Dual-function anti-PD-1 / IL-7 molecules

Abstract

The present invention relates to bifunctional molecules comprising an anti-PD-1 antibody and IL-7 and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to the field of immunotherapy. The present invention provides a bifunctional molecule comprising an anti-PD1 antibody or an antibody fragment thereof. [Background technology]

[0002] Targeting T cell inhibitory checkpoints with therapeutic antibodies for disinhibition is an area of ​​intense research (for review, see Pardoll, Nat Rev Cancer. 2012;12:253-264). Targeting immune checkpoints in adaptive immunity has demonstrated excellent therapeutic efficacy in many cancers, albeit in a limited percentage of patients. Combining immune checkpoint therapy with other immunotherapeutic strategies has shown excellent efficacy in preclinical models but remains challenging in the clinic.

[0003] Immune cell activation is controlled by integrating the balance of costimulatory and co-inhibitory signals. T cell receptor (TCR)-mediated T cell activation is modulated by both costimulatory and co-inhibitory signals. Antigen-independent secondary signals modify the primary signal provided by the interaction of the antigen peptide-MHC complex with the TCR, conferring specificity to the response. T cell costimulatory and co-inhibitory pathways have broad immunoregulatory functions, controlling effector, memory, and regulatory T cells, as well as naive T cells. Therapeutic modulation of these pathways is translating into effective new strategies for treating cancer (for a review, see Schildberg et al., Vol. 44 (No. 5), Immunity, 2016). Ongoing research into the regulation of immune responses has led to the identification of multiple immunological pathways that can be targeted for the development of cancer therapies. Such molecules are referred to herein as immune checkpoint co-activators or co-inhibitors (for reviews see Sharma et al., Cell, 161(2), 2015 and Pardoll, Nature Reviews Cancer, 12(4), 2012).

[0004] Programmed cell death protein 1 (PD-1, also known as CD279) is a cell surface protein molecule belonging to the immunoglobulin superfamily. It is expressed on T and B lymphocytes and macrophages and plays a role in cell fate and differentiation. Two ligands for PD-1, PD-L1 and PD-L2, have been identified, which have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). The interaction of PD-1 with its ligands results in a reduction of tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancerous cells. In particular, PD1 ligation inhibits T cell responses by reducing signals downstream of TCR stimulation to T cells, resulting in reduced activation and cytokine production.

[0005] PD-1 / PD-L1 therapy is approved by the FDA for first- and second-line treatment of a wide range of hematologic and solid tumors, but objective responses based on a greater than 30% reduction in tumor size, as defined by RECIST criteria, vary widely across cancer subtypes.

[0006] High response rates were observed in refractory Hodgkin lymphoma (65-85%) (Borcherding N et al., J Mol Biol., 2018 July 6; 430(14):2014-2029), microsatellite instability-high colon cancer (MSI-H, 25%-80%), or Merkel cell carcinoma (56%).

[0007] Moderate objective response rates are observed in patients with melanoma (24-44%) and non-small cell lung cancer (12.8-43.7%) in whom anti-PD-1 therapy is used as first-line treatment. Although only a proportion of patients benefit from the therapy, PD-1 / PD-L1 therapy has improved overall survival compared with chemotherapy, the older standard of care.

[0008] In some solid tumors, particularly pancreatic cancer, non-MSI colorectal cancer, gastric cancer, and some breast cancers, clinical responses have been low or nonexistent (Borcherding N et al., J Mol Biol., 2018 July 6; 430(14):2014-2029).

[0009] Multiple mechanisms have been described that can explain these differences in efficacy and resistance to PD-1 / PD-L1 checkpoint therapy, particularly several of which relate to T cell biology, such as (1) impaired formation of memory T cells, (2) impaired T cell infiltration, (3) insufficient generation of tumor-specific T cells, (4) inadequate T cell function, and (5) an immunosuppressive microenvironment induced by regulatory T cells. Combination with therapies targeting IL-7 signaling may be a good strategy to overcome anti-PD-1 resistance in patients by stimulating T cell infiltration, sustaining T cell effector capacity, and promoting long-lasting memory T cell responses without stimulating the expansion and survival of regulatory T cells.

[0010] Interleukin-7 (IL-7), a member of the IL-2 superfamily of immunostimulatory cytokines, plays a key role in the adaptive immune system, promoting immune responses mediated by B and T cells. This cytokine activates immune function by stimulating the survival and differentiation of T and B cells, lymphocyte survival, and 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 and memory T cells. IL-7 also enhances human immune responses by promoting the secretion of IL-2 and interferon-γ. The IL-7 receptor is a heterodimer consisting of IL-7Rα (CD127) and the common γ chain (CD132). While the γ chain is expressed by all hematopoietic cell types, IL-7Rα is primarily expressed by lymphocytes, including B and T lymphocyte precursors, naive T cells, and memory T cells. IL-7Rα expression on regulatory T cells has been observed to be low compared with effector / naive T cells, which express higher levels, and therefore CD127 is used as a surface marker to distinguish these two populations. IL-7Rα is also expressed on innate lymphoid cells such as NK cells and gut-associated lymphoid tissue (GALT)-derived T cells. The IL-7Rα (CD127) chain is shared with TSLP (tumor stromal lymphopoietin), and CD132 is shared with IL-2, IL-4, IL-9, IL-15, and interleukin-21. Two major signaling pathways are induced by CD127 / CD132: (1) the Janus kinase / STAT pathway (i.e., Jak-Stat-3 and 5) and (2) the phosphatidylinositol-3 kinase pathway (i.e., PI3K-Akt). IL-7 administration is well tolerated by patients and is associated with an expansion of CD8 and CD4 cells and a relative decrease in CD4+ T regulatory cells. Recombinant naked IL-7 or IL-7 fused to the N-terminal Fc domain of an antibody is being tested in the clinic. The latter is based on the rationale that fusion to the Fc domain extends the half-life of IL-7, enhancing the long-term efficacy of the treatment.Because IL-2 acts on both Tregs and T effector cells, whereas IL-7 selectively activates T effector cells, targeting IL-7 signaling should be more promising than targeting IL-2 signaling.

[0011] To increase the efficacy of anti-PD-1 immunotherapy and overcome potential anti-PD-1 resistance in patients, developing combination therapies targeting IL-7 signaling may be a good strategy for stimulating T cell infiltration, sustaining T cell effector activity, and promoting long-lasting memory T cell responses without stimulating the expansion and survival of regulatory T cells. Indeed, anti-PD-1 therapy increases CD127 expression on exhausted T cells, thereby enhancing their ability to respond to IL-7 and simultaneously enhancing the production of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) (Pauken et al., Science. 2016 December 2;354(6316):1160-1165; Shi et al., Nat Commun. 2016 August 8;7:12335). [Prior art documents] [Patent documents]

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[0014] However, the validation and development of combination immunotherapies has been significantly limited by the cost of biological therapies and the limited availability of such immunotherapies. Thus, there remains a significant need in the art for new and improved agents for safe immunotherapy, particularly for cancer, that target T cells and have effective and positive effects on adaptive immune responses, particularly T cell immune responses. The inventors have achieved significant progress with the invention disclosed herein. [Means for solving the problem]

[0015] The present inventors provide a bifunctional molecule comprising an anti-hPD-1 antibody and human IL-7, which is promising for numerous therapeutic applications, particularly in the treatment of cancer. The present invention is based on the development of an antibody that specifically targets human PD-1, exhibiting high binding affinity to PD-1 and potently competing with its ligands, PD-L1 and PD-L2. Surprisingly, fusing the N-terminus of IL-7 to the C-terminus of the Fc region of an anti-hPD-1 antibody preserves its high affinity for CD127 (the IL-7 receptor) to a similar level as endogenous IL-7, suggesting potent IL-7R activation. Furthermore, fusing the Fc domain to IL-7 extends the half-life of the product. Furthermore, the bifunctional anti-PD1 / IL-7 molecule disclosed herein allows IL-7 accumulation in PD-1+ T cell infiltrates and relocalization of IL-7 on PD-1+ T cells. In particular, anti-PD-1 / IL-7 bifunctional molecules induce the proliferation and activation of naive, partially exhausted, and fully exhausted T cell subsets, as reflected by cytokine (e.g., IFNγ) secretion and integrin (e.g., alpha4 and beta7 and LFA-1) expression. Such anti-hPD-1 / IL-7 bifunctional molecules have the potential to overcome associated resistance mechanisms and improve the efficacy of anti-PD-1 immunotherapy.

[0016] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) (i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain (VL) comprising LCDR1, LCDR2, and LCDR3; an anti-human PD-1 antibody or an antigen-binding fragment thereof comprising: (b) human interleukin-7 (IL-7) or a fragment thereof A bifunctional molecule comprising: The bifunctional molecule relates to an antibody or fragment thereof covalently linked to human IL-7 or a fragment thereof, preferably by a peptide linker, as a fusion protein.

[0017] In particular, the N-terminus of human IL-7 or a fragment thereof is attached to the C-terminus of the heavy chain or light chain, or both, of an anti-human PD-1 antibody or antigen-binding fragment thereof.

[0018] In one aspect, the antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a human antibody.

[0019] In a particular aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain (VL) comprising LCDR1, LCDR2, and LCDR3; A bifunctional molecule comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising or consisting of: - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1; - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, in which X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N and E; the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, wherein X is G or T; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15; - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16; Concerning bifunctional molecules.

[0020] In particular, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: (a) a 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 (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T.

[0021] More specifically, the present invention relates to a bifunctional molecule comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 24; and (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28. The present invention relates to a bifunctional molecule comprising or consisting of:

[0022] Alternatively, the anti-PD1 antibody is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, PDR001, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4.

[0023] In particular, the IL-7 or variant thereof comprises or consists of an amino acid sequence having at least 75% identity to wild-type human IL-7 (wth-IL-7). In a particular embodiment, the IL-7 comprises or consists of the amino acid sequence set forth in SEQ ID NO:51.

[0024] Alternatively, the IL-7 is an IL-7 variant that exhibits at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 51, wherein the variant comprises at least one amino acid mutation that i) reduces the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R, and ii) improves the pharmacokinetics of a bifunctional molecule comprising the IL-7 variant compared to a bifunctional molecule comprising wth-IL-7.

[0025] In particular, the at least one mutation may be an amino acid substitution or group of amino acid substitutions selected from the group consisting of: (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F, or W142Y, (iii) D74E, D74Q, or D74N, iv) Q11E, Y12F, M17L, Q22E, and / or K81R; or any combination thereof.

[0026] In one aspect, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, and C47S-C92S and C34S-C129S.

[0027] In another aspect, the IL-7 variant comprises an amino acid substitution selected from the group consisting of W142H, W142F, and W142Y.

[0028] In another aspect, the IL-7 variant comprises an amino acid substitution selected from the group consisting of D74E, D74Q, and D74N.

[0029] Preferably, the IL-7 variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 53 to 66. Even more preferably, the IL-7 variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 54, 56, or 63.

[0030] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain, preferably an IgG1 or IgG4 heavy chain constant domain.

[0031] In a more specific aspect, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and optionally, a light chain constant domain selected from the group consisting of T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S23 and K444A, preferably with a substitution or combination of substitutions selected from the group consisting of N297A, and L234A / L235A, optionally in combination with M252Y / S254T / T256E.

[0032] In another more specific aspect, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain, optionally with a substitution or combination of substitutions selected from the group consisting of S228P; L234A / L235A, S228P+M252Y / S254T / T256E, and K444A.

[0033] Optionally, the antibody or fragment thereof is linked to IL-7 or a variant thereof by a linker sequence preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, more preferably by (GGGGS)3 or (GGGS)3.

[0034] In a very specific aspect, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N, and the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and optionally, one of the following substitutions: T250Q / M428L; M252Y / S254T / T256E+H433K / N434F; E233P / L234V / L235A / G236A+A327G / A330S / P331S; E33 and K444A, preferably with a substitution or combination of substitutions selected from the group consisting of N297A, and L234A / L235A, optionally in combination with M252Y / S254T / T256E, wherein the antibody or fragment thereof is linked to the IL-7 variant by the linker (GGGGS)3.

