [Table 10]

[0359] Furthermore, PD-L1 / PD-1 antagonist bioassays (Figure 2B) demonstrate that anti-PD-1 IL7 molecules with one or two anti-PD-1 arms are highly effective in blocking the binding of PD-L1 to the PD-1 receptor. Even with the removal of one anti-PD-1 arm from constructs 3 and 4, all anti-PD-1*1 IL7 constructs exhibited high antagonistic properties. The EC50 of constructs 3 and 4 showed only a 1 / 25 reduction in activity compared to the anti-PD-1*2 IL7 construct (Table 2 (Table 11)).

[0360] [Table 11]

[0361] Next, the inventors evaluated the affinity of different constructs to the CD127 receptor using Biacore assays and ELISA assays. Since one IL-7 molecule was removed from constructs 2 and 3, it was expected that these molecules would have lower binding ability to the CD127 receptor and lower pSTAT5 activation compared to the IL-7 heterodimer construct. However, the inventors observed that the anti-PD-1*2 IL-7 W142H*1 molecule had similar affinity to the CD127 receptor compared to anti-PD-1*2 IL-7 W142H*2 (BICKI-IL-7 W142H), and, as expected, lower affinity compared to anti-PD-1 IL7 bifunctional molecules including the wild-type IL-7 form (Table 3 (Table 12)). Surprisingly, the anti-PD-1*2 IL7 W142H*1 and anti-PD-1*1 IL7 W142H*1 molecules exhibited high pSTAT5 activity similar to that of PD-1 IL7 bifunctional molecules, including the wild-type form of IL-7 (Figure 3). Based on these observations, it is possible that monomeric IL-7 combined with the W142H IL-7 mutation enables the optimal three-dimensional structure of the IL-7 molecule for promoting IL-7 signaling to human T cells. Even with only one IL7, molecules with the W142H IL-7 mutation exhibited good activation (pSTAT5) similar to molecules with IL7 wt containing two cytokines. This result is surprising in the context of IL-7 variants with lower receptor affinity than wild-type IL-7.

[0362] Similar conclusions were drawn for anti-PD-1 IL7 molecules constructed with a single anti-PD-1 arm fused to a single IL-7 W142H mutation. Similar and equivalent high pSTAT5 activity was obtained with the anti-PD-1*2 IL-7 WT*2 construct, the anti-PD-1*2 IL-7 W142H*1 construct, and the anti-PD-1*1 IL-7 W142H*1 construct (Figure 3C).

[0363] [Table 12]

[0364] In vivo experiments were conducted to determine the efficacy of different anti-PD-1 IL-7 constructs. A single dose of anti-PD-1 IL-7 molecule was injected into mice at an isovalent molar concentration (34 nM / kg). Four days post-treatment, CD4 T cell proliferation and CD8 T cell proliferation were quantified by flow cytometry using the Ki67 marker. Figure 4 shows that anti-PD-1 IL-7 molecules with a single W142H mutation (anti-PD-1*2 IL-7 W142H*1 and anti-PD-1*1 IL-7 W142H*1) or anti-PD-1 IL-7 molecules with a single PD-1 titer and two IL7 W142H cytokines (anti-PD-1*1 IL7 W142H*2) showed high efficacy in promoting CD8 T cell proliferation and lower efficacy in promoting CD4 T cell proliferation.

[0365] NFAT bioassays were performed to determine the ability of bifunctional molecules containing an anti-PD1 antibody (monovalent or bivalent) and one or two IL7 mutant cytokines to reactivate TCR-mediated signaling. Figure 5A shows that bifunctional molecules constructed with two anti-PD-1 arms and one IL-7 cytokine enhance NFAT activation compared to anti-PD-1 antibody alone. This indicates that the synergistic activity of drugs that enhance TCR-mediated signaling is conserved in anti-PD-1 IL-7 bifunctional molecules constructed with only one IL-7 cytokine. As seen in Figure 9A, treating cells with the anti-PD1 and IL7 combination did not produce such a synergistic effect.

[0366] In addition, the inventors then evaluated the activity of anti-PD-1 IL-7 molecules designed with only anti-PD-1 titer (anti-PD-1*1) and demonstrated that while the anti-PD-1*1 IL-7 W142H constructs (anti-PD-1*1 IL7 W142H*1 and *2) maintain synergistic activity, the combination of PD-1*1 + isotype IL-7 W142H*2 treatment shows lower efficacy in stimulating TCR signaling (NFAT activation) (Figure 5B).

[0367] Finally, the specific cis-targeting and cis-activity of different anti-PD-1 IL-7 constructs were analyzed using a co-culture assay. U937 PD-1+CD127+ cells were mixed with PD-1-CD127+ cells (1:1 ratio) and then incubated with escalating 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 for each PD-1+ and PD-1- cell population (Figures 6A and 6B). The inventors confirmed 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 bind to IL-7R and transmit signals into PD-1+ cells, significantly activating IL7R signaling pSTAT5 into PD-1+ cells. Importantly, the construct PD-1*1 IL7 W142H*1 demonstrated the highest activity in stimulating pSTAT5 signaling to PD-1+ cells compared to the other constructs (anti-PD-1*2 IL7 W142H*1 and anti-PD-1*1 IL7 W142H*2). These data suggest that a bifunctional molecule constructed with one anti-PD-1 arm and one IL-7 possesses the optimal conformation and activity to enable preferential activation of IL-7R in PD-1+ activated T cells in the context of cancer.

[0368] (Example 2: Anti-PD-1 IL-7 molecules constructed with one or two anti-PD-1 arms and one or two IL7 W142H cytokines exhibit a favorable pharmacokinetic profile in vivo.) Pharmacokinetic studies of anti-PD-1 IL-7 bifunctional molecular constructs 2, 3, and 4, as shown in Figure 1, were evaluated. Humanized PD1 KI mice were intraperitoneally injected with a single dose of anti-PD-1 IL-7 molecules (34.4 nM / kg). Plasma drug concentrations were analyzed by human IgG-specific ELISA (Figure 7). The area under the curve was also calculated (see Table 4 (Table 13)). The area under the curve represents the total drug exposure over time for each construct. The 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 showed significantly enhanced PK properties compared to anti-PD-1*2 IL7WT*1. A Cmax 2.8 to 19 times higher was observed compared to anti-PD-1*1 IL7WT*2. Importantly, the anti-PD-1*1 IL7 W142H*1 and anti-PD-1*1 IL7 W142H*2 molecules maintain high drug concentrations (11-15 nM) corresponding to sufficient PK values ​​in vivo for at least 96 hours, while the anti-PD-1*2 IL7WT*2 molecule is undetectable in plasma at only 2 nM. The 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 enable long-term drug exposure after a single injection. While 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 demonstrated similar favorable PK profiles in vivo, Figure 6B demonstrates that the construct anti-PD-1*1 IL-7 W142H*1 possesses a superior ability to activate PD-1+ cells.

[0369] [Table 13]

[0370] (Example 3. The bifunctional antibody constructed with anti-PD-1 titer and one fusion protein X of 1 demonstrated higher productivity by mammalian cells compared to the bifunctional antibody constructed with anti-PD-1 titer and one fusion protein X of 2.) The productivity of bifunctional antibodies in formats B and C using mammalian cells was evaluated and compared. Complete heavy chains containing IL-7-fused Fc were transiently co-transfected with light chains into CHO suspension cells. The amount of antibody obtained after production and purification was quantified using sandwich ELISA (chromogenesis with immobilized donkey anti-human Fc antibody and mouse anti-human kappa + peroxidase conjugate goat anti-mouse antibody). Concentration was determined using human IVIgG standards. Productivity was calculated as the amount of purified antibody per liter of collected culture supernatant.

[0371] Results: The bifunctional antibodies, anti-PD-1*2 / IL-7*1 (format B) and antibody PD-1*1 IL-7*1 (format C), were produced in CHO mammalian cells, and the results are shown in Figure 8. Surprisingly, the anti-PD-1*1 / IL-7*1 construct (format C) had a significantly better production yield (mg / L) than anti-PD-1*2 / IL-7*1 (format B). A significant 1.7-fold (+ / -0.7; n=5) higher productivity was obtained. These results indicate that anti-PD-1*1 / IL-7*1 (format C) exhibited excellent manufacturability, which is crucial for the next steps in clinical development and therapeutic application.