[0035] In another aspect, the present invention relates to an isolated nucleic acid sequence or a group of isolated nucleic acid molecules encoding a bifunctional molecule as disclosed herein, a vector comprising a nucleic acid or a group of nucleic acid molecules as disclosed herein, and / or a host cell comprising a vector comprising a nucleic acid or a group of nucleic acid molecules as disclosed herein.

[0036] In another aspect, the present invention relates to a method for producing a bifunctional molecule, the method comprising culturing a host cell as disclosed herein and optionally isolating the bifunctional molecule.

[0037] In another aspect, the invention relates to a pharmaceutical composition comprising a bifunctional molecule, a nucleic acid or a group of nucleic acid molecules, a vector, or a host cell as disclosed herein, and a pharmaceutically acceptable carrier.

[0038] Optionally, the pharmaceutical composition preferably further comprises an additional therapeutic agent selected from the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antivirals, Aurora kinase inhibitors, proapoptotic agents (e.g., Bcl-2 family inhibitors), activators of the death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunological agents, inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics epitopes or neoepitopes derived from tumor antigens, such as antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNA, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent 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 inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, and peptide vaccines, as well as combinations of one or more of these substances.

[0039] In particular, the pharmaceutical composition, the bifunctional molecule, the nucleic acid or group of nucleic acid molecules, the vector, or the host cell are intended for use as a medicament.

[0040] Finally, the present invention relates to a pharmaceutical composition, a bifunctional molecule, a nucleic acid or a group of nucleic acid molecules, a vector, or a host cell as disclosed herein for use as a medicament, preferably for use in the treatment of cancer, preferably a cancer selected from the group consisting of: hematological malignancies or solid tumors with expression of PD-1 and / or PD-L1, such as cancers selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndromes, and acute myeloid leukemia, cancers induced by viruses or associated with immunodeficiency, such as Kaposi's sarcoma (e.g., associated with Kaposi's sarcoma herpesvirus); cervical cancer, anal cancer, penile cancer, and vulvar squamous cell carcinoma, and oropharyngeal cancer (e.g., associated with human papillomavirus); B-cell non-Hodgkin's lymphoma, including diffuse large B-cell lymphoma. cancers selected from the group consisting of non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classical Hodgkin's 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 virus); Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)); and cancers associated with human immunodeficiency virus infection (HIV), as well as cancers selected from the group consisting of 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, and cervical cancer.

[0041] Optionally, the bifunctional molecule, pharmaceutical composition, isolated nucleic acid molecule or group of isolated nucleic acid molecules, vector, or host cell is for use in combination with radiation therapy or an additional therapeutic agent preferably selected from the group consisting of: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antivirals, Aurora kinase inhibitors, proapoptotic agents (e.g., Bcl-2 family inhibitors), activators of cell death pathways, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapy. epitopes or neoepitopes derived from tumor antigens, such as methods, immunological agents, inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent 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 inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, and peptide vaccines, as well as combinations of one or more of these substances.

[0042] The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell, or use as disclosed herein is for use to inhibit the suppressive activity of T regulator cells, to activate T effector cells, and / or to stimulate the proliferation of naive, partially exhausted, and fully exhausted T cells.

[0043] The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell, or use as disclosed herein may also be for use in the treatment of an infectious disease, preferably a chronic infectious disease, even more preferably a chronic viral infection. Preferably, the infectious disease is caused by a virus selected from the group consisting of HIV, hepatitis virus, herpes 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 virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus. [Brief explanation of the drawings]