[0372] In particular, with regard to bifunctional antibodies containing two different arms, one major problem is mispairing of the chains and incorrect association of chain A (knob chain) and chain B (hole chain). In fact, undesirable homodimerization (chain A + chain A, or chain B + chain B) commonly occurs. This usually results in lower yield and purity of heterodimer bifunctional antibodies (formats B and C) compared to the production of homodimer bifunctional antibodies (format A), which is a significant disadvantage. The important question remains: how to produce homogeneous bifunctional antibodies of high quality with limited or negligible by-products and impurities.

[0373] However, the inventors have surprisingly demonstrated that by using an optimized strategic design of format C, the bifunctional antibody induces higher production compared to homodimer format B. Moreover, the production yield of anti-PD-1*1-IL-7*1 is within a similar range to that of anti-PD-1 alone (anti-PD-1*1 or anti-PD-1*2), and its productivity is equivalent to 45 mg / L (n=5) under similar production conditions.

[0374] Another major problem with the production of heterodimer antibodies is their purity. The knob-in-hole strategy is favorable for heterodimer production (chain A + chain B) and reduces the production of homodimer chain A or homodimer chain B, but this strategy is not 100% effective, and additional purification is required to isolate the heterodimer construct (Wang et al., 2019, Antibodies, Vol. 8, p. 43).

[0375] However, the inventors observed that high yields of heterodimers were obtained after the production of anti-PD-1*1 / IL-7*1 format C. Figure 9 shows size exclusion chromatography of anti-PD-1*1 / IL-7wt*1 (Figure 9A) and anti-PD-1*1 / IL-7v*1 (Figure 9B) after protein A purification. One major peak corresponding to the heterodimer forms chains A and B, but the homodimeric type Fc / Fc (chain A + chain A) was not detected, and the homodimeric type (chain A + chain A) was present in very small amounts (less than 2%). These data suggest that the anti-PD-1*1 / IL-7*1 of the present invention is optimized in terms of productivity and prevents mispairing. Compared to the prior art, yields of approximately 70-75% of the heterodimers were obtained with other bispecific antibody backbones using the same KIHs-s strategy.

[0376] To achieve such purity, the inventors optimized the molecular design. In fact, the inventors observed that this high purity was obtained only when chain A was an Fc domain and chain B was anti-PD-1*1 IL-7*1. Co-transfection of the sole chain B containing the VL+ hole mutation (anti-PD-1*1 / IL-7v*1) in CHO mammalian cells did not induce the production of homodimers of chain B (0 mg / L). Conversely, when anti-PD-1*1 / IL-7v*1 was chain A containing the knob mutation, high production of homodimer chain A (88 mg / L) was obtained after co-transfection of the sole VL+ chain A (anti-PD-1*1 / IL-7v*1). Based on these data, the inventors chose to design a molecule having Fc as chain A and anti-PD-1*1 / IL-7*1 as chain B in order to avoid the production of homodimers of chain A.

[0377] Overall, these data indicate that anti-PD-1*1 / IL-7*1 of Format C according to the present invention, having chain A (Fc domain with a knob mutation) and chain B (anti-PD-1*1 / IL-7* with a hole mutation), is the best construct for high productivity and purity of the product. This facilitates the development of therapeutic agents for large-scale production.

[0378] (Example 4. Anti-PD-1 / cytokine bifunctional antibodies can activate pSTAT5 signaling into primary human T cells.) The inventors then evaluated the biological activity of proteins fused to anti-PD-1 antibodies and tested the ability of all anti-PD-1 / cytokine bifunctional molecules to activate primary T cells. For this purpose, human peripheral blood T cells were treated with different concentrations of anti-PD-1*1 / IL-7wt*1, anti-PD-1*1 / IL-7v*1, anti-PD-1*1 / IL-15*1, and anti-PD-1*1 / IL-21*1 constructs at 37°C for 15 minutes. After incubation, the cells were fixed, permeabilized, and stained with anti-pSTAT5 antibody.

[0379] Results: Figure 10A shows that anti-PD-1*1 / IL-7wt*1, anti-PD-1*1 / IL-7v*1, anti-PD-1*1 / IL-15*1, and anti-PD-1*1 / IL-21*1 effectively induce pSTAT5 signaling to primary T cells (CD3+ T cells), suggesting that the cytokine fused to the Fc domain of the anti-PD-1 molecule in format C preserves its ability to stimulate human T cells. We then compared the efficiency of activating pSTAT5 signaling between anti-PD-1 / IL-7 format A (anti-PD-1*2 / IL-7v*2) and anti-PD-1*1 / IL-7v*1 format C. Since the construct anti-PD-1*1 / IL-7v*1 contains only one IL-7v cytokine, it was expected that the pSTAT5 activation of this molecule would be lower compared to format A. However, as shown in Figure 10B, surprisingly, higher pSTAT5 activation was observed for the anti-PD-1*1 / IL-7v*1 (format C) versus the anti-PD-1*2 / IL-7v*2 construct (format A), suggesting that the anti-PD-1 / IL-7*1 construct (format C) of the present invention enables the optimal three-dimensional structure of the IL-7 molecule to promote the activation of IL-7 signaling in primary T cells.

[0380] (Example 5. The anti-PD-1 bifunctional molecule enables preferential binding to PD-1+ cells rather than PD-1- cells, and the anti-PD-1 / IL7 molecule enables synergistic activation of TCR signaling in PD-1+ T cells.) The inventors evaluated the ability of the anti-PD-1 bifunctional molecule to target PD-1+ T cells and to enable preferential delivery and cis-binding of cytokines or proteins fused to PD-1+ cells. U937 PD-1- cells and U937 PD1+ CD127+ cells were co-cultured (1:1 ratio) and incubated with escalating doses of anti-PD1 / IL-7 molecules. Binding and IL-7R signaling (pSTAT5) were quantified by flow cytometry. The EC50 (nM) and pSTAT5 activation of binding were determined for each construct and for each PD-1+ and PD-1- cell population. In parallel, binding of the bifunctional antibody was detected by anti-IgG-PE (Biolegend, clone HP6017) and analyzed by flow cytometry.

[0381] Results: Figures 11A and 11B show the binding of anti-PD-1*1 / IL-7wt*1 and anti-PD-1*1 / IL7v*1 molecules to cells expressing only CD127+ or co-expressing CD127 and the PD-1 receptor. The data indicate that both molecules preferentially bind to PD-1+CD127+ cells rather than PD-1-CD127+ cells and have comparable efficacy to anti-PD-1 alone (anti-PD-1*2). Simultaneously, activation of PSTAT5 signaling between PD-1+ cells and PD-1- cells was also evaluated in detail in Figure 11C. Strong activation of IL7R signaling pSTAT5 was observed in PD-1+CD127+ cells versus PD-1-CD127+ cells after treatment with anti-PD-1*1 / IL-7wt or IL7v*1 antibodies (58-315 times higher activation). However, isotype / IL7 antibodies exhibited similar efficacy in PD-1+ and PD-1- cells, confirming that the anti-PD-1 domain of the anti-PD-1*1 / IL-7*1 molecule enables preferential binding of IL-7 on PD-1+ cells, thus enabling drug targeting and activation in the same cells. Since anti-PD-1 IL-7 enriches IL-7 fused to bifunctional molecules or other molecules on PD-1+ tumor-specific T cells in the tumor microenvironment compared to PD-1-negative naive T cells, this aspect is related to the bioactivity of the drug in vivo. Overall, these data indicate that a single arm of anti-PD-1 is sufficient to enable selective delivery of fused cytokines on PD-1+ cells.