[0044] [Figure 1]PD-1 binding ELISA assay. Human recombinant PD-1 (rPD1) protein was immobilized, and antibodies were added at different concentrations. Color development was performed with a peroxidase-conjugated anti-human Fc antibody. Colorimetric analysis was determined at 450 nm using TMB substrate. (A) Anti-PD1 (■), anti-PD1VL-IL7 (o), and anti-PD1VH-IL7 (●) were compared for their binding to recombinant PD1 (rPD1). (B) Comparison of chimeric Bicki (●) versus humanized Bicki (■) derived from an anti-PD1 antibody fused to IL7 on its heavy and / or light chain: anti-PD1VH-IL7 (left graph), anti-PD1VL-IL7 (middle graph), or anti-PD1VH and VL-IL7 (right graph). Molecules were added at different concentrations onto plates coated with human PD1 recombinant protein. (C) PD-1 binding of Bicki anti-PD-1 / IL-7 constructed with Keytruda (●) and Opdivo (■) scaffolds was tested at different concentrations on plates coated with human PD1 recombinant protein. [Figure 2] Antagonistic ability of Bicki anti-PD1-IL7 molecules to block PD-1 / PD-L1 and PD1 / PDL2 interactions. (A) ELISA assay: PD-L1 was immobilized on a Maxisorp plate, and conjugated antibody plus biotinylated recombinant human PD-1 was added. The conjugates were prepared at a fixed concentration of PD1 (0.6 μg / mL), and different concentrations of anti-PD1 (■), anti-PD1VH-IL7 (●), or anti-PD1VL-IL7 (o) antibodies were tested. (B) Biacore affinity assay of PD-1 recombinant protein on human PD-L2 recombinant protein pre-incubated with anti-PD1, anti-PD1VH-IL7, or anti-PD1VL-IL7 antibodies. Human recombinant PD-L2 was immobilized on a CM5 biochip, and conjugated antibody (200 nM) plus recombinant human PD-1 (100 nM) was added. Data are expressed as % of the relative response of the interaction measured by Biacore: 100% = PD-1 relative response. [Figure 3]The Bicki anti-PD1-IL7 molecule stimulates the IL-7R signaling pathway, as measured by ex vivo STAT5 phosphorylation in human PBMCs. PBMCs isolated from peripheral blood of healthy volunteers were incubated with recombinant IL-7 (rIL7) (gray ●) + / - anti-PD1 (gray ▽), anti-PD1VH-IL7 (■), or anti-PD1VL-IL7 (●) for 15 minutes. Cells were then fixed, permeabilized, and stained with AF647-conjugated anti-pSTAT5 (clone 47 / Stat5(pY694)). Data were obtained by calculating MFI pSTAT*%pSTAT5+ population, normalized to a standard (100% = rIL-7 57.5 nM) and represent the average of three different donors in two independent experiments. [Figure 4]Bicki anti-PD1-IL7 enhances T cell activation in vitro. (A) Discover'x PD-1 Path Hunter bioassay: Jurkat T cells stably expressing an engineered PD-1 receptor (ED) fused to a beta-gal fragment and an engineered SHP1 (EA) fused to a complementary beta-gal fragment. Addition of an anti-PD1 antagonist antibody blocks PD-1 signaling, leading to a loss of bioluminescent signal (RLU). Anti-PD1 (■) or anti-PD1VH-IL7 (●) were tested at different molar concentrations. Data are expressed as RLU (relative luminescent signal). (B) Promega PD-1 / PD-L1 bioassay: (1) effector T cells (Jurkat mice stably expressing PD-1, NFAT-inducible luciferase) and (2) activated target cells (CHO K1 cells stably expressing PDL1 and surface proteins engineered to activate the cognate TCR in an antigen-independent manner) were co-cultured. After addition of BioGlo™ luciferin, luminescence was quantified and reflects T cell activation. Serial molar concentrations of anti-PD1 antibodies + / - recombinant IL-7 (rIL-7) or Bicki anti-PD1VH-IL7 or anti-PD1VL-IL7 antibodies were tested. Each dot represents the EC50 of one experiment. (C) The ability of Bicki anti-PD-1 / IL-7 constructed in the Keytruda or Opdivo backbone to stimulate NFAT. T cell activation was also examined using the Promega PD-1 / PD-L1 bioassay. Keytruda alone or Opdivo alone (●) versus pembrolizumab VH IL-7 or nivolumab VH IL-7 (○) were tested at different concentrations. [Figure 5] Bicki anti-PD1-IL7 molecules enhance IFNγ secretion. T cells isolated from peripheral blood of healthy volunteers were stimulated on OKT3 / PDL1-coated plates (2 and 5 μg / mL, respectively) in the presence of isotype control, anti-PD1+ / -rIL7, anti-PD1VH-IL7, anti-PD1VL-IL7, or isotype VH-IL7 at a final antibody concentration of 5 μg / mL. Five days after stimulation, secreted IFNγ was determined by sandwich ELISA. Results are representative of four donors (n=2 experiments). [Figure 6] Bicki anti-PD1-IL7 stimulates T cell proliferation to a similar extent as recombinant soluble IL-7. PBMCs isolated from peripheral blood of healthy volunteers were stimulated with anti-CD3 / CD28. 24 hours after stimulation, PBMCs were harvested and restimulated in the presence of rIL-7 or Bicki anti-PD1VH-IL7 on OKT3 / PDL1-coated plates (2 and 5 μg / mL, respectively). (A) Fixed doses (29 nM Bicki or 3.2 nM rIL-7) or (B) multiple doses of rIL-7 (□), anti-PD1 alone, or anti-PD1VH-IL7 (●), anti-PD1VL-IL7 (■), or isotype VH IL-7 (▲) were tested. Five days after stimulation, T cell proliferation was assessed by H-thymidine incorporation. Data are normalized (100% = rIL7 10 nM) and represent the average obtained with three different donors. EC50 (pM) refers to the concentration required to reach 50% of T cell proliferation. [Figure 7] Bicki anti-PD1VH-IL7 stimulates integrin expression on the T cell surface. Human PBMCs were incubated for 3 days without the molecule (gray histogram), with rIL-7 / rIL-2 or rIL-7 (50 ng / mL), or with anti-PD-1 or anti-PD1VH-IL7 (5 μg / mL). (A) Alpha4 and beta7 integrin cell surface expression analysis. FACS was analyzed by LSR, and data are presented as fold change normalized to 1, corresponding to the mean control (untreated) fluorescence for each donor. (B) LFA-1 cell surface expression analysis (CD11a and CD18) by FACS using LSR. Results are presented as mean fluorescence. Each dot represents one donor from three independent experiments. [Figure 8]Modeling chronic antigen stimulation of T cells resulting in exhausted T cells. Human PBMCs were repeatedly stimulated every 3 days on CD3CD28-coated plates (3 μg / mL OKT3 and 3 μg / mL CD28.2 antibody). (A) 24 hours after stimulation, T cells were stained for PD-1, Lag3, and Tim3 inhibitory receptors. Expression was analyzed by flow cytometry and FACS LSRII using fluorochrome-conjugated antibodies. Data are expressed as the percentage of positive cells for three donors (one donor = one curve). (B) T cell proliferation capacity was determined by thymidine 3H incorporation 5 days after each stimulation. (C) 24 hours after each stimulation, supernatant IFNg secretion was analyzed by ELISA (pg / ml). [Figure 9] IL7 pathway activation in exhausted T cells: Response of exhausted T cells to a 15-minute incubation of cells with rIL-7 or Bicki anti-PD1VH-IL7 by measuring STAT5 phosphorylation 48 hours after each stimulation. Cells were then fixed, permeabilized, and stained with AF647-conjugated anti-pSTAT5 (clone 47 / Stat5(pY694)). (A) Gray histograms represent cells treated with rIL7, and black histograms represent cells treated with Bicki anti-PD1VH-IL7. Data are normalized (MFI pSTAT*%pSTAT5 population) and are representative of four different donors. (B) The ED50 for pSTAT5 was determined in pM for each stimulation and refers to the concentration of rIL-7 (■ gray) or Bicki anti-PD1VH-IL7 (■ black) required to achieve 50% pSTAT5 activation. [Figure 10A]Proliferation of exhausted T cells upon IL-7 stimulation. Human PBMCs were repeatedly stimulated with CD3 CD28-coated plates (3 μg / mL OKT3 and 3 μg / mL CD28.2 antibody). 24 hours after each stimulation, T cells were restimulated on OKT3-coated plates (2 μg / mL) in the presence of anti-PD1, rIL-7, or Bicki anti-PD1-IL7 (anti-PD1 VH-IL7 or anti-PD1 VL-IL7). H3 uptake assays were performed on day 5 to determine T cell proliferation. Raw proliferation data from one donor after 3, 4, and 5 stimulations (STIM) are shown (H3 uptake (cpm)). [Figure 10B] Proliferation of exhausted T cells upon IL-7 stimulation. Human PBMCs were repeatedly stimulated with CD3 CD28-coated plates (3 μg / mL OKT3 and 3 μg / mL CD28.2 antibody). T cells stimulated three times were either not stimulated further (no stimulation) or restimulated on anti-CD3 (StimOKT3), anti-CD3 + recombinant PDL1 (StimOKT3 / PDL1), or anti-CD3 + recombinant PDL2 (StimOKT3 / PDL2)-coated plates (2 and 5 μg / mL, respectively). H3 uptake assays were performed on day 5, and data were normalized (1 = H3 uptake with isotype antibody). n = 4 donors and 2 separate experiments. [Figure 11]Treg suppressive activity on CD8+ effector T cell proliferation. CD8+ effector T cells and CD4+CD25highCD127low Tregs were isolated from peripheral blood of healthy donors and stained with a cell proliferation dye (CPDe450 for CD8+ T cells). Treg / CD8+ Teffs were then co-cultured at a 1:1 ratio on OKT3-coated plates (2 μg / mL) for 5 days in the presence or absence of rIL-7, anti-PD-1, anti-PD-1+rIL-7, or anti-PD1VH-IL7. Effector T cell proliferation was analyzed by cytofluorometry. (A) Data represent the % of proliferating Teffs alone (black histogram) or co-cultured with Tregs (gray histogram) + / - SEM based on the loss of CPD markers in the CD8 T effector cell population (n = 4 donors in 4 separate experiments). (B) Treg proliferation was assessed using the CPD proliferation dye after incubation with different equimolar doses of IL-7 (▲), IL-2 (●), IL-15 (■), or anti-PD1VH-IL7 (▼). [Figure 12] In vivo efficacy of Bicki anti-PD1-IL7 antibody in a humanized mouse model. Mice were intraperitoneally injected with human PBMCs. Treatment with anti-PD1 antibody alone or Bicki anti-PD1VH-IL7 (5 mg / kg) was performed twice weekly. Sixteen days after injection, blood was collected and mice were sacrificed. (A) The percentage of peripheral human CD3 T cells was analyzed by flow cytometry in the human CD45+ cell population. Each dot represents one mouse. (B) Human IFNg was dosed in plasma by ELISA. Each dot represents one mouse. (C) Human CD3+ cell infiltration was quantified in the colon, liver, and lung by immunohistofluorescence. Proximal and distal colon, liver, and lung were embedded in TissueTek® OCT and stained for Dapi and human CD3. Each dot represents one mouse, and for the colon, each dot represents the average CD3+ count of three sections. [Figure 13]Immunophenotyping of human tumor-infiltrating lymphocytes. T cells were extracted from renal cancer (△) (▽), metastatic colorectal (□), pancreatic cancer (○), and hepatocellular carcinoma (●) (◇) and stained for CD3, CD4, CD8, PD-1, CD127, and CD132. Immunofluorescence was analyzed by FACS LSRII. Data are presented for the CD4+CD3+ or CD8+CD3+ populations. [Figure 14] STAT5 activation in intratumoral regulatory or effector T cells in ex vivo tumors. Cells were extracted from schwannoma (▼), kidney (○), hepatocellular carcinoma (□), (■), metastatic colorectal (●), or pancreatic cancer (▲) patient tumors and treated with rIL-7 or Bicki anti-PD1VH-IL7 (29 nM) for 15 minutes. Cells were then fixed, permeabilized, and stained for pSTAT5 (clone 47 / Stat5(pY694)), CD3, and Foxp3. Histograms represent the mean pSTAT5 fluorescence in the CD3+FoxP3+ population (T regulatory cells) or CD3+FoxP3- (effector T cells). [Figure 15] In vitro study of IFNγ secretion in human cancer biopsies after treatment with Bicki anti-PD1-IL7: Human tumor biopsies were crushed in complete medium to separate the cells. Cells were resuspended in complete medium with 5 μg / mL of isotype control, anti-PD1, B12-IL7 isotype control antibody (isotype-VH IL-7), anti-PD-1 + recombinant IL-7, or anti-Bicki anti-PD1 VH-IL7. After 48 hours, supernatants were collected and IFNγ secretion was analyzed using MSD technology (Mesoscale Discovery). A. Results are shown for colorectal cancer cells; B. Results are shown for individual tumors (CC: colorectal cancer biopsy, HCC: hepatocellular carcinoma biopsy, KC: renal carcinoma). [Figure 16]STAT5 activation in intratumoral regulatory T cells or effector T cells after treatment with Bicki anti-PD1VH-IL7. (A) Percentage of intratumoral FoxP3 Treg cells that integrated into colorectal cancer, schwannoma, renal cancer, or hepatocellular carcinoma tumors. (B) Cells from colorectal cancer (●), schwannoma (○), and pancreatic cancer (□) were analyzed for STAT5 activation in FoxP3-CD3+ effector T cells versus FoxP3-CD3+ Treg cells after treatment (15 min incubation) with rIL7 or anti-PD1VH-IL7 (29 nM). Cells were then fixed, permeabilized, and stained for pSTAT5 (clone 47 / Stat5(pY694)), CD3, and Foxp3. [Figure 17] PD-1 binding ELISA assay of bicki IL-7 mutants. Human recombinant PD-1 (rPD1) protein was immobilized, and antibodies were added at different concentrations. Color development was performed with a peroxidase-conjugated anti-human Fc antibody. Colorimetric analysis was determined at 450 nm using TMB substrate. (A) PD-1 binding of bifunctional molecules containing anti-PD1 antibodies and IL-7 mutated at amino acids D74, Q22, Y12F, M17, Q11, and K81. (B) PD-1 binding of bifunctional molecules containing IL-7 mutated at amino acid W142. (C) PD-1 binding of bifunctional molecules containing mutated disulfide bonds of IL-7 (SS1, SS2, and SS3 mutations). All molecules tested in this figure were constructed with the IgG4m isotype and a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. [Figure 18]CD127-binding ELISA assay of IgG fused to mutated IL-7. PD-1 recombinant protein was immobilized on a plate. Then, bifunctional anti-PD-1 IL-7 molecules pre-incubated with CD127 recombinant protein (histidine tag, Sino reference 10975-H08H) were added to the wells. Color development was performed with a mixture of anti-histidine antibody conjugated to biotin and streptavidin conjugated to peroxidase. Colorimetric analysis was performed at 450 nm using TMB substrate. (A) CD127 binding of bifunctional molecules containing IL-7 mutated at amino acids D74, Q22, M17, Q11, Y12F, and K81. (B) CD127 binding of bifunctional molecules containing IL-7 mutated at amino acid W142. [Figure 19] IL-7-7R signaling pathway of different bifunctional molecules measured by STAT5 phosphorylation. Human PBMCs isolated from peripheral blood of healthy volunteers were incubated with bifunctional anti-PD-1 IL-7 molecules for 15 minutes. Cells were then fixed, permeabilized, and stained with AF647-conjugated anti-pSTAT5 (clone 47 / Stat5(pY694)). Data were obtained by calculating MFI pSTAT5 in CD3 T cells. (A) pSTAT5 activation of anti-PD-1 IL-7 bifunctional molecules containing IL-7 mutated at amino acids D74, Q22, M17, Y12F, Q11, and K81. (B) pSTAT5 activation of anti-PD-1 IL-7 bifunctional molecules containing IL-7 mutated at amino acid W142. (C) pSTAT5 activation of anti-PD-1 IL-7 bifunctional molecules containing IL-7 mutations at the SS2 (black) and SS3 (▲) disulfide bonds compared to anti-PD-1 IL-7 WT (gray). All molecules tested in this figure were constructed with the IgG4m isotype and a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. [Figure 20]Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules in mice. Mice were intravenously injected with a single dose of IgG fused to wild-type or mutant IL-7. Serum concentrations of the molecules were assessed by ELISA at multiple time points after injection. (A) Injection of IgG4-G4S3 IL7 WT (■ gray); IgG4-G4S3 IL7 D74E (● black). (B) Injection of IgG4-G4S3 IL7 WT (■ gray) or IgG4-G4S3 IL7 W142H (● black). (C) Injection of IgG4-G4S3 IL7 WT (■ gray); IgG4-G4S3 IL7 SS2 (●) or IgG4-G4S3 IL7 SS3 (▲). (D) Correlation between the PK of each molecule and the area under the curve (AUC) calculated from the ED50 pSTAT5 (nM). All molecules tested in this figure were constructed with the IgG4m isotype and a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. [Figure 21] The addition of a disulfide bond between anti-PD-1 and IL-7 decreases pSTAT5 activation while increasing drug exposure in vivo. (A) IL7R signaling measured by pSTAT5 activation in human PBMCs after treatment with the anti-PD-1 IL-7 bifunctional molecule WT (gray ●) or an anti-PD-1 IL-7 bifunctional molecule with an additional disulfide bond (black ●). (B) Pharmacokinetics in mice of the anti-PD-1 IL-7 bifunctional molecule WT (gray ●) or an anti-PD-1 IL-7 bifunctional molecule with an additional disulfide bond (black ●). Mice were intravenously injected with a single dose containing the anti-PD-1 IL-7 bifunctional molecule. Serum concentrations of the molecules were assessed by ELISA at multiple time points post-injection. All molecules tested in this figure were constructed with the IgG4m isotype and a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. [Figure 22]PD-1 binding ELISA assay. Human recombinant PD-1 (rPD1) protein was immobilized, and antibodies were added at different concentrations. Color development was performed with a peroxidase-conjugated anti-human Fc antibody. Colorimetric analysis was performed using TMB substrate at 450 nm. (A) PD-1 binding of the anti-PD-1 IL-7 WT bifunctional molecule with IgG4m isotype (gray ●), the anti-PD-1 IL-7 WT bifunctional molecule with IgG1m isotype (black ▲), the anti-PD-1 IL-7 D74E bifunctional molecule with IgG1m isotype (■), or the anti-PD-1 IL-7 W142H bifunctional molecule with IgG1m isotype (◇). (B) In a separate experiment, PD-1 binding of the anti-PD-1 IL-7 SS2 bifunctional molecule with IgG4m isotype (■) or the anti-PD-1 IL-7 SS2 bifunctional molecule with IgG1m isotype (▲) was tested. [Figure 23]CD127-binding ELISA assay of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1N298A or IgG4 isotype. Recombinant proteins targeted by the antibody scaffold were immobilized, and then antibodies fused to IL-7 were pre-incubated with CD127 recombinant protein (histidine tag, Sino reference 10975-H08H). Color development was performed with a mixture of anti-histidine antibody conjugated to biotin and streptavidin conjugated to peroxidase. Colorimetric analysis was performed at 450 nm using TMB substrate. (A) CD127 binding of anti-PD-1 IL-7 W142H bifunctional molecules containing the IgG4m isotype (gray ●), anti-PD-1 IL-7 W142H bifunctional molecules containing the IgG1m isotype (black ▲), or anti-PD-1 IL-7 WT bifunctional molecules containing the IgG1m isotype (black ●). (B) CD127 binding of the anti-PD-1 IL-7 SS2 bifunctional molecule with the IgG4m isotype (● gray), the anti-PD-1 IL-7 SS2 bifunctional molecule with the IgG1m isotype (▲ black), or the anti-PD-1 IL-7 WT bifunctional molecule with the IgG1m isotype (● black). (C) CD127 binding of the anti-PD-1 IL-7 SS3 bifunctional molecule with the IgG4m isotype (● gray), the anti-PD-1 IL-7 SS3 bifunctional molecule with the IgG1m isotype (▲ black), or the anti-PD-1 IL-7 WT bifunctional molecule with the IgG1m isotype (● black). (D) CD127 binding of the anti-PD-1 W142H bifunctional molecule with the IgG1m isotype (● black) or the isotype IgG1m + YTE (● gray). CD127 binding of anti-PD-1 D74E bifunctional molecules containing isotype IgG1m (black) or isotype IgG1m + YTE (gray) was also tested. All molecules tested in this figure were constructed with a GGGGSGGGSGGGGGS linker between the Fc and IL-7 domains. [Figure 24]IL-7R signaling analysis of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1N298A or IgG4 isotype. Human PBMCs or Jurkat PD1+CD127+ cells were incubated with the anti-PD-1 IL-7 bifunctional molecule for 15 minutes. Cells were then fixed, permeabilized, and stained with AF647-conjugated anti-pSTAT5 (clone 47 / Stat5(pY694)). Data were obtained by calculating the percentage of pSTAT5 in CD3 T cells. (A) pSTAT5 signaling on human PBMCs after treatment with the bifunctional molecule anti-PD-1 IL-7 with the D74E mutation containing the IgG4m isotype (●gray) or the IgG1m isotype (▲black). (B) pSTAT5 signaling on human PBMCs after treatment with anti-PD-1 IL-7 SS2 containing the IgG4m isotype (●gray) or anti-PD-1 IL-7 SS2 containing the IgG1m isotype (▲black). (C) pSTAT5 signaling in human PBMCs after treatment with anti-PD-1 IL-7 SS3 (● gray) containing IgG4m isotype or IgG1m (▲ black). (D) (Left panel) pSTAT5 signaling in Jurkat PD1+CD127+ cells after treatment with anti-PD-1 IL-7 WT constructed with IgG4m (● gray) or IgG1m (▲ black) isotype. (Right panel) pSTAT5 signaling after treatment with anti-PD-1 IL-7 SS2 (● gray) containing IgG4m isotype or anti-PD-1 IL-7 SS2 (▲ black) containing IgG1m. [Figure 25]Anti-PD-1 IL-7 mutant bifunctional molecules enhance T cell activation in vitro. Promega PD-1 / PD-L1 bioassay: (1) effector T cells (Jurkat mice stably expressing PD-1 and NFAT-inducible luciferase) and (2) activated target cells (CHO K1 cells stably expressing PDL1 and a surface protein designed to activate the cognate TCR in an antigen-independent manner) were cocultured. After addition of BioGlo™ luciferin, luminescence was quantified and represents T cell activation. Serial molar concentrations of anti-PD1 antibodies + / - recombinant IL-7 (rIL-7) or anti-PD1 IL-7 bifunctional molecules were tested. Each dot represents the EC50 of a single experiment. (A) NFAT activation of anti-PD-1 IL-7 WT bifunctional molecules with IgG4m isotype (gray circles), anti-PD-1 (▲), or anti-PD-1 + rIL-7 (○). (B) NFAT activation by anti-PD-1 IL-7 D74E IgG4m (●), PD-1 IL-7 D74E IgG1m (▲ dotted line), and anti-PD-1 alone (black ▲). (C) NFAT activation by anti-PD-1 IL-7 W142H bifunctional molecule with IgG4m (●), PD-1 IL-7 W142H bifunctional molecule with IgG1m (▲ dotted line), and anti-PD-1 alone (black ▲). (D) NFAT activation by anti-PD-1 IL-7 SS2 bifunctional molecule with IgG4m (●) and anti-PD-1 alone (black ▲). [Figure 26]Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1m or IgG4m isotypes. Mice were intravenously injected with a single dose containing IgG fused to wild-type or mutant IL-7. Serum drug concentrations were assessed by ELISA at multiple time points post-injection. (A) Pharmacokinetics of the anti-PD-1 IL-7 WT bifunctional molecule with IgG4m (● gray flat line), anti-PD-1 IL-7 WT bifunctional molecule with IgG1m (● gray dashed line), anti-PD-1 IL-7 D74E bifunctional molecule with IgG1m (▲ black dashed line), anti-PD-1 IL-7 W142H bifunctional molecule with IgG4m (○ black flat line), anti-PD-1 IL-7 W142H bifunctional molecule with IgG1m (○ dashed black flat line), anti-PD-1 IL-7 SS3 bifunctional molecule with IgG4 (■ flat line), and anti-PD-1 IL-7 SS3 bifunctional molecule with IgG1m (■ dashed line). (B) Pharmacokinetics of the anti-PD-1 IL-7 D74E, D74Q, W142H, and D74E+W142H mutant bifunctional molecules with IgG1m. [Figure 27] Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules constructed with the IgG1 N298A+K444A isotype. Mice were intravenously injected with a single dose of anti-PD-1 IL-7 D74E bifunctional molecules containing the IgG1 N298A isotype (■) or the IgG1 m+K444A mutant isotype (●). Antibody concentrations were assessed by ELISA at multiple time points post-injection. [Figure 28]Linker length does not significantly affect pharmacokinetics but does decrease stimulation of IL-7R signaling. (A) Pharmacokinetics of anti-PD-1 IL-7 WT bifunctional molecules constructed with different linkers (GGGGS), (GGGGS)2, and (GGGGS)3). (B) Pharmacokinetics of anti-PD-1 IL-7 D74 bifunctional molecules constructed with different linkers (GGGGS), (GGGGS)2, and (GGGGS)3). (C) Pharmacokinetics of anti-PD-1 IL-7 W142H bifunctional molecules constructed with different linkers ((GGGGS)2 and (GGGGS)3). Mice were intravenously injected with a single dose containing IgG fused to wild-type or mutant IL-7. The concentration of IgG fused to IL-7 was assessed by ELISA at multiple time points post-injection. (D) pSTAT5 signaling of anti-PD-1 IL-7 bifunctional molecules constructed with no linker or GGGGS, (GGGGS)2, and (GGGGS)3 linkers. [Figure 29] The anti-PD-1 IL-7 variant selectively targets PD-1+CD127+ cells over PD-1-CD127+ cells. Jurkat cells expressing CD127+ or co-expressing CD127+ and PD-1+ were stained with 45 nM anti-PD-1 IL-7 bifunctional molecule and revealed with anti-IgG-PE (Biolegend, clone HP6017). Data represent the ratio of the mean fluorescence obtained on PD-1+CD127+ Jurkat cells to the mean fluorescence obtained on PD1-CD127+ Jurkat cells. The following bifunctional molecules were tested in this assay: anti-PD-1 IL-7 WT bifunctional molecule IgG1m, anti-PD-1 IL-7 D74E bifunctional molecule IgG1m, anti-PD-1 IL-7 W142H bifunctional molecule IgG1m, anti-PD-1 IL-7 SS2 bifunctional molecule IgG4m, and anti-PD-1 IL-7 SS3 bifunctional molecule IgG1m. [Figure 30-1]Anti-PD-1 IL-7 molecules enhance T cell proliferation and demonstrate preclinical safety in cynomolgus monkeys. They target PD-1+CD127+ cells over PD-1-CD127+ cells. Cynomolgus monkeys were intravenously injected with a single dose of Bicki anti-PD-1 IL-7WT (6.87 nM / kg (n=2)) or 34.35 nM (n=1)). Blood analysis was performed up to 15 days or 4 hours after injection. (A) Lymphocyte counts were assessed in peripheral blood at multiple time points following injection of Bicki anti-PD-1 IL-7WT at 6.87 nM / kg (n=2). (B) CD4 / CD8 or B cell proliferation was assessed by flow cytometry using Ki67 / CD4 / CD8 and CD19 markers after injection of Bicki anti-PD-1 IL-7WT at 6.87 nM / kg (n=2). (C) pSTAT5 expression in CD3+ T cells was analyzed by FACS after injection of Bicki anti-PD-1 IL-7WT at 6.87 nM / kg (n=2) at multiple time points. (D / E / F / G / H) Biochemistry and cellular hematology analyses were assessed at multiple time points. [Figure 30-2] See Figure 30-1. [Figure 30-3] See Figure 30-1. [Figure 30-4] See Figure 30-1. [Figure 30-5] See Figure 30-1. [Figure 30-6] See Figure 30-1. [Figure 31] Description of the mechanism of action of Bicki anti-PD1-IL-7 according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0045] Introduction The antibodies of the present invention are bifunctional because they combine a specific anti-PD-1 effect with the effect of human interleukin-7 fused to the anti-PD-1 antibody. Indeed, the present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody and IL-7, wherein the interleukin is covalently linked to a polypeptide chain of the anti-PD-1 antibody, either to the light chain or the heavy chain, or both, or fragments thereof, of the antibody. The anti-PD-1 antibody or fragment thereof chain and IL-7 are prepared as a fusion protein. In this particular embodiment, the N-terminus of the IL-7 is linked to the C-terminus of the anti-PD-1 antibody or fragment thereof chain, optionally via a peptide linker.