[0382] Next, the inventors evaluated the biological effects of cis-targeting of anti-PD-1 bifunctional molecules on PD-1+ T cells. The Promega PD-1 / PD-L1 kit (reference J1250) assay was used. Briefly, two cell lines were used: (1) effector T cells (Jarcat cells stably expressing PD-1 and NFAT-induced luciferase), and (2) activated target cells (CHO K1 cells stably expressing PDL1 and surface proteins designed to activate the cognitive TCR in an antigen-independent manner). When the cells were co-cultured, the PD-L1 / PD-1 interaction inhibited TCR-mediated activation, thereby blocking NFAT activation and luciferase activity. Addition of an anti-PD-1 antibody blocked the PD-1-mediated inhibitory signal, restoring TCR-mediated signaling, which in turn led to NFAT activation, luciferase synthesis, and the release of bioluminescent signals.

[0383] The results of the bioassay are shown in Figure 12A, which demonstrate that the bifunctional anti-PD-1*1 / IL7wt*1 molecule is better at activating TCR-mediated signaling (NFAT) than anti-PD1*1 or anti-PD1*1 + untargeted isotype-IL7 (as separate compounds), demonstrating the synergistic effect of the bifunctional molecule on PD1+ T cells. The anti-PD-1*1 / IL-7v*1 molecule containing an IL-7 variant also showed a significant synergistic effect in reactivating NFAT signaling in T cells (Figure 12B). These data indicate that fusing one IL-7 cytokine to one anti-PD-1*1 favorably induces higher activation of TCR signaling than combination strategies in which the two separate compounds do not induce such efficacy.

[0384] (Example 6.1, the bifunctional molecule with anti-PD-1 titer demonstrated good in vivo pharmacokinetics.) The pharmacokinetics and pharmacology of the product were evaluated in mice after a single injection. C57bl6JRj mice (female, 6-9 weeks old) were administered a single dose (34 nmol / kg) of anti-PD-1 or a bifunctional antibody intravenously or intraperitoneally. Plasma drug concentrations were determined by ELISA using immobilized anti-human light chain antibody (clone NaM76-5F3), and then serum containing the antibody was added. Detection was performed by adding peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; inquiry 709-035-149) and developing it using a conventional method. The area under the curve corresponding to drug exposure was calculated for each construct.

[0385] Results: The inventors first compared the pharmacokinetics of all different bifunctional formats (Formats A, B, and C). Figure 13 shows the pharmacokinetic profiles of anti-PD-1 / IL-7v constructed with one or two IL-7v cytokines and monovalent or dual anti-PD-1 valents after a single intravenous (Figure 13A) or intraperitoneal (Figure 13B) injection. Anti-PD-1*1 / IL-7v*1 (Format C) demonstrated the best pharmacokinetic profile compared to anti-PD-1*2 / IL-7v*2 (Format A) and anti-PD-1*2 / IL-7v*1 (Format B). Both intravenous and intraperitoneal injections demonstrated that anti-PD-1*1 / IL-7*1 (Format C) is the optimal construct for enhancing the pharmacokinetics of the bifunctional molecule.

[0386] Next, the inventors investigated whether a bifunctional molecule having one anti-PD-1 arm (anti-PD-1*1) and IL-7 exhibited a better in vivo pharmacokinetic profile compared to the same bifunctional molecule constructed with two anti-PD-1 arms. Figure 14 shows the results for each construct. The data show that all anti-PD-1*1 / IL-7 (Format C) bifunctional molecules exhibited a significantly better pharmacokinetic profile compared to the corresponding bifunctional molecule, anti-PD-1*2 / IL-7*1 (Format B).

[0387] In another experiment, the pharmacokinetics of anti-PD-1*2 or anti-PD-1*1 antibodies alone were also evaluated to understand whether anti-PD-1 constructs alone enable a favorable pharmacokinetic profile, or whether this finding is applicable only to bifunctional molecules. Figure 15 shows that both anti-PD-1*2 and anti-PD-1*1 have similar profiles after intravenous (Figure 15A) or intraperitoneal (Figure 15B) injection, surprisingly suggesting that anti-PD-1*1 constructs induce a favorable pharmacokinetic profile only with respect to bifunctional molecules.

[0388] Poor pharmacokinetic profiles are a well-known problem with bifunctional antibodies. Their rapid excretion and short in vivo half-lives limit their clinical use. Good drug concentrations (20 and 100 nM) corresponding to satisfactory in vivo PK values ​​are maintained for at least 48–72 hours with anti-PD-1*1 / IL-7*1 constructs, whereas only 2 nM of anti-PD-1*2 IL-7*2 molecules are detected in serum. The Format C anti-PD-1*1 / IL-7*1 of the present invention enables improved in vivo pharmacokinetic profiles during prolonged exposure compared to other formats of bifunctional antibodies (Formats B and C).

[0389] (Example 7. The anti-PD-*1 / IL-7*1 bifunctional molecule promotes the proliferation of T cells in vivo and induces significant antitumor efficacy compared to anti-PD-1*2 / IL*7*2 or anti-PD-1*2 / IL*7*1 constructs.) In vivo T cell proliferation was evaluated after a single dose of a bifunctional molecule (34 nM / kg) was administered intraperitoneally to mice with subcutaneous MC38 tumors. Four days post-treatment, blood and tumor samples were collected, T cells were stained with anti-Ki67 antibody, and proliferation was quantified by flow cytometry.

[0390] In vivo efficacy was evaluated in two different orthotopic syngeneic models: a liver cancer model and a mesotopic mesotopic model. Immunocompetent mice genetically modified to express human PD-1 (exon 2) were used for these experiments. For the mesothelioma model, AK7 mesothelioma cells were injected intraperitoneally (3*10). 6 (Individual cells / mouse). For the Hepa 1.6 model, 2.5*10 6 The cells were injected into the portal vein. In Experiment 1, mice were treated with similar drug exposure concentrations of PBS, an anti-PD-1 control (anti-PD-1*2), and anti-PD-1*1 / IL*7v*1. In Experiment 2, mice were treated with similar drug concentrations of PBS, an anti-PD-1 control (anti-PD-1*2), and anti-PD-1*1 / IL*7wt*1. AK7 cells stably express luciferase, enabling in vivo quantification of tumor volume after D-luciferin injection. Data were analyzed in photons / second per cm² per steradian, representing the mean values ​​of dorsal and ventral signals.

[0391] Results: Figure 16A shows that the anti-PD-1*1 / IL7wt*1 or IL7v*1 bifunctional molecule (Format C) promotes significant proliferation of CD4 and CD8 T cells to a greater extent than the anti-PD-1 antibody (anti-PD-1*1 or anti-PD-1*2). Significantly superior CD4 T cells were also observed with these two constructs compared to the anti-PD-1*2 / IL7*2 (Format A) and anti-PD-1*2 / IL7*1 (Format B) constructs. Similarly, higher proliferation was observed after treatment with anti-PD-1*1 / IL7wt*1 or anti-PD-1*1 / IL-7v*1 compared to the anti-PD-1*2 / IL7*2 (Format A) and anti-PD-1*2 / IL7*1 (Format B) constructs. These data support the fact that different constructs are more efficient at activating pSTAT5 signaling in T cells, with the anti-PD-1*1 / IL7*1 construct inducing higher pSTAT5 signaling compared to the anti-PD-1*2 / IL7*2 construct (Figure 10A).

[0392] Interestingly, the inventors observed that anti-PD-1*1 / IL7wt*1 or IL7v*1 significantly induced the proliferation of stem cell-like effector memory CD8 T cells in tumors more effectively than anti-PD-1*2 IL-7*2 and anti-PD-1*2 molecules (Figure 16B). The ability of anti-PD-1*1 IL-7*1 to enhance the TCF1+ stem cell-like CD8 T cell population is particularly interesting because it is crucial for the immune regulation of cancer. These cells are capable of self-renewal to generate a pool of tumor-specific T cells with high effector function.

[0393] Figures 17A and 17B show the in vivo efficacy of anti-PD-1*1 IL-7wt*1 and anti-PD-1*1 IL-7v*1 in a hepatotopic model of liver cancer. In two different experiments, anti-PD-1*1 / IL*7*1 (both wt and variant) (format C) demonstrated significantly superior efficacy compared to anti-PD-1*2 antibody. 85% of complete tumor eradication (complete response) was achieved after treatment with anti-PD-1*1 / IL7v*, compared to only 16% of mice treated with anti-PD-1*2.