[0046] As known to those skilled in the art, tumor cells may not be sufficiently 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 linked to overexpression of inhibitory receptors, reduced effector cytokine production, and reduced cytolytic activity, leading to failure of cancer elimination and, in general, immune evasion of cancer. Therefore, restoring exhausted T cells is an emerging clinical strategy for cancer treatment.

[0047] PD-1 is a major inhibitory receptor that regulates T cell exhaustion. In fact, T cells with high PD-1 expression exhibit a reduced ability to eliminate cancer cells. Anti-PD1 therapeutic compounds, particularly anti-PD1 antibodies, are clinically used in cancer treatment to block the inhibitory effect of PD1-PDL1 interaction (PD1 on T cells and PDL1 on tumor cells) and T cell exhaustion. However, anti-PD1 antibodies are not always efficient enough to enable "reactivation" of exhausted T cells.

[0048] Applicants demonstrate herein that bifunctional anti-PD1-IL-7 molecules according to the present invention enhance the activation (NFAT-mediated activation) of T cells, particularly exhausted T cells, compared to anti-PD-1 alone. In particular, anti-PD1-IL-7 bifunctional molecules induce the proliferation and activation of naive, partially exhausted, and fully exhausted T cell subsets, as reflected by cytokine (e.g., IFNγ) secretion. Such anti-PD1-IL-7 bifunctional molecules have the potential to overcome associated resistance mechanisms and improve the efficacy of anti-PD-1 immunotherapy.

[0049] In particular, the applicant shows that the interaction of an anti-PD1-IL-7 bifunctional molecule with a single T cell expressing i) PD1 and ii) IL-7 receptors leads to the unexpected activation of the NFAT pathway (TCR signaling), which has a positive effect on T cell activation, especially on exhausted T cells, and enhances the ability of T cells to eliminate neoplastic cells.

[0050] This means that, on the one hand, the IL-7 in the bifunctional molecule of the present invention targets the IL-7 receptor and activates the PSTAT5 pathway, and, on the other hand, the anti-PD1 portion of the bifunctional molecule blocks PD-1 / PD-L1 interaction. The BICKI molecule targets both IL-7 and PD-1 on the same cells. This results in synergistic activation of TCR (NFAT) signaling, which is never observed when the combination of anti-PD1 antibody and IL-7 is used separately (as two separate compounds). This activation cannot be provided by a bifunctional molecule targeting PD-L1. In fact, it is known in the art that PD-L1 is expressed on tumor cells but not on immune cells such as T cells.

[0051] In addition, bifunctional anti-PD1 / IL-7 molecules allow the accumulation of IL-7 in PD-1+ T cell infiltrates and the relocalization of IL-7 on PD-1+ T cells. This accumulation of IL-7 near PD-1+ T cells is particularly interesting in the context of exhausted T cells, which require high doses of IL-7 to activate or reactivate these T cells.

[0052] A synergistic effect on T cell activation has been observed not only with the specific anti-PD-1 antibodies of the present invention, but also with two other reference anti-PD-1 antibodies, namely Opdivo and Keytruda.

[0053] In addition, bifunctional anti-PD1 / IL-7 molecules have the ability to promote T-cell infiltration into tumors, which is advantageous for optimizing anti-PD1 antibody therapy, considering that currently, lack of T-cell infiltration at tumor sites is a major obstacle to the efficacy of anti-PD1 antibody therapy.

[0054] Furthermore, the bifunctional anti-PD1 / IL-7 molecule blocks Treg-mediated inhibitory effects. Thus, the bifunctional molecule can specifically activate T effector cells but not Treg cells, whereas anti-PD1 antibodies cannot inhibit Treg suppressive activity on T effector cells. Thus, the inventors have demonstrated that the bifunctional anti-PD1-IL7 molecule favors T cell effectors over the T regulatory immune balance by stimulating the proliferation and survival of effector T cells while sparing regulatory T cells.

[0055] In addition, the IL7 anti-PD-1 bifunctional molecule has other advantages.

[0056] Furthermore, we demonstrate that the IL7 anti-PD-1 bifunctional molecule activates T effector cells (Teff) preferentially over T regulatory cells (Treg). The IL7 anti-PD-1 bifunctional molecule has the advantages of not promoting Treg proliferation, inducing Treg inactivation, and inducing activation of T cells, especially exhausted T cells.

[0057] IL7-anti-PD-1 bifunctional molecules allow for very favorable dosing and exhibit a favorable therapeutic index (ratio of lethal dose (DL50) to therapeutically effective dose). In particular, IL7 can be administered to patients typically in the range of 10 to 1500 μg / kg, preferably 200 to 1200 μg / kg, and even high doses, such as 1200 μg / kg, are well tolerated by patients. Therefore, IL7-anti-PD-1 bifunctional molecules enable the production of therapeutic compounds with appropriate dosages of both the component compounds and the final product. In fact, a well-tolerated high dose of IL7 (e.g., approximately 1.2 mg / kg for IL7) corresponds to approximately 2 mg / kg of the antibody, which is sufficient for administration to patients.

[0058] The IL7 anti-PD-1 bifunctional molecule has the advantage of being able to essentially target only partially exhausted exhausted T cell precursors, which are important targets for meeting the medical needs mentioned above. In addition, in the case of viral infections, which are also associated with T cell exhaustion, chronic activation is important, and therefore viral pathologies are within the scope of pathologies targeted by the new products of the present application.

[0059] Finally, in certain embodiments, the inventors have designed bifunctional molecules comprising IL-7 mutants or variants. The IL-7 mutants or variants are characterized by i) reduced affinity for the IL-7 receptor (IL-7R) compared to that of wild-type IL-7, and ii) improved pharmacokinetics of the bifunctional molecules comprising the IL-7 variant compared to bifunctional molecules comprising wild-type IL-7. First, the use of IL-7 variants in bifunctional molecules is important for increasing the in vivo pharmacokinetics of the bifunctional molecules. Second, reducing the affinity of the IL-7 variant for its receptor not only increases the ability of the bifunctional molecules to selectively bind to target T cells via the anti-PD-1 antibody portion of the bifunctional molecule and provide specific effects on those cells, but also enhances the ability to exploit synergistic effects associated with the action of the two portions of the bifunctional molecule on the same T cells. Bifunctional molecules comprising IL-7 variants have good PD-1 binding and antagonist activity. In addition, the bifunctional molecule provides a favorable balance between its affinity for PD-1 and its affinity for IL-7R. Surprisingly, the inventors observed that bifunctional molecules with an IgG1 heavy chain constant domain exhibit improved activity (pStat5 signaling, synergy, and CD127 binding) of IL-7 variants compared to the same molecules with an IgG4 heavy chain constant domain. In addition, the use of a linker (GGGGS)3 between the antibody and IL-7 maximizes the activity (pStat5 signaling and CD127 binding) of the IL-7 variants.

[0060] The bifunctional molecules of the present invention have, in particular, one or several of the following advantages:

[0061] - The bifunctional molecules induce proliferation of naive, partially exhausted, and fully exhausted T cell subsets, but not exclusively partially exhausted T cells, as occurs with anti-PD1 / PDL1 therapy. More specifically, the bifunctional molecules have a synergistic effect on T cell activation. The bifunctional molecule specifically localizes IL-7 near or on exhausted PD-1+ T cells that have entered tumors, enabling targeting of cells that require higher concentrations of IL-7. The bifunctional molecule specifically induces the accumulation of IL-7 in PD-1+ T cell infiltrates and the relocalization of IL-7 on PD-1+ T cells. - Anti-PD-1 blockade fails to reprogram exhausted T cells into active memory T cells, limiting long-term tumor clearance, whereas bifunctional molecules promote the formation, survival, and proliferation of memory T cells through the presence of IL-7. Thus, bifunctional molecules induce sustained antitumor immunity through sustained and expanded memory T cell responses. - Anti-PD1 / PD-L1 therapeutic efficacy is associated with pre-existing T cell infiltrates and T cell effector functions, particularly the IFNγ signature, whereas bifunctional molecules synergistically increase the proliferation of effector T cells and their ability to secrete IFNγ. The bifunctional molecules can reduce the immunosuppressive microenvironment by reducing the Treg population and inhibiting the secretion of TGFβ (an inhibitory cytokine). More specifically, the bifunctional molecules specifically stimulate effector T cells without stimulating Tregs. In bifunctional molecules, IL-7 may be fused to the C-terminal portion of the heavy and / or light chain, preserving the high affinity of IL-7 for CD127 as in naked / native IL-7. The bifunctional molecules may be more potent in terms of IL-7R activation and half-life. - The bifunctional molecule is produced as a bifunctional molecule with high production yield. The bifunctional molecules reduce the immunosuppressive activity of Treg cells that have entered the tumor microenvironment by reducing the number of Tregs. More specifically, the bifunctional molecules specifically stimulate effector T cells without stimulating Tregs. - The bifunctional compounds increase the expression of integrins (i.e., alpha4 and / or beta7 and LFAT) and promote T cell infiltration into tissues and / or tumors compared to anti-PD1 responses alone. In particular, the bifunctional compounds promote T cell migration and tumor infiltration. The bifunctional molecule may comprise an IL-7 variant or mutant as identified by the inventors to maximize in vivo pharmacokinetics while maintaining a favorable affinity balance between IL-7 and IL-7R and anti-PD-1 and PD-1, and maintaining IL-7 activity and the antagonist activity of the anti-PD-1 antibody.

[0062] definition In order that the present invention may be more readily understood, certain terms are defined herein below. Additional definitions are set forth throughout the detailed description.

[0063] Unless otherwise defined, all technical terms, notation, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some cases, for clarity and / or ease of reference, terms having commonly understood meanings are also defined herein. The inclusion of such definitions herein should not be construed as necessarily representing a meaning different from that commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using conventional methodology.

[0064] As used herein, the terms "interleukin-7," "IL-7," and "IL-7" refer to a mammalian endogenously secreted glycoprotein, particularly an IL-7 polypeptide, derivatives, and analogs thereof, that have substantial amino acid sequence identity to wild-type mammalian IL-7 and have substantially equivalent biological activity, e.g., in a standard bioassay or assay for IL-7 receptor binding affinity. For example, IL-7 refers to the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of: i) a native or naturally occurring allelic variant of an 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) a biologically active variant of an IL-7 polypeptide. IL-7 may include or lack its peptide signal. Alternative names for this molecule are "pre-B cell growth factor" and "lymphopoietin-1." Preferably, the term "IL-7" refers to human IL-7. For example, the human IL-7 amino acid sequence is approximately 152 amino acids (without the signal peptide), has Genbank accession number NP_000871.1, and the gene is located on chromosome 8q12-13. Human IL-7 is described in UniProtKB-P13232.

[0065] As used herein, the terms "wild-type interleukin-7," "wt-IL-7," and "wt-IL7" refer to a mammalian endogenously secreted glycoprotein, particularly an IL-7 polypeptide, derivatives, and analogs thereof, that have substantial amino acid sequence identity with wild-type functional mammalian IL-7 and have substantially equivalent biological activity, e.g., in a standard bioassay or assay for IL-7 receptor binding affinity. For example, wt-IL-7 refers to the amino acid sequence of i) a native 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) a recombinant or non-recombinant polypeptide having the amino acid sequence of a biologically active IL-7 polypeptide. IL-7wt may include or lack its peptide signal. Alternative names for this molecule are "pre-B cell growth factor" and "lymphopoietin-1." Preferably, the term "wt-IL-7" refers to human IL-7 (wth-IL7). For example, the human wt-IL-7 amino acid sequence is approximately 152 amino acids (without the signal peptide), has Genbank accession number NP_000871.1, and the gene is located on chromosome 8q12-13. Human IL-7 is described, for example, in UniProtKB-P13232.

[0066] 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 "hPD-1" are used interchangeably and refer to the programmed death-1 receptor, also known as CD279, including variants and isoforms of human PD-1 and analogs that share at least one epitope with PD-1. PD-1 is a key regulator of immune responses and the threshold of peripheral immune tolerance. PD-1 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 in GenBank accession number NP_005009. Four splice variants of PD1 are expressed on human peripheral blood mononuclear cells (PBMCs). Thus, PD-1 proteins include 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, these terms include any variants and isoforms of human PD-1 naturally expressed by PBMCs or expressed by cells transfected with the PD-1 gene.

[0067] As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Unless specifically indicated otherwise, the term "antibody" includes intact immunoglobulins and any other modified configurations of immunoglobulin molecules that contain an antigen recognition site of the required specificity, including "antibody fragments" or "antigen-binding fragments" (Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), variants thereof, molecules containing antibody portions, diabodies, linear antibodies, single chain antibodies, and glycosylation variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term antibody refers to humanized antibodies.

[0068] As used herein, an "antigen-binding fragment" of an antibody refers to a molecule corresponding to a portion of the antibody's structure, presumably in its native form, that exhibits antigen-binding ability for PD-1. In particular, such fragments exhibit the same or substantially the same antigen-binding specificity for the antigen as that of the corresponding four-chain antibody. Advantageously, the antigen-binding fragment has a similar binding affinity to that of the corresponding four-chain antibody. However, antigen-binding fragments with reduced antigen-binding affinity compared to the corresponding four-chain antibody are also encompassed within the present invention. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. Such antigen-binding fragments can also be referred to as "functional fragments" of antibodies. An antigen-binding fragment of an antibody is a fragment that includes the recognition site of the antigen, i.e., the extracellular domain of PD1, and thus includes the hypervariable domains, or portions thereof, called CDRs (complementarity-determining regions), that define the antigen recognition specificity.

[0069] A "Fab" fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 fragments are produced by cleavage of the disulfide bond between the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, are cleared more rapidly from the animal's circulation, and may have less nonspecific tissue binding than intact antibodies (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0070] An "Fv" fragment is the minimum fragment of an antibody which contains a complete target recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. In many cases, the six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can have the ability to recognize and bind to a target, albeit with lower affinity than the entire binding site.

[0071] "Single-chain Fv" or "scFv" antibody-binding fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for target binding.

[0072] A "single domain antibody" is composed of a single VH or VL domain that exhibits sufficient affinity for PD-1. In a specific embodiment, a single domain antibody is a camelized antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0073] In terms of structure, an antibody may have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). Each heavy and light chain contains a constant region and a variable region (or "domain"). The light and heavy chain variable regions contain a "framework" region separated by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs has been defined (see Kabat et al., "Sequences of Proteins of Immunological Interest," and US Department of Health and Human Services, 1991, which is incorporated herein by reference). Preferably, the CDRs are defined by the Kabat method. The framework regions act to form a scaffold that provides the CDRs with the correct orientation through interchain non-covalent interactions. The CDRs are primarily responsible for binding to an antigen epitope. The CDRs of each chain are typically referred to as "complementarity-determining region 1" or "CDR1," "CDR2," and "CDR3," numbered sequentially from the N-terminus. The VL and VH domains of antibodies according to the invention may comprise four framework regions or "FRs," which are referred to in the art and herein as "framework region 1" or "FR1," "FR2," "FR3," and "FR4," respectively. These framework regions and complementarity-determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (amino-terminus to carboxy-terminus). The term "antibody framework," as used herein, refers to a portion of either the VL and / or VH variable domain that serves as a scaffold for the antigen-binding loops (CDRs) of that variable domain.

[0074] "Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in an antibody conformation. The CDRs of an antibody heavy chain are typically referred to as "HCDR1," "HCDR2," and "HCDR3." The framework regions of an antibody heavy chain are typically referred to as "HFR1," "HFR2," "HFR3," and "HFR4."

[0075] "Antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in an antibody conformation. Kappa light chain and lambda light chain refer to the two major antibody light chain isotypes. The CDRs of an antibody light chain are typically referred to as "LCDR1," "LCDR2," and "LCDR3." The framework regions of an antibody light chain are typically referred to as "LFR1," "LFR2," "LFR3," and "LFR4."