[0394] In comparison, the inventors also tested the anti-PD-1*2 / IL-7*2 construct and observed lower antitumor efficacy for the anti-PD-1*2 / IL-7*2 construct in the same liver cancer model, which underlies the superior in vivo activity of the anti-PD-1*1 / IL-7*1 construct compared to the anti-PD-1*1 / IL-7*1 construct.

[0395] In orthotopic mesothelioma models (Figures 18A and B), anti-PD-1*1 / IL7v*1 demonstrated high antitumor efficacy, with a >85% complete response rate similar to anti-PD-1*2 antibody treatment. These data indicate that anti-PD-1*1 / IL7v*1 is highly effective in anti-PD-1 sensitive models, suggesting that the drug exhibits similar efficacy to bivalent anti-PD-1, even when containing only one anti-PD-1 arm (anti-PD-1*1) in format C.

[0396] Overall, these data highlight the critical importance of designing bifunctional antibodies to achieve in vivo antitumor efficacy. A fusion of one cytokine or protein with anti-PD-1*1 (Format C) showed the best antitumor efficacy and T cell in vivo proliferation, while bifunctional molecules constructed with two anti-PD-1 arms and one or two cytokines or proteins did not induce efficient T cell proliferation or antitumor effect in vivo.

[0397] (Example 8. The anti-PD-1*1 IL-7v*1 construct inactivates the inhibitory function of Tregs in vitro to a greater extent than IL-7 cytokines and the anti-PD-1*1 IL7WT*1 bifunctional antibody.) Anti-PD1 therapy stimulates T-cell effector function, but immunosuppressive molecules (TGFB, IDO, IL-10, etc.) and regulatory cells (Tregs, MDSCs, M2 macrophages) create an adversarial microenvironment that limits the full potential of the therapy. Treg cells express low levels of IL-7R (CD127), but they can still stimulate pSTST5 after IL-7 treatment, and it is known that IL-7 can reverse the Treg inhibitory function (Allgauer A et al., J. Immunol. 2015, vol. 195, pp. 3139-3148; Liu W et al., J Exp Med. 2006, vol. 203, pp. 1701-1711; Seddiki N et al., J exp Med 2006, vol. 203, pp. 1693-1700; Codarri L et al., J exp Med 2007, vol. 204, pp. 1533-1541; Heninger AK et al., J immunol 2012, vol. 189, pp. 5649-5658). To evaluate the effectiveness of anti-PD-1 / IL7 constructs in deactivating Treg function compared to IL-7, an inhibitory assay was performed by co-culturing Treg and effector T cells. The inventors observed in Figure 19 that IL-7 or anti-PD1-IL7 treatment blocked the Treg-mediated inhibitory effect, allowing Teff cell proliferation even in the presence of Treg cells. Anti-PD1 antibodies were unable to inhibit Treg inhibitory activity in effector T cells.

[0398] Surprisingly, anti-PD-1*1 IL7 W142H*1 demonstrated the highest efficacy (**p<0.05) in suppressing Treg function compared to IL-7 cytokines and similarly compared to the anti-PD-1*1 IL7WT*1 construct. These data highlight the advantages of using the anti-PD-1*IL7W142H*1 construct over naked IL-7 cytokines or non-mutant anti-PD-1*1 IL7*1 bifunctional antibodies. The dual effect of the monovalent variant, affecting both Treg deactivation and strong T cell proliferation simultaneously, was unexpected given that the selected IL7 variant W142H exhibited lower affinity for IL7R compared to the wild-type IL-7 cytokine.

[0399] Methods: Evaluation of the in vitro inhibitory activity of Tregs on CD8 effector T cell proliferation. CD8+ effector T cells and autologous CD4+ CD25-high CD127-low Tregs were selected from the peripheral blood of healthy donors and stained with a cell proliferation dye (CPDe450 for CD8+ T cells). Next, Tregs / CD8+ Tregs were co-cultured in a 1:1 ratio for 5 days on OKT3 coated plates (2 μg / mL), and Treg cell proliferation was quantified by flow cytometry by loss of the CPD marker.

[0400] (Example 9. Anti-PD-1*1 IL-7v*1 demonstrated superior in vivo efficacy compared to the anti-PD-1*1 IL7WT*1 construct in two different tumor models.) In vivo efficacy was evaluated in two different orthotopic syngeneic models: a liver cancer model and a mesotopic mesotopic model. Immunocompetent mice genetically modified to express human PD-1 (exon 2) were used for these experiments. For the mesothelioma model, AK7 mesothelioma cells were injected intraperitoneally (3*10). 6 (Individual cells / mouse). For the Hepa 1.6 model, 2.5*10 6The cells were injected into the portal vein. In Experiment 1, mice were treated with PBS, an anti-PD-1 control (anti-PD-1*2), anti-PD-1*1 / IL7v*1 (anti-PD-1*1 IL7W142H*1), or similar drug exposure concentrations of anti-PD-1*1 IL7wt*1. AK7 cells and Hepa1.6 stably express luciferase, enabling in vivo quantification of tumor volume after D-luciferin injection. Data were analyzed in photons / second per cm² per steradian, representing the mean values ​​of dorsal and ventral signals.

[0401] The AK7 intraperitoneal model is highly sensitive to PD-1 antibody treatment, as evidenced by the observation of high CD4+ and CD8+ T cell infiltration expressing PD-1 in the tumor microenvironment, enabling a favorable response to anti-PD-1 antibodies, as demonstrated in Figure 18. In the same experiment, the efficacy of anti-PD-1*1 IL7v*1 was compared to that of its wild-type IL7 homolog construct (anti-PD-1*1 IL7wt*1). The anti-PD-1*1 IL7v*1 construct induced a 92% complete response (n=1 death / 14 mice), demonstrating superior efficacy compared to the anti-PD-1*1 IL7wt*1 construct, which induced moderate antitumor efficacy (62% complete response) (Figure 20A). Bioluminescence analysis of tumors showed that anti-PD-1*1 IL7v*1 induced tumor clearance within 11–18 days post-treatment, while in the anti-PD-1*1 IL7wt*1 group, tumors shrank after treatment but eventually recurred (data not shown), indicating that treatment with the IL-7 wild-type construct may have transient efficacy compared to a bifunctional antibody constructed with low-affinity IL-7 (IL7W142H).

[0402] To evaluate the memory response induced by anti-PD-1*1 IL7v*1 treatment, all cured mice treated with anti-PD-1*1 IL7v*1 were re-challenged with a second injection of AK7 mesothelioma cells. As shown in Figure 20B, tumor bioluminescence was not detected after tumor re-challenge, but high bioluminescence signals were detected at multiple time points in challenged naive mice. These data demonstrate that anti-PD-1*1 IL7v*1 induced a strong, long-lasting specific memory antitumor response in the absence of any novel treatment.

[0403] Although anti-PD-1*1 IL7v*1 exhibits low affinity for IL-7R, this construct demonstrated unexpectedly higher efficiency than the anti-PD-1*IL-7wt*1 construct in a PD-1-sensitive tumor model, and preserved its antagonist anti-PD-1 activity-like anti-PD-1*2 antibody. These data highlight that anti-PD-1*1 IL7v*1 is a preferred construct for maintaining PD-1 inhibitory receptor blocking activity in vivo. We hypothesize that mutations in IL-7 balance the affinity of the bifunctional antibody to PD-1+ tumor-specific T cells (shown in Figure 11C) rather than PD-1-CD127+ non-tumor-specific T cells, resulting in better drug efficacy in vivo.