[0076] With respect to antibody binding to a target molecule, the term "bind" or "binding" refers to peptides, polypeptides, proteins, fusion proteins, molecules, and antibodies (including antibody fragments) that recognize and contact the antigen. Preferably, the term refers to antigen-antibody type interactions. The terms "specific binding," "specifically binds," "specific for," "selectively binds to," and "selective for," with respect to a particular antigen (e.g., PD-1) or an epitope on a particular antigen (e.g., PD-1), mean that the antibody recognizes and binds to the specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically (or preferentially) binds to PD-1 or a PD-1 epitope is an antibody that binds to this PD-1 epitope, e.g., with higher affinity, avidity, more readily, and / or for a longer period of time, than it binds to other PD-1 epitopes or non-PD-1 epitopes. Preferably, the term "specific binding" refers to a specific binding activity of 10 -7 In certain embodiments, the antibody has a binding affinity of 10 or less than 10 M. -8 M, 10 -9 M or 10 -10binds with an affinity equal to or lower than M.

[0077] As used herein, the terms "PD-1 antibody," "anti-PD-1 antibody," "PD-1 Ab," "PD-1-specific antibody," and "anti-PD-1 Ab" are used interchangeably and refer to an antibody, as described herein, that specifically binds to PD-1, particularly human PD-1. In some embodiments, the antibody binds to the extracellular domain of PD-1. In particular, an anti-PD-1 antibody is an antibody capable of binding to the PD-1 antigen and inhibiting the PD-1-mediated signaling pathway, thereby enhancing an immune response, such as T cell activation.

[0078] As used herein, the terms "bifunctional molecule," "bifunctional compound," "bifunctional protein," "Bicki," "Bicki antibody," "bifunctional antibody," and "bifunctional checkpoint inhibitor molecule" have the same meaning and can be used interchangeably. These terms refer to an antibody that recognizes one antigen by having at least one region specific for that antigen (e.g., derived from the variable region of the antibody) and at least a second region that is a polypeptide. More specifically, a bifunctional molecule is a fusion protein of an antibody or portion thereof, preferably an antigen-binding fragment thereof, with another polypeptide or polypeptide fragment thereof.

[0079] The term "chimeric antibody," as used herein, refers to an antibody or antigen-binding fragment in which a portion of the heavy and / or light chain is derived from one species and the remainder of the heavy and / or light chain is derived from a different species. In an illustrative example, a chimeric antibody may contain a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0080] The term "humanized antibody," as used herein, is intended to refer to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., a chimeric antibody containing minimal sequence derived from a non-human antibody). A "humanized antibody," e.g., a non-human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs have been replaced by residues from at least one CDR of a non-human antibody (donor antibody), while maintaining the desired specificity, affinity, and capacity of the original antibody. The donor antibody may be any suitable non-human antibody, such as a mouse antibody, rat antibody, rabbit antibody, chicken antibody, or non-human primate antibody, that has the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody have been replaced by framework region residues from the donor antibody. Alternatively, selected framework region residues of the donor antibody have been replaced by framework region residues from a human antibody or humanized antibody. Additional framework region modifications may be made within the human framework sequences. Thus, humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. Such amino acid modifications can be made to further refine antibody function and / or enhance the humanization process. "Amino acid change" or "amino acid modification," as used herein, refers to a change in the amino acid sequence of a polypeptide. "Amino acid modification" includes substitutions, insertions, and / or deletions in a polypeptide sequence. "Amino acid substitution" or "substitution," as used herein, refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. "Amino acid insertion" or "insertion" refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. "Amino acid deletion" or "deletion" refers to the removal of an amino acid at a particular position in a 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") with similar chemical properties (e.g., charge, bulk, and / or hydrophobicity).As used herein, "amino acid position" or "amino acid position number" are used interchangeably and refer to the position of a particular amino acid in an amino acid sequence, generally designated by the single-letter code for the amino acid. The first amino acid in an amino acid sequence (i.e., starting from the N-terminus) shall be considered to be position 1.

[0081] 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 change the functional properties of a protein. Conservative substitutions and corresponding rules are well described in the state of the art. For example, conservative substitutions can be defined by substitutions within the group of amino acids reflected in the table below.

[0082] [Table 1]

[0083] [Table 2]

[0084] [Table 3]

[0085] As used herein, an "isolated antibody" is an antibody that has been separated and / or recovered from a component of its natural environment. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. In some embodiments, the antibody is purified to homogeneity and / or to greater than 90%, 95%, or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC) under reducing or non-reducing conditions.

[0086] The terms "derive from" and "derived from," as used herein, refer to a compound that has a structure that is derived from the structure of a parent compound or protein, and that is sufficiently similar in structure to that disclosed herein that one of skill in the art would expect, based on that similarity, to exhibit the same or similar properties, activity, and utility as the claimed compound. For example, a humanized antibody derived from a murine antibody refers to an antibody or antibody fragment that shares similar properties as the murine antibody, e.g., recognizes the same epitope, and shares a similar VH and VL with modified residues that contribute to and / or enhance the humanization of the antibody.

[0087] The term "treatment" refers to any action intended to improve the well-being of a patient, such as the cure, prevention, prophylaxis, and slowing of a disease or disease symptoms. The term refers to both curative and / or prophylactic treatment of a disease. Curative treatment is defined as treatment that results in a cure or treatment that alleviates, improves, and / or eliminates, reduces, and / or stabilizes a disease or disease symptoms or the suffering it causes directly or indirectly. Prophylactic treatment includes both treatment that results in the prevention of a disease and treatment that reduces and / or delays the progression and / or onset of a disease or the risk of its occurrence. In certain embodiments, such terms refer to the improvement or eradication of a disease, disorder, infection, or symptoms associated therewith. In other embodiments, the term refers to minimizing the spread or worsening of cancer. Treatment according to the present invention does not necessarily imply a 100% or complete cure. Rather, there are various degrees of treatment that one of skill in the art will recognize as having potential benefit or therapeutic effect. Preferably, the term "treatment" refers to the application or administration of a composition comprising one or more active substances to a subject having, for example, a disorder / disease associated with the PD-1-mediated signaling pathway.

[0088] As used herein, the term "disorder" or "disease" refers to the improper functioning of an organ, part, structure, or system of the body due to genetic or developmental error, infection, toxin, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, the term refers to a health impairment or disease, e.g., a disease that interferes with normal physical or mental function. More preferably, the term disorder refers to an immune and / or inflammatory disease that affects animals and / or humans, such as cancer.

[0089] The term "immune disease," as used herein, refers to a condition in a subject characterized by cell, tissue, and / or organ injury caused by a subject's immunological response against the subject's own cells, tissues, and / or organs. The term "inflammatory disease" refers to a condition in a subject characterized by inflammation, e.g., chronic inflammation. Autoimmune disorders may or may not be associated with inflammation. Furthermore, inflammation may or may not be caused by an autoimmune disorder.

[0090] The term "cancer," as used herein, is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0091] As used herein, the terms "PD-1 associated or related disease," "PD-1 positive cancer," or "PD-1 positive infectious disease" are intended to refer to a cancer or infectious disease (e.g., caused by a virus and / or bacteria) that results from PD-1 expression or has symptoms / characteristics of PD-1 expression, i.e., any condition caused, exacerbated, or otherwise linked to increased or decreased PD-1 expression or activity.

[0092] As used herein, the terms "subject," "host," "individual," or "patient" refer to humans, including adults and children.

[0093] As used herein, a "pharmaceutical composition" refers to one or more preparations of an active substance, such as one comprising a bifunctional molecule according to the present invention, together with optional other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active substance to an organism. The compositions of the present invention may be in a form suitable for any conventional route of administration or use. In one embodiment, a "composition" typically refers to a combination of an active substance, e.g., a compound or composition, with an inert (e.g., detectable substance or label) or active naturally occurring or non-naturally occurring carrier, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, or adjuvant, including a pharmaceutically acceptable carrier. An "acceptable vehicle" or "acceptable carrier," as referred to herein, refers to any known compound or combination of compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0094] "Effective amount" or "therapeutically effective amount," as used herein, refers to the amount of an active agent, either alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject, e.g., the amount of active agent required to treat a target disease or disorder or to produce a desired effect. An "effective amount" will vary depending on the agent, the disease and its severity, characteristics of the subject being treated, including age, physical condition, size, sex, and weight, the duration of treatment, the nature of concurrent therapy (if applicable), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. Such factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. In general, it is preferable to use the maximum dose of the individual components or combinations thereof, i.e., the highest safe dose according to sound medical judgment.

[0095] As used herein, the term "medicine" refers to any substance or composition that has curative or preventative properties for a disorder or disease.

[0096] The term "in combination," as used herein, refers to the use of more than one therapies (e.g., prophylactic and / or therapeutic agents). The use of the term "in combination" does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject with a disease or disorder.

[0097] The terms "polynucleotide," "nucleic acid," and "nucleic acid sequence" are equivalent and refer to a polymeric form of nucleotides of any length, e.g., RNA or DNA or analogs thereof. Nucleic acids (e.g., components or portions of nucleic acids) of the invention can be naturally occurring, modified, or genetically engineered, isolated, and / or non-naturally occurring. Genetically engineered nucleic acids include recombinant and synthetic nucleic acids.

[0098] An "isolated nucleic acid encoding an anti-PD1 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell. As used herein, the terms "nucleic acid construct," "plasmid," and "vector" are equivalent and refer to a nucleic acid molecule that serves to transfer a passenger nucleic acid sequence, such as DNA or RNA, into a host cell.

[0099] As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be or has been a recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antibody constructs of the present invention and / or the antibody construct itself. Introduction of the respective substances into a cell can be accomplished by transformation, transfection, and the like. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Host cells include, for example, bacterial cells, microbial cells, plant cells, and animal cells.

[0100] "Immune cells," as used herein, refer to cells involved in innate and adaptive immunity, such as, for example, white blood cells (leukocytes) 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 (NKT)), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). In particular, immune cells can be selected in a non-exhaustive list including B cells, T cells, particularly CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells, and monocytes. "T cells," as used herein, include, for example, CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and inhibitory T cells.

[0101] As used herein, the terms "T effector cells," "Teff," or "effector cells" describe a group of immune cells, including several T cell types, that actively respond to stimuli, such as costimulation. This term specifically includes 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 specifically includes CD4+ cells, CD8+ cells, Treg cells, cytotoxic T cells, and helper T cells (Th1 and Th2).

[0102] As used herein, the terms "regulatory T cells," "Treg cells," or "Treg" refer to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. 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 believed to be derived from the same lineage as naive CD4 cells.

[0103] The term "exhausted T cells" refers to a population of T cells that are in a state of dysfunction (i.e., "exhaustion"). T cell exhaustion is characterized by a progressive loss of function, an altered transcriptional profile, and persistent expression of inhibitory receptors. Exhausted T cells lose their ability to produce cytokines, proliferate, and cause cytotoxicity, ultimately leading to their elimination. Exhausted T cells typically display higher levels of CD43, CD69, and inhibitory receptors, along with lower expression of CD62L and CD127.

[0104] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver that result in the selective damage, destruction, or elimination from the human body of invading pathogens, pathogen-infected cells or tissues, cancerous cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0105] The term "antagonist," as used herein, refers to a substance that blocks or reduces the activity or functionality of another substance. In particular, this term refers to an antibody that binds to a cellular 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., generation of an immunosuppressive microenvironment). The antagonist activity of an antibody according to the present invention can be assessed by competitive ELISA.

[0106] As used herein, the term "isolated" indicates that a described material (e.g., antibody, polypeptide, nucleic acid, etc.) has been substantially separated from or enriched relative to other materials that naturally accompany it. In particular, an "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its natural environment. For example, an isolated antibody is one that has been purified to (1) greater than 75% by weight of the antibody as determined by the Lowry method, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions. Isolated antibody includes antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0107] The term "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually indicated.

[0108] The terms "a" or "an" can refer to one or more of the element that it modifies (e.g., "a reagent" can mean one or more reagents), unless the context makes it clear that either one of the elements or more than one of the elements is being described.