[0404] To evaluate the effectiveness of an anti-PD(L)1 refractory model for mimicking primary resistance in cancer patients, we selected the mouse liver cancer Hepa1.6 model. This is an orthotopic syngeneic model performed in immunocompetent mice (expressing human PD-1). This model is particularly important for the exclusion of tumor T cells from the described tumor (Gauttier V et al., 2020, Clin Invest, Vol. 130, pp. 6109-6123). The effectiveness of anti-PD-1*1 IL7v*1 with low affinity for IL7R versus anti-PD-1*1 IL7wt*1 with high affinity for IL7R was compared side-by-side in the same experiment. In separate groups, mice were treated with the same drug exposure concentrations of PBS (control), anti-PD-1*2, or isotype*1 IL7*v*1 (a homologous construct of a bispecific antibody targeting the antiviral protein envelope, used as the isotype control in the experiment). Anti-PD-1*1 IL7v*1 achieved a 60% complete tumor response, which is clearly superior to anti-PD-1*1 IL7wt*1 constructed with high-affinity wild-type IL7 (only 47% complete response), as shown in Figure 21. In this model, anti-PD1 antibodies are, as expected, ineffective. Furthermore, isotype*1 IL7v*1 is ineffective in this model, and combining anti-PD-1 and IL-7 treatment using anti-PD-1 / IL7 constructs is a good therapeutic strategy for enhancing T cell activation and antitumor response in PD-1 refractory models.

[0405] Memory responses induced by anti-PD-1*1 IL7v*1 treatment were also tested in this model, and the absence of the same tumor was observed.

[0406] Overall, these data confirm the superior efficacy of anti-PD-1 / IL-7 constructs with a low affinity for the CD127 receptor, an anti-PD-1 titer of 1, and one IL-7 cytokine mutation (W142H).

[0407] (Example 10. Demonstrated in vivo efficacy of anti-PD-1*1 IL-7v*1 in an anti-PD-1 refractory model correlates with strong transcriptional activity of the anti-PD-1 receptor and intratumoral proliferation of stem cell-like memory CD8 T cell subpopulation (TCF1+Tox- cells)) To better understand the effects of anti-PD-1*1 IL7v*1 in the tumor microenvironment, whole-tumor transcriptome analysis was also performed. Gene expression was detected and quantified using Nanostring technology (nCounter® PanCancer Immune profiling panel). Background was thresholded to the geometric mean of the negative control, and data were normalized to multiple reference genes included in the panel. Differential gene expression (DEG) was analyzed using the R package. The heatmap of unsupervised hierarchical cluster analysis of DEGs from DESeq2 analysis in Figure 22 shows that transcriptional expression patterns were very similar between the anti-PD-1 and anti-PD-1*1 IL7W14H*1 groups and significantly different from the PBS group, suggesting that the anti-PD-1 domain of the anti-PD-1*1 IL7v*1 construct conserved its antagonist bioactivity in vivo despite its anti-PD-1 titer. Protein-protein interaction network functional enrichment analysis by stringing of genes upregulated after treatment with anti-PD-1*2 or anti-PD-1*1 IL7W142H*1 identified several gene clusters involved in chemotactic immune receptor activity, Jak-STAT cytokine signaling, and antigen presentation (MHC protein complex binding and TCR signaling) compared to PBS conditions. Among the genes differentially expressed between anti-PD-1*2 and anti-PD-1*1 IL7W142H*1, we observed that CD8 or CD4 T early activation / memory stem cell-like T cell signatures associated with the expression of TCF7, CCR7, SELL, and IL7R genes were significantly upregulated in the anti-PD-1*1 IL7W142H*1 group compared to the anti-PD-1 and PBS groups (Figure 22B), using the single-sample GSEA (ssGSEA) signature algorithm from the R package GSVA. In contrast, as expected, upregulation of exhausted CD8 T cell genes (LAG3, PRF1, CD8A, HAVRC2, GZMB, CD8B1, KLRD1, TNFRSF9, TIGIT, CTSW, CCL4, CD63, IFNG, CXCR6, FASL, CSF1) was observed in the anti-PD-1 treated group.We adapted the genetic signatures of exhausted T cells and naive-like / stem cell-like memory T cells from a study that defined different T cell subsets in cancer using single-cell transcriptome analysis (Andreatta et al., Nature comm 2021).

[0408] Phenotypic analysis of CD8 T-cell-infiltrating lymphocytes by flow cytometry was also performed ex vivo to further characterize the population induced by anti-PD-1*1 IL7v*1 treatment. Despite the T-cell exclusion from tumors initially described in this resistance model, the tumor-infiltrating lymphocyte (TIL) composition was strongly increased after anti-PD-1*1 IL7W142H*1 treatment, and the product dramatically altered the T-cell subset (Figures 22B and C, 23C). Flow cytometry analysis demonstrated that anti-PD-1*1 IL7 W142H*1 alters the composition of the tumor microenvironment, favoring the accumulation of CD8 T cells over CD4 cells without affecting Tregs (Figure 23A). A high percentage increase in CD8+ CD44+ activated T cells with the stem cell-like memory T cell phenotype (CD3+CD8+CD44+TCF1+TOX-) that also express the Ki67 proliferation marker (Figure 23C) was observed after treatment (Figure 23B). Anti-PD-1 treatment induces the accumulation of TOX-TCF1- or TOX+ TCF1--related exhaustion phenotypes in tumors (Utzschneider et al., Immunity 2016, Vol. 45, pp. 415-427; Mann et al., 2019, Nature Immunology, Vol. 20, pp. 1092-1094). These data support transcriptome analysis and further determine that T cells activated by the anti-PD-1*1 IL7W142H*1 molecule express the CD44 activation marker, suggesting that this T cell subset is not a naive T cell subset but an early activated stem cell (steam)-like memory T cell subset (TCF1+TOX-). These data also confirm Example 9, which describes the efficacy of anti-PD-1*1 IL7W142H*1 in vivo in promoting the accumulation and proliferation of stem cell-like memory T cells in another tumor model.

[0409] (Example 11. Anti-PD-1*1 IL7v*1 maintains the survival of chronically stimulated human T cells and induces proliferation of TCF1+ T cells.) To confirm the effect of anti-PD-1*1IL7v*1 on human T cells, the inventors tested the effects of anti-PD-1 / IL7 constructs in a chronic antigen stimulation model in vitro. Human PBMCs were repeatedly stimulated every 3 days in CD3 CD28 coated plates (3 μg / mL, OKT3 and 3 μg / mL, CD28.2 antibody). After each stimulation, anti-PD-1*1 IL7v*1 (anti-PD-1*1 IL7W142H*1) construct, isotype control, or anti-PD-1*1 antibody was added to the culture. T cell viability and phenotype were evaluated by flow cytometry 24 hours after the 5th stimulation.

[0410] Figure 24A shows that anti-PD-1*1 IL7W142H*1 maintains chronically exhausted T cell survival compared to anti-PD-1 treatment. Phenotypic analysis of T cells (Figure 24B) demonstrated that anti-PD-1*1 IL7v*1 promotes specific proliferation and maintenance of a TCF1+ CD8 T cell subset. The TCF1+ T cell population is described as a stem cell-like T cell population capable of self-renewal and long-term effective responses. These results allow for the prediction of long-term effects in solid tumors by reactivating TILs with anti-PD-1*1 IL7v*1 in the early stages of cancer (adjuvant or neoadjuvant situations) and in various immunotumor evasion situations, preventing exhausted T cell proliferation in primary or secondary resistance to immuno-oncological treatment or other cancer treatments.

[0411] (Example 12. Anti-PD-1*1 IL-7v*1 demonstrated in vivo monotherapy efficacy in human models with varying resistance to PD-1 treatment.) In a triple-negative breast cancer (TNBC) model (immunodeficient mice subcutaneously transplanted with breast cancer cells MDA-MB231), mice were humanized with human peripheral blood mononuclear cells (PBMCs) from four different donors and then treated with either anti-PD-1*2 or anti-PD-1*1 IL7W142H*1 bifunctional antibodies. In all PBMC donors tested, anti-PD-1*1 IL7v*1 reduced tumor growth, while anti-PD-1*2 alone was ineffective (Figure 25).

[0412] In another humanized mouse model, a lung cancer model (A549), anti-PD-1*1 IL7v*1 was identified compared to anti-PD-1*1, which is associated with increased serum IFNg secretion in anti-PD-1*1 treated mice (day 34) (Figure 26). Both models demonstrate that anti-PD-1*1 IL7v can modulate the human immune-mediated antitumor response in vivo to a greater extent than anti-PD-1*2.

[0413] (Example 13. Anti-PD-1*1 IL7v*1 demonstrated a favorable pharmacokinetic profile compared to the anti-PD-1*1 IL7wt*1 molecule in cynomolgus monkeys.) Cynomolgus monkeys were intravenously injected with one dose of anti-PD-1*1 IL7wt*1 (0.8 mg / kg, 4.01 mg / kg) or one dose of anti-PD-1*1 IL7v*1 (anti-PD-1*1 IL7 W142H*1) (0.8 mg / kg, 4.01 mg / kg, or 25 mg / kg). Serum was collected at multiple time points after injection, and the anti-PD-1 IL7 construct was quantified by ELISA immunoassay using MSD technology. Briefly, human PD1 protein was immobilized, and anti-PD-1*1 IL7*1 antibody was added to serum. The ELISA was developed using sulfotaged anti-human kappa light chain monoclonal antibody.

[0414] Pharmacokinetic data were linear and dose-dependent for both constructs. However, the anti-PD-1*1 IL7v*1 construct showed a better pharmacokinetic profile compared to the anti-PD-1*1 IL7wt*1 construct (Figure 27) (at a dose of 4.01 mg / kg, area under the curve 29.6 vs. 10⁸, IL7wt vs. IL7v). Interestingly, anti-PD-1*1 IL7v*1, which has low affinity for the IL7 receptor, induced CD8 T cell proliferation in vivo for 10–14 days, demonstrating that the bioeffect of the drug persists beyond pharmacokinetic exposure. These data enable a novel pharmacokinetic model for measuring long-term effects on non-human primate T cell subsets and are applicable to human settings, namely CD8+ T cell proliferation after a single injection of the anti-PD-1*1 IL7v*1 bifunctional antibody.

[0415] (Example 14. Anti-PD-1*1 IL7v *1 constructed with the IgG1 N297A isotype or the LALA PG IgG1 isotype has the same ability to activate pSTAT5 signaling in human T cells.) In Examples 1-13, format IgG1 N297A was used for the anti-PD-1*1 / cytokine construct. The inventors tested another Fc silent format with additional LALA PG mutations, and it was noted that these mutations completely inactivate ADCC, ADCP, and CDC activity because the LALA PG mutation impairs binding to the FcR receptor.

[0416] The IL-7R activity of anti-PD-1*1 IL7W142H*1 was evaluated by pSTAT5 activity (Figure 28). No difference was observed between the two constructs in terms of activity in CD4 and CD8 human T cells, indicating that the present invention can be constructed with different Fc silent isotypes.

[0417] (Example 15. Anti-PD-1*1 IL7v*1 constructed with the IgG1 N297A isotype or a variant to improve FcRn binding, or a variant with a low charge pHi, has the same ability to activate the same pSTAT5 signaling in human T cells.) The inventors designed and compared the bioactivity of multiple structures of bifunctional molecules, including novel anti-PD-1*1 IL7W142H*1 variants: mutations in the Fc domain to improve FcRn binding (YTE, LS, DHS), or mutations in the light chain anti-PD-1*1, as shown in Figure 29. All anti-PD-1 / IL7 W142H constructs possess high affinity for the PD-1 receptor similar to that of the anti-PD-1*1 IL7W142H*1 N297A antibody, as demonstrated by ELISA assays (Figure 29). Such anti-PD-1*1 IL7W142H*1 variants include VH as defined in SEQ ID NO: 24, IL-7 as defined in SEQ ID NO: 5, and VL as defined in SEQ ID NOs: 28, 88, or 99.

[0418] The anti-PD-1 / IL7 W142H mutant molecule demonstrated high pSTST5 activity similar to that of the anti-PD-1*1 IL7W142H*1 N297A bifunctional molecule, but exhibited lower activity than anti-PD-1*1 IL-7wt*1 in naive T lymphocytes (PD1- cells) (Figure 30). Based on these findings, mutations in the Fc domain or VL domain support a useful alternative scaffold in the context of the present invention.

[0419] Materials and methods PD1 binding ELISA For the activity ELISA assay, recombinant hPD1 (Sino Biologicals, Beijing, China; inquiry 10377-H08H) was immobilized on plastic at 0.5 μg / ml in carbonate buffer (pH 9.2), and purified antibody was added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; inquiry 709-035-149) was added and elucidated using conventional methods.

[0420] ELISA antagonist: Competition between PDL1 and humanized anti-PD1 A competitive ELISA assay was performed using the PD-1:PD-L1 inhibitor screening ELISA assay pair (AcroBiosystems; USA; inquiry EP-101). In this assay, recombinant hPDL1 was immobilized on plastic at 2 μg / ml in PBS pH 7.4 buffer. Purified antibodies (at different concentrations) were mixed with biotinylated human PD1 (AcroBiosystems; USA; inquiry EP-101) at a final (immobilized) concentration of 0.66 μg / ml, and competitive binding was measured at 37°C for 2 hours. After incubation and washing, peroxidase-labeled streptavidin (Vector Laboratories; USA; inquiry SA-5004) was added to detect biotin-PD-1Fc binding, which was then developed using conventional methods.

[0421] pSTAT5 analysis PBMCs isolated from the peripheral blood of healthy human volunteers were incubated with anti-PD-1 / IL-7 molecules at 37°C for 15 minutes.

[0422] To determine cis activity, U937 cells transduced with CD127 and PD-1 were mixed with U937 cells transduced with only CD127+. Cells were stained with a 1:1 mixture of cell proliferation dye (CPDe450 or CPDe670, Thermofisher) and treated with the test molecule at 37°C for 15 minutes. Each cell subset was labeled with the cell proliferation dye (CPDe450 or CPDe670) and co-cultured. Next, the cells were fixed, permeabilized, and stained with AF647-labeled anti-pSTAT5 (clone 47 / Stat5 (pY694), BD Bioscience). For human PBMCs, pSTAT5 activation was evaluated in the CD3+ T cell population. For the U937 assay, pSTAT5 activation was evaluated in U937 PD1+ CD127+ cells and U937 CD127+ cells.

[0423] Cell binding analysis U937 cells transduced with CD127 and PD-1 were mixed with U937 cells transduced with CD127+ only. The cells were stained with a 1:1 mixture of cell proliferation dye (CPDe450 or CPDe670, Thermofisher). After staining the cells with Yellow / live dead fixable stain (Thermofisher), they were stained with human Fc block (BD Bioscience) diluted in 2% human serum PBS. Next, the cells were stained according to the series concentrations of the test molecules, and antibody development was performed using anti-human IgG-PE antibody (Biolegend, clone HP6017), followed by analysis by flow cytometry.

[0424] In vivo pharmacokinetics of anti-PD-1 / IL7 To analyze pharmacokinetics, a single dose of the molecule was injected intraorbitally, intraperitoneally, or intravenously (posterior orbital) into C57bl6JrJ mice (female, 6-9 weeks old). Plasma drug concentrations were determined by ELISA using immobilized anti-human light chain antibody (clone NaM76-5F3) diluted with serum containing IgG-fused Il67. Detection was performed using peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; inquiry 709-035-149), and development was performed using conventional methods.

[0425] 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 (inquiry J1250). Two cell lines were used: (1) effector T cells (Jarcut cells stably expressing PD-1 and NFAT-induced luciferase) and (2) activation target cells (CHO K1 cells stably expressing PDL1 and surface proteins designed to stimulate the cognitive TCR in an antigen-independent manner). When the cells were co-cultured, the PD-L1 / PD-1 interaction inhibited TCR-mediated activation, thereby blocking NFAT activation and luciferase activity. The addition of anti-PD-1 antibodies blocked the PD-1-mediated inhibitory signal, which led to NFAT activation, luciferase synthesis, and the release of bioluminescent signals. Experiments were conducted according to the manufacturer's recommendations. Serial dilutions of the test molecules were tested. Four hours after co-culturing PD-L1+ target cells, PD-1 effector cells, test molecules, and BioGlo® luciferin substrate were added to the wells, and the plates were read using a Tecan® luminometer.