[0109] The term "about," when used herein in connection with any and all values ​​(including the lower and upper limits of a numerical range), means any value with an acceptable range of 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%). Use of the word "about" at the beginning of a value string modifies each of the values ​​(i.e., "about 1, 2, and 3" refers to about 1, about 2, and about 3). Additionally, when a list of values ​​is set forth herein (e.g., approximately 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate and fractional values ​​thereof (e.g., 54%, 85.4%).

[0110] Anti-PD-1 antibody The bifunctional molecule according to the present invention comprises a first entity comprising an anti-hPD-1 antibody or an antigen-binding fragment thereof.

[0111] Specifically provided herein are antibodies that bind to human PD-1. In some aspects, the antibodies specifically bind to human PD-1, preferably to the extracellular domain of human PD-1. In some aspects, the antibodies selectively bind to one or more of full-length human PD-1, PD-1Aex2, PD-1Aex3, PD-1Aex2,3, and PD-1Aex2,3,4.

[0112] In some aspects, the anti-PD1 antibody is an isolated antibody, particularly a non-naturally occurring isolated antibody. Such isolated anti-PD1 antibodies can be prepared by at least one purification step. In some embodiments, the isolated anti-PD1 antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the isolated anti-PD1 antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% by weight of the antibody, with the remaining weight comprising other solutes dissolved in a solvent.

[0113] Preferably, such antibodies are capable of blocking or inhibiting the interaction of PD-1 with at least one of its ligands (e.g., PD-L1 and / or PD-L2). The ability to "block binding" or "block interaction" or "inhibit interaction," as used herein, refers to the ability of an antibody or antigen-binding fragment to prevent, to any detectable extent, the binding interaction between two molecules (e.g., PD-1 and its ligands PD-L1 and / or PD-L2).

[0114] Preferably, the anti-PD1 antibody or antigen-binding fragment thereof is an antagonist of the binding of human PD-L1 and / or PD-L2 to human PD-1, more preferably of the binding of human PD-L1 and PD-L2 to human PD-1.

[0115] In certain embodiments, the anti-hPD1 antibody or antigen-binding fragment inhibits the binding interaction of PD-1 with at least one of its ligands (e.g., PD-L1 and / or PD-L2, preferably PD-L1 and PD-L2) by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0116] Anti-hPD1 antibodies according to the invention may comprise any class of immunoglobulin, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), an immunoglobulin chain or fragment thereof (such as Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human (e.g., murine) immunoglobulin that targets human PD-1. Preferably, anti-hPD-1 antibodies according to the invention are derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG4 or IgG1.

[0117] In one embodiment, an antigen-binding fragment of an antibody comprises a heavy chain comprising a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, a light chain comprising a variable domain comprising LDCR1, LDCR2, and LDCR3, and a fragment of the heavy chain constant domain. Thus, by fragment of the heavy chain constant domain, it should be understood that the antigen-binding fragment comprises at least a portion of the complete heavy chain constant domain. By way of example, the heavy chain constant domain may comprise at least the C H 1 domain, or at least C of the heavy chain H 1 and C H 2 domains, or at least C of the heavy chain H 1. C H 2, and C H The heavy chain constant domain may comprise or consist of three domains. A fragment of a heavy chain constant domain can also be defined as comprising at least a portion of the Fc domain of a heavy chain. Thus, an antigen-binding fragment of an antibody includes the Fab portion of an intact antibody, the F(ab')2 portion of an intact antibody, and the Fab' portion of an intact antibody. The heavy chain constant domain may also comprise or consist of, for example, a complete heavy chain constant domain as exemplified herein; several complete heavy chain constant domains are described herein. In specific embodiments of the present invention, when an antigen-binding fragment of an antibody comprises a fragment of a heavy chain constant domain comprising or consisting of a portion of a complete heavy chain constant domain, the heavy chain constant domain fragment may consist of at least 10 amino acid residues, or may consist of 10 to 300 amino acid residues, particularly 210 amino acid residues.

[0118] Preferably, the antibody against human PD-1 is a monoclonal antibody. The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope. Preferably, such monoclonal antibodies (mAbs) are derived from a mammal, such as a mouse, rodent, rabbit, goat, primate, non-human primate, or human. Techniques for preparing such monoclonal antibodies can be found, for example, in Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.), Lange Medical Publications, Los Alamos, CA, USA, and references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL, CSH Press; and Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.), Academic Press, New York, NY, USA.

[0119] In certain embodiments, the anti-hPD1 antibodies provided herein are chimeric antibodies. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0120] In certain embodiments, the anti-hPD1 antibody is a humanized antibody. Humanized antibodies typically comprise one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human or humanized antibody sequences. Alternatively, some FR residues may be substituted to restore or improve antibody specificity, affinity, and / or humanization. Humanized antibodies will also optionally comprise at least a portion of a human or humanized constant region (Fc).Methods for antibody humanization are well known in the art, see, for example, Winter and Milstein, Nature, 1991, 349:293-299; Riechmann et al., Nature, 332, 323 (1988); Verhoeyen et al., Science, 239, 1534 (1988); Rader et al., Proc. Nat. Acad. Sci. USA, 1998, 95:8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275:36073-36078; Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86:10029-10033; Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) pp. 1619-1633; Kashmiri, SV et al., Methods 36 (2005) pp. 25-34 (describing SDR (a-CDR) grafting); Padlan, EA, Mol. Immunol. 28 (1991) pp. 489-498 (describing "resurfacing"); Dall'Acqua, WF et al., Methods 36 (2005) pp. 43-60 (describing "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) pp. 61-68, and Klimka, A. et al., Br. J. Cancer 83 (2000) pp. 252-260 (describing "guided selection" for FR shuffling). and 6,180,370. Preferably, the humanized antibody against human PD-1 is a monoclonal antibody.

[0121] In particular, a humanized antibody has a T20 humanness score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90%, and most preferably a T20 humanness score comprised between 85% and 95%, preferably between 88% and 92%.

[0122] "Humanity" is generally measured using a T20 score analyzer to quantify the humanity of the variable regions of monoclonal antibodies, as described in Gao SH, Huang K, Tu H, Adler A S., BMC Biotechnology. 2013, vol. 13, p. 55. The T20 humanity score is a parameter widely used in the field of antibody humanization, first disclosed by Gao et al. (BMC Biotechnol., 2013, vol. 13, p. 55). The T20 humanity score is commonly used in patent applications to define humanized antibodies (e.g., WO 15161311, WO 17127664, WO 18136626, WO 18190719, WO 19060750, or WO 19170677).

[0123] A web-based tool is provided to calculate the T20 score of an antibody sequence using the T20 Cutoff Human Database: http: / / abAnalyzer.lakepharma.com. To calculate the T20 score, the input VH, VK, or VL variable region protein sequence is first assigned a Kabat numbering scheme to identify the CDR residues. The full-length sequence or framework-only sequence (with the CDR residues removed) is compared to all sequences in the respective antibody database using the blastp protein-protein BLAST algorithm. After extracting the sequence identity between each pairwise comparison and analyzing all sequences in the database, the sequences are sorted from highest to lowest based on sequence identity to the input sequence. The percent identity of the top 20 matching sequences is averaged to obtain the T20 score.

[0124] For each chain type (VH, VK, VL) and sequence length (full-length or framework only) in the "All Human Databases," each antibody sequence was scored against its corresponding database using the T20 score analyzer. After excluding the input sequence itself, the T20 scores of the top 20 matching sequences were obtained (since sequence 1 itself is always the input antibody, the percent identity of sequences 2-21 was averaged). The T20 scores for each group were sorted from highest to lowest. The score decrease was approximately linear for most of the sequences, but the T20 scores for the bottom approximately 15% of antibodies began to decrease sharply. Therefore, the bottom 15% of sequences were removed, and the remaining sequences formed the T20 cutoff human database. The T20 score cutoff indicates the lowest T20 score of a sequence in the new database.

[0125] Thus, the humanized anti-PD1 antibody contained in the bifunctional molecule according to the present invention has a T20 humanity score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90%, most preferably between 85% and 95%, preferably between 88% and 92%.

[0126] In one embodiment, the anti-PD1 antibody can be selected from the group consisting of: pembrolizumab (keytruda lambrolizumab, also known as MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pidilizumab (CT-011), cemiplimab (Libtayo), camrelizumab, AUNP12 , AMP-224, AGEN-2034, BGB-A317 (tisleizumab), PDR001 (spartalizumab), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, genolimuzumab (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. Oncol. 10:136 (2017)), BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI-1110 (see WO 2014 / 194302), AGEN2034 (WO 2014 / 194302), 7 / 040790), MGA012 (see WO 2017 / 19846), or IBI308 (see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in WO 2006 / 121168. Other known bifunctional or bispecific molecules that target PD-1 include RG7769 (Roche), XmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda), and XmAb23104 (Xencor).

[0127] In certain embodiments, the anti-PD1 antibody may be pembrolizumab (keytruda lambrolizumab, also known as MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538).

[0128] Specific examples of humanized anti-hPD1 antibodies are described herein below, along with their CDRs, framework regions, and Fc and hinge regions.

[0129] CDR "Complementarity determining region" or "CDR" is known in the art to refer to noncontiguous sequences of amino acids within an antibody variable region that confer antigen specificity and binding affinity. The precise amino acid sequence boundaries of a given CDR can be readily determined using any of several well-known systems, including those described in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed. (1991) "Kabat" numbering system); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 ("Chothia" numbering system); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering system); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering system); and Honegge and Pluckthun, J. Mol. Biol. 2001, 309:657-70 ("AHo" numbering system). Unless otherwise specified, the numbering system used to identify particular CDRs herein is the Kabat numbering system.

[0130] In one embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or antigen-binding fragment thereof. The CDR regions of the humanized antibody may be derived from a murine antibody, and are designed to: i) provide a safe humanized antibody with a very high level of humanization (greater than 85%) and stability, and ii) have a binding affinity (KD) for human PD-1 of 10 or greater. -7 Less than M, preferably 10-8 The antibody may be optimized to enhance antibody properties, more particularly, greater manufacturability when produced in mammalian cells, and higher production yields in mammalian cells such as COS cells and HCO cells, while preserving antagonist activity (i.e., inhibition of human PD-L1 binding to human PD-1), so that the antibody has a nucleotide sequence less than M.

[0131] In very particular embodiments, the bifunctional molecule is an anti-human PD-1 antibody or antigen-binding fragment thereof, preferably (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 a humanized anti-human PD-1 antibody or an antigen-binding fragment thereof comprising: - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with 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: 1; - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with 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: 2; - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, in which X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N, E, and optionally with 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: 3; - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, wherein 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: 12; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with 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.

[0132] In one aspect, the bifunctional molecule comprises: (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising: - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with 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: 1; - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with 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: 2; - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, wherein either Xi is D and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N and E, or Xi is E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N, E and S, and optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than 2, 3, 7 and 8 of SEQ ID NO: 3; - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, wherein 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: 12; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with 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.

[0133] In another embodiment, the bifunctional molecule is (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising: - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with 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: 1; - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with 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: 2; - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11, optionally with 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: 4, 5, 6, 7, 8, 9, 10, or 11; - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, optionally with 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: 13 or SEQ ID NO: 14; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with 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.

[0134] In another embodiment, the bifunctional molecule comprises: (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising: (a) the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 13, optionally with 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: 13; (b) the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof; (c) the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with 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; (d) the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with 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: 1; (e) the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with 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: 2; and (f) the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 4, optionally with 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: 4; or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 5, optionally with 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: 5, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 6, optionally with 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: 6, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 7, optionally with 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: 7, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 8, optionally with 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: 8, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 9, optionally with 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: 9; or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 10, optionally with 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: 10, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 11, optionally with 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: 11.

[0135] In another embodiment, the bifunctional molecule comprises: (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising: (a) the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 14, optionally with 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: 14; (b) the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof; (c) the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with 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; (d) the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with 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: 1; (e) the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with 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: 2; and (f) the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 4, optionally with 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: 4; or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 5, optionally with 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: 5, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 6, optionally with 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: 6, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 7, optionally with 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: 7, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 8, optionally with 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: 8, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 9, optionally with 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: 9; or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 10, optionally with 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: 10, or - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 11, optionally with 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: 11.

[0136] In a particular embodiment, the modification is a substitution, particularly a conservative substitution.