[0426] in vivo proliferation A single dose (34 nM / kg) of the bifunctional molecule was administered intraperitoneally to C57bl6JrJ mice (female, 6-9 weeks old) with subcutaneous MC38 tumors. Mice were treated with a single dose (34 nM / kg) via intraperitoneal injection. Four days after treatment, blood was collected, and T cells were stained with anti-CD45, CD3, anti-CD8, anti-CD4 antibodies and anti-ki67 antibodies. Proliferation was quantified by flow cytometry.

[0427] In vivo humanized PD1 knock-in mouse model The efficacy of anti-PD-1 / IL-7 molecules was evaluated in vivo in syngeneic immunocompetent mice genetically modified to express human PD-1 (exon 2). For a mesothelioma orthotopic model, AK7 mesothelioma cells were injected intraperitoneally (3e6 cells / mouse) and then treated with equivalent drug exposure doses [anti-PD-1*2 (1 mg / kg), anti-PD-1*1 / IL-7*1, 4 mg / kg] on days 4, 6, and 8. The injected AK7 cells stably expressed luciferase, enabling the generation of in vivo bioluminescence signals after intraperitoneal injection of D-luciferin (3 μg / mouse, GoldBio, Saint Louis MO, USA, inquiry 115144-35-9). Ten minutes after luciferin injection, bioluminescence signals were measured for 1 minute on the dorsal and ventral sides of mice using a Biospace Imager. The data were analyzed in terms of photons / second per cm² per steradian, and the mean values ​​for dorsal and ventral signals are represented. Each group represents the mean + / - SEM of 5-7 mice per group. For the liver cancer model, Hepa1.6 liver cancer cells were subcutaneously injected into the portal vein at a dose of 2.5e6 cells. The mice were then treated on days 4, 6, and 8 with equivalent drug exposure doses [anti-PD-1*2 (1 mg / kg), anti-PD-1*1 / IL-7*1, 4 mg / kg].

[0428] Antibodies and bifunctional molecules The following antibodies and bifunctional molecules were used in the different experiments disclosed herein: pembrolizumab (Keytrudra, Merck), nivolumab (Opdivo, Bristol-Myers Squibb), and bifunctional molecules disclosed herein, including anti-PD1 humanized antibodies containing 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, 88, or 99, or anti-PD1 chimeric antibodies containing a heavy chain as defined in SEQ ID NO: 71 and a light chain as defined in SEQ ID NO: 72.

[0429] [Table 14]

[0430] 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 contains an anti-PD1 chimeric antibody containing 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 a light chain as defined in SEQ ID NO: 72. This molecule also contains an IL7 variant such as the one described in SEQ ID NO: 5.

[0431] In the examples, the control molecule, called BICKI-IL-7 WT, corresponds to construct 1 but has wild-type IL-7. It contains 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.

[0432] Another control molecule is anti-PD1*2 (which does not contain any IL7). This molecule contains a heavy chain as defined in SEQ ID NO: 79 and a light chain as defined in SEQ ID NO: 80.

[0433] 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 contains 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 contains, in particular, a heavy chain (hole) bound to IL-7 W142H as defined in SEQ ID NO: 83, or a heavy chain (knob) as defined in SEQ ID NO: 81 and a light chain as defined in SEQ ID NO: 80.

[0434] 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 contains 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 contains 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.

[0435] A control construct called anti-PD-1*1 is similar to structure 3 but lacks the IL-7 variant. Such a control contains 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 contains 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.

[0436] 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 contains 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 contains 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.

[0437] Constructs 2, 3, and 4 were genetically engineered to possess the IgG1 N298A isotype, and the amino acid sequences of the Fc region were mutated to create knobs in CH2 and CH3 of heavy chain A and holes in CH2 and CH3 of heavy chain B. All anti-PD-1 IL-7 and anti-PD-1*1 constructs contain the IgG1N298A mutant isotype, with the exception of the anti-PD-1*2 construct (lacking IL-7) and anti-PD-1*2 IL7wt*2 (BICKI-IL-7 WT) constructed with the IgG4 S288P isotype.

[0438] Description of the structures used in Examples 3-8 Different constructs of the bifunctional antibody were tested and compared. The following formats were tested: (1) Format A (anti-PD-1*2 / IL-7*2), (2) Format B (anti-PD-1*2 / IL-7*1), and (3) Format C (anti-PD-1*1 / IL-7*1 fused to the heavy chain). For Format C, the Fc domain contains CH1, CH2, and a hinge region. All constructs were manipulated using the IgG1 N298A isotype, with amino acid sequences mutated at the Fc region to create knobs at CH2 and CH3 of heavy chain A, and holes at CH2 and CH3 of heavy chain B. All constructs contain the GGGGSGGGGSGGGGS linker (SEQ ID NO: 70) between the Fc domain and the fused IL-7m protein.

Claims

1. A bifunctional molecule comprising a single antigen-binding domain and a single IL-7 variant, the bifunctional molecule comprises a first monomer comprising an antigen-binding domain covalently linked via its C-terminus to the N-terminus of a first Fc chain, optionally via a peptide linker, and a second monomer comprising a complementary second Fc chain lacking the antigen-binding domain and an IL-7 variant; i) the IL-7 variant is covalently linked to the C-terminus of the first Fc chain, optionally via a peptide linker; or ii) the single antigen-binding domain comprises a variable heavy chain and a variable light chain, and the IL-7 variant is covalently linked to the C-terminus of the light chain; the antigen-binding domain binds to PD-1; and 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 set forth in SEQ ID NO:1, and 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 the IL-7R, and ii) improves the pharmacokinetics of a bifunctional molecule comprising the IL-7 variant compared to a bifunctional molecule comprising wth-IL-7. Bifunctional molecules.

2. 2. The bifunctional molecule of claim 1, wherein the IL-7 variant comprises at least one amino acid mutation selected from the group consisting of: (i) W142G, W142A, W142V, W142C, W142L, W142I, W142M, W142H, W142Y and W142F, preferably 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.

3. The bifunctional molecule of claim 1 or 2, wherein the IL-7 variant is linked, preferably by its N-terminus, at the C-terminus of the first Fc chain.

4. 4. The bifunctional molecule of any one of claims 1 to 3, wherein the IL-7 variant comprises an amino acid substitution selected from the group consisting of W142H, W142F, and W142Y, with amino acid numbering as shown in SEQ ID NO:

1.

5. 4. The bifunctional molecule of any one of claims 1 to 3, wherein the IL-7 variant comprises the amino acid substitution W142H.

6. The bifunctional molecule according to any one of claims 1 to 3, wherein the IL-7 variant comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 2 to 15.

7. 4. The bifunctional molecule of claim 1, wherein the IL-7 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:

5.

8. 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;L234A / L235A;N297A+M252Y / S254T / T256E;N297A+N298A+ M252Y / S254T / T256E+ 8. The bifunctional molecule of any one of claims 1 to 7, comprising a heavy chain constant domain of human IgG1, preferably an Fc domain, with a substitution or combination of substitutions selected from the group consisting of K444A, K322A, K444A, K444E, K444D, K444G, K444S, P329G, L234A / L235A / P329G, M428L, L309D, Q311H, N434S, M428L+N434S and L309D+Q311H+N434S, preferably N297A optionally in combination with M252Y / S254T / T256E, and L234A / L235A or L234A / L235A / P329G.

9. Bifunctional molecules according to the invention may optionally comprise 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;L234A / L235A 8. The bifunctional molecule according to any one of claims 1 to 7, comprising a heavy chain constant domain of human IgG1, preferably an Fc domain, with a substitution or combination of substitutions selected from the group consisting of: N297A+M252Y / S254T / T256E; K322A and K444A, preferably N297A optionally in combination with M252Y / S254T / T256E, and L234A / L235A.

10. 8. The bifunctional molecule of any one of claims 1 to 7, comprising a heavy chain constant domain of human IgG4, preferably an Fc domain, optionally with a substitution or combination of substitutions selected from the group consisting of S228P; L234A / L235A, S228P+M252Y / S254T / T256+K444A, P329G, K444E, K444D, K444G, K444S, and L234A / L235A / P329G.