[0137] In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, wherein X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, and E; and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, wherein X is G or T.

[0138] In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, wherein Xi is D and X2 is selected from the group consisting of T, H, A, Y, N, and E, preferably in the group consisting of H, A, Y, N, and E, or Xi is E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and S; and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, wherein X is G or T.

[0139] In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, where X1 is D and X2 is selected from the group consisting of T, H, A, Y, N, and E, preferably in the group consisting of H, A, Y, N, and E; and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, where X is G or T.

[0140] In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, wherein X1 is E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and S; and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, wherein X is G or T.

[0141] In another embodiment, the anti-human PD-1 antibody, or antigen-binding fragment thereof, comprises or consists essentially of (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16.

[0142] In another embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof is (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 4, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 5, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 6, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 7, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 8, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 9, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 10, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 11, and (ii) a light chain comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16. comprising or consisting essentially of

[0143] In another embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof is (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 4, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 5, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 6, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 7, and (ii) a light chain comprising CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 8, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 9, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 10, and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 11, and (ii) a light chain comprising CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16. comprising or consisting essentially of

[0144] Framework In one embodiment, an anti-PD1 antibody or antigen-binding fragment according to the invention comprises framework regions, in particular 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.

[0145] Preferably, the anti-PD1 antibodies or antigen-binding fragments according to the invention comprise human or humanized framework regions. A "human acceptor framework," for purposes of this specification, 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 a human consensus framework, as defined below. A human acceptor framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes 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 identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence. A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.

[0146] In particular, the anti-PD1 antibody or antigen-binding fragment comprises heavy chain variable framework regions (HFRs) HFR1, HFR2, HFR3, and HFR4 comprising the amino acid sequences of SEQ ID NOs: 41, 42, 43, and 44, respectively, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof in HFR3, i.e., at any position other than positions 27, 29, and 32 of SEQ ID NO: 43. Preferably, the anti-PD1 antibody or antigen-binding fragment comprises HFR1 of SEQ ID NO: 41, HFR2 of SEQ ID NO: 42, HFR3 of SEQ ID NO: 43, and HFR4 of SEQ ID NO: 44.

[0147] Alternatively or additionally, the anti-PD1 antibody or antigen-binding fragment comprises light chain variable region framework regions (LFRs) LFR1, LFR2, LFR3, and LFR4 comprising the amino acid sequences of SEQ ID NOs: 45, 46, 47, and 48, respectively, and optionally with one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the humanized anti-PD1 antibody or antigen-binding fragment comprises LFR1 of SEQ ID NO: 45, LFR2 of SEQ ID NO: 46, LFR3 of SEQ ID NO: 47, and LFR4 of SEQ ID NO: 48.

[0148] VH-VL The VL and VH domains of an anti-hPD1 antibody comprised in a bifunctional molecule according to the invention may comprise four framework regions separated by three complementarity determining regions, preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (amino-terminus to carboxy-terminus).

[0149] In a first embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof contained in the bifunctional molecule is (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein Xi is D or E and X is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N and E, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position except 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, wherein 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.

[0150] In a second embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof contained in the bifunctional molecule is (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein either X1 is D and X2 is selected from the group consisting of T, H, A, Y, N, E, preferably in 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 in the group consisting of H, A, Y, N, E, and S; a heavy chain variable region (VH) 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 NO: 17; (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein 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.

[0151] In a third embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof contained in the bifunctional molecule is (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein Xi is D and X is selected from the group consisting of T, H, A, Y, N, E, preferably in the group consisting of H, A, Y, N, E, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position except 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, wherein 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.

[0152] In another embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule is: (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein Xi is E and X is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and S, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position except 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, wherein 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.

[0153] In another embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule 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, optionally with 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: 18, 19, 20, 21, 22, 23, 24, or 25, 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, optionally with 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. Includes.

[0154] In another embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule is: (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 18, 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 NO: 18; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, optionally with 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 (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 19, 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 NO: 19; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, optionally with 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 (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 20, 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 NO: 20; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, optionally with 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 (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 21, 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 NO: 21; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, optionally with 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 (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 22, 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 NO: 22; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, optionally with 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 (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 23, 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 NO: 23; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, optionally with 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 (a) comprising or consisting of the amino acid sequence of SEQ ID NO:24, 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 NO:24; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, optionally with 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 (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 25, 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 NO: 25; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, optionally with 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 (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 18, 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 NO: 18; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, optionally with 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: 28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 19, 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 NO: 19; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, optionally with 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: 28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 20, 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 NO: 20; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, optionally with 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: 28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 21, 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 NO: 21; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, optionally with 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: 28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 22, 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 NO: 22; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, optionally with 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: 28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 23, 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 NO: 23; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, optionally with 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: 28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:24, 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 NO:24; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, optionally with 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: 28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:25, optionally containing 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:25; and (b) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with 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:28. Includes.

[0155] In a particular embodiment, the modification is a substitution, particularly a conservative substitution.

[0156] CH-CL In one embodiment, the heavy (CH) and light (CL) chains comprise the VL and VH sequences as described herein above.

[0157] In certain embodiments, the anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule is (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, optionally with 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; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, optionally with 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.

[0158] In another embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule is: (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:29, optionally containing one or more amino acid 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:29. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with 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 (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 30, optionally containing one or more amino acid 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: 30; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with 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 (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 31, optionally containing one or more amino acid 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: 31; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with 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 (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 32, optionally containing one or more amino acid 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: 32. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with 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 (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, optionally containing one or more amino acid 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: 33. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with 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 (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 34, optionally containing one or more amino acid 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: 34. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with 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 (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 35, optionally containing one or more amino acid 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: 35. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with 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 (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 36, optionally containing one or more amino acid 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: 36. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with 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 (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:29, optionally containing one or more amino acid 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:29. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with 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: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 30, optionally containing one or more amino acid 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: 30; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with 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: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 31, optionally containing one or more amino acid 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: 31; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with 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: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 32, optionally containing one or more amino acid 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: 32. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with 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: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, optionally containing one or more amino acid 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: 33. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with 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: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 34, optionally containing one or more amino acid 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: 34. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with 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: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 35, optionally containing one or more amino acid 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: 35. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with 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: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 36, optionally containing one or more 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: 36. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with 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: 38. Includes.

[0159] Preferably, the modifications are substitutions, particularly conservative substitutions.

[0160] Fc and hinge regions Several studies to develop therapeutic antibodies have led to the optimization of antibody properties through genetic engineering of the Fc region, enabling the generation of molecules better suited for their required pharmacological activity. The Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L and M252Y / S254T / T256E+H433K / N434F, have been shown to increase the binding affinity to FcRn and the half-life of IgG1 in vivo. However, there is not always a direct correlation between increased FcRn binding and improved half-life. One approach to improving the efficacy of therapeutic antibodies is to increase their serum persistence, allowing for higher circulating levels, less frequent administration, and reduced doses. It may be desirable to genetically engineer the Fc region to either reduce or increase the effector function of an antibody. For antibodies targeting cell surface molecules, particularly those on immune cells, effector function must be abrogated. Conversely, for antibodies intended for oncological use, increasing effector function can improve therapeutic activity. The four human IgG isotypes bind with different affinities to activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIIa), inhibitory FcγRIIb receptors, and the first component of complement (C1q), resulting in very different effector functions. Binding of IgG to FcγR or C1q depends on residues located in the hinge region and CH2 domain. Two regions of the CH2 domain are important for FcγR and C1q binding, and IgG2 and IgG4 have unique sequences.

[0161] Antibodies according to the present invention optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region (Fc) of a mammalian immunoglobulin, even more preferably of a human immunoglobulin or humanized immunoglobulin. Preferably, the Fc region is a portion of the anti-hPD-1 antibody described herein. The anti-hPD1 antibody or antigen-binding fragment thereof included in the bifunctional molecules of the present invention may comprise an immunoglobulin constant region, or a fragment, analog, variant, mutant, or derivative of the constant region. As will be appreciated by those skilled in the art, the choice of IgG isotype of the heavy chain constant domain is a central concern, depending on whether a particular function is required and on the need for suitable in vivo half-life. For example, antibodies designed for the selective eradication of cancer cells typically require an active isotype that enables effector-mediated cell killing by complement activation and antibody-dependent cell-mediated cytotoxicity. Both human IgG1 and IgG3 (shorter half-life) isotypes, particularly the human IgG1 isotype (wild-type and variants), meet these criteria. In particular, depending on the IgG isotype of the heavy chain constant domain (particularly human wild-type and variant IgG1 isotypes), the anti-hPD1 antibodies of the invention may be cytotoxic to cells expressing PD-1 by CDC, ADCC, and / or ADCP mechanisms. Indeed, the fragment crystallizable (Fc) region interacts with various accessory molecules to mediate indirect effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0162] In preferred embodiments, the constant region is derived from a human immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4, and other classes. In a further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. Preferably, the anti-PD1 antibody comprises an IgG1 Fc region or an IgG4 Fc region. Even more preferably, the anti-hPD1 antibody comprises an IgG4 Fc region with S228P, which stabilizes IgG4.

[0163] In one embodiment, the anti-PD1 antibody comprises a truncated Fc region or a fragment of a truncated Fc region. In one embodiment, the constant region comprises a CH2 domain. In another embodiment, the constant region comprises a CH2 domain and a CH3 domain, or comprises a hinge-CH2-CH3 domain. Alternatively, the constant region may comprise all or part of the hinge region, CH2 domain, and / or CH3 domain. In a preferred embodiment, the constant region contains a CH2 domain and / or a CH3 domain derived from a human IgG4 heavy chain. In some embodiments, the constant region contains a CH2 domain and / or a CH3 domain derived from a human IgG4 heavy chain.

[0164] In another embodiment, the constant region comprises a CH2 domain and at least a portion of a hinge region. The hinge region may be derived from an immunoglobulin heavy chain, for example, IgG1, IgG2, IgG3, IgG4, or other class. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable class, with or without mutation. More preferably, the hinge region is derived from a human IgG1 heavy chain. In one embodiment, the constant region comprises a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In a specific embodiment, the CH2 domain is derived from a human IgG2 or IgG4 heavy chain and the hinge region is derived from an altered human IgG1 heavy chain.

[0165] In one embodiment, the constant region contains a mutation that reduces affinity for an Fc receptor or reduces an Fc effector function, for example, the constant region may contain a mutation that eliminates a glycosylation site in the constant region of an IgG heavy chain.

[0166] In another embodiment, the constant region comprises at least a portion of a CH2 domain and a hinge region. The hinge region may be derived from an immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4, or another class. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or another suitable class. The IgG1 hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of the immunoglobulin. These same cysteines enable efficient and consistent disulfide bond formation between the Fc portions. Therefore, a 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. The IgG2 isotype hinge region has four disulfide bonds that tend to promote oligomerization and the possibility of incorrect disulfide bonding during secretion in recombinant systems. A suitable hinge region may be derived from an IgG2 hinge, with each of the first two cysteines preferably mutated to another amino acid. The hinge region of IgG4 is known to be inefficient at forming interchain disulfide bonds. However, a suitable hinge region of the present invention may be derived from an IgG4 hinge region, preferably containing mutations that enhance the correct formation of disulfide bonds between heavy chain-derived moieties (Angal S et al. (1993) Mol. Immunol. 30:105-8). More preferably, the hinge region is derived from a human IgG4 heavy chain.

[0167] In one embodiment, the constant region comprises a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype, hi a specific embodiment, the CH2 domain is derived from a human IgG4 heavy chain and the hinge region is derived from an altered human IgG1 heavy chain.

[0168] According to the present invention, the constant region may contain CH2 and / or CH3 domains and hinge regions derived from different antibody isotypes, i.e., hybrid constant regions. For example, in one embodiment, the constant region contains CH2 and / or CH3 domains derived from IgG2 or IgG4 and a mutated hinge region derived from IgG1. Alternatively, a mutated hinge region derived from another IgG subclass may be used in the hybrid constant region. For example, a mutated form of the IgG4 hinge that allows efficient disulfide bonding between the two heavy chains may be used. Alternatively, the mutated