11. 8. The bifunctional molecule according to claim 1, comprising a heavy chain constant domain of human IgG4, preferably an Fc domain, optionally with a substitution or combination of substitutions selected from the group consisting of S228P; L234A / L235A, S228P+M252Y / S254T / T256E, and K444A.

12. 8. The bifunctional molecule of claim 1, wherein the Fc domain is an IgG1 or IgG4 containing the mutations LALA (L234A / L352A) or LALA PG (L234A / L235A / P329G).

13. 13. The bifunctional molecule of claim 1, wherein the first Fc chain and the second Fc chain form a heterodimeric Fc domain, in particular a knobs-into-holes heterodimeric Fc domain.

14. The bifunctional molecule of claim 13, wherein the first Fc chain is a hole chain or H chain and comprises substitutions T366S / L368A / Y407V / Y349C and N297A, and the second Fc chain is a knob chain or K chain and comprises substitutions T366W / S354C and N297A.

15. The bifunctional molecule of claim 13, wherein the second Fc chain comprises or consists of SEQ ID NO: 75 and / or the first Fc chain comprises or consists of SEQ ID NO:

77.

16. 16. The bifunctional molecule of any one of claims 1 to 15, comprising a first monomer comprising an antigen-binding domain covalently linked via its C-terminus to the N-terminus of a first heterodimeric Fc chain, optionally via a peptide linker, wherein the first heterodimeric Fc chain is covalently linked by its C-terminus to the N-terminus of an IL-7 variant, optionally via a peptide linker, and a second monomer comprising a complementary second heterodimeric Fc chain lacking an antigen-binding domain.

17. 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 GGGGSGGGSGGGGGS (SEQ ID NO: 70), preferably (GGGGS) 3 16. The bifunctional molecule of claim 1 , wherein the antigen-binding domain or the Fc domain is fused to the antigen-binding domain or the Fc domain via a peptide linker which is

18. 16. The bifunctional molecule of any one of claims 1 to 15, wherein the IL-7 variant is fused to the antigen-binding domain or the Fc domain by a peptide linker of SEQ ID NO:

70.

19. 19. The bifunctional molecule of any one of claims 1 to 18, wherein the antigen-binding domain is a Fab domain, a Fab', a single chain variable fragment (scFV), or a single domain antibody (sdAb).

20. 19. The bifunctional molecule of any one of claims 1 to 18, wherein the antigen-binding domain is a Fab domain or a Fab'.

21. The antigen-binding domain is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317, spartalizumab, MK-3477, SCH-900475, PF-06801591, JNJ-63723283, genolimuzumab, LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103, 21. The bifunctional molecule of any one of claims 1 to 20, derived from an antibody selected from the group consisting of MEDI-0680, MEDI0608, JS001, BI-754091, CBT-501, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, AGEN2034, MGA012, IBI308, 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4.

22. 21. The bifunctional molecule of any one of claims 1 to 20, wherein the antigen-binding domain is derived from an antibody selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, camrelizumab, spartalizumab, BI-7540991, and genolimuzumab.

23. 21. The bifunctional molecule of any one of claims 1 to 20, wherein the antigen-binding domain is derived from pembrolizumab or nivolumab.

24. 21. The bifunctional molecule of any one of claims 1 to 20, wherein the antigen-binding domain comprises or essentially consists of: (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) a light chain comprising CDR1 of SEQ ID NO: 64, 65, 89, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO: 16 or 90.

25. 21. The bifunctional molecule of any one of claims 1 to 20, wherein the antigen-binding domain comprises or essentially consists of: (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) a light chain comprising CDR1 of SEQ ID NO: 65, CDR2 of SEQ ID NO: 66, and CDR3 of SEQ ID NO:

16.

26. wherein 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; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 88, or SEQ ID NO:

99.

21. The bifunctional molecule of any one of claims 1 to 20, comprising or consisting essentially of:

27. 21. The bifunctional molecule of any one of claims 1 to 20, wherein the antigen-binding domain comprises or essentially consists of a heavy chain variable region (VH) of SEQ ID NO: 24 and a light chain variable region (VL) of SEQ ID NO:

28.

28. 28. The bifunctional molecule of any one of claims 1 to 20, 26 or 27, wherein 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, and wherein the IL-7 variant comprises the amino acid substitution W142H, with amino acid numbering as shown in SEQ ID NO: 1, preferably comprising the amino acid substitution as set forth in SEQ ID NO:

5.

29. (i) 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; (ii) the IL-7 variant comprises or consists essentially of a sequence as set forth in SEQ ID NO:5; (iii) the second Fc chain comprises or consists of SEQ ID NO: 75 and / or the first Fc chain comprises or consists of SEQ ID NO: 77; and 29. A bifunctional molecule according to any one of claims 1 to 20 or 26 to 28.

30. 30. The bifunctional molecule of claim 29, further comprising a peptide linker of SEQ ID NO:

70.

31. 21. The bifunctional molecule of any one of claims 1 to 20, comprising a first monomer of SEQ ID NO: 83, a second monomer of SEQ ID NO: 75, and a third monomer of SEQ ID NO: 37, 38, 80, 100, or 101, preferably SEQ ID NO: 38 or 80.

32. 21. The bifunctional molecule of any one of claims 1 to 20, comprising a first monomer comprising or consisting of SEQ ID NO: 83, a second monomer comprising or consisting of SEQ ID NO: 75, and a third monomer comprising or consisting of SEQ ID NO:

80.

33. 33. An isolated nucleic acid sequence or a group of isolated nucleic acid molecules encoding a bifunctional molecule according to any one of claims 1 to 32.

34. 34. A host cell comprising the isolated nucleic acid of claim 33.

35. 35. A pharmaceutical composition comprising the bifunctional molecule of any one of claims 1 to 32, the isolated nucleic acid of claim 33, or the host cell of claim 34, optionally together with a pharma- ceutically acceptable carrier.

36. A bifunctional molecule according to any one of claims 1 to 32, an isolated nucleic acid according to claim 33, a host cell according to claim 34 or a pharmaceutical composition according to claim 35 for use as a medicament, preferably for use in the treatment of cancer or an infectious disease.

37. 37. The bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition of claim 36 in the treatment of cancer or viral infection by stimulating effector memory stem cell-like T cells.

38. Cancer is cancer of the hematopoietic system, solid tumors, carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, urethral cancer, environmentally induced cancer and any combination of such cancers, metastatic or non-metastatic, melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric or gastroesophageal cancer, cervical cancer, hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndromes, acute myeloid leukemia, Kaposi's sarcoma; cervical, anal, penile and vulvar cancers associated with human papillomavirus 38. The bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition of claim 36 or 37, selected from the group consisting of squamous cell carcinoma of the nasal cavity and oropharyngeal carcinoma; B-cell non-Hodgkin's lymphoma (NHL) including diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, and classical Hodgkin's lymphoma; lymphoproliferative disorders associated with Epstein-Barr virus (EBV) and / or Kaposi's sarcoma herpesvirus; hepatocellular carcinoma associated with Hepatitis B and / or C virus; Merkel cell carcinoma associated with Merkel cell polyomavirus (MPV); and cancers associated with human immunodeficiency virus (HIV) infection.

39. 38. The bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition of claim 36 or 37, wherein the viral infection is caused by a virus selected from the group consisting of HIV, a hepatitis virus, such as hepatitis A, B, or C, a herpes virus, such as VZV, HSV-1, HAV-6, HSV-II, CMV, or Epstein-Barr virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

40. 40. The bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition of claim 36, 37, 38, or 39 for use in combination with a therapeutic agent or treatment selected from the group consisting of chemotherapy, radiation therapy, targeted therapy, anti-angiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes derived from tumor antigens, myeloid checkpoint inhibitors, immunotherapeutic agents, and histone deacetylase (HDAC) inhibitors.

41. 41. The bifunctional molecule, nucleic acid, host cell, or pharmaceutical composition of claim 40, wherein the therapeutic agent is an immune checkpoint blockade agent or an activator of adaptive immune cells (T and B lymphocytes) selected from the group consisting of anti-CTLA4, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonists, CD40-L, TLR agonists, anti-ICOS, ICOS-L, and B cell receptor agonists.

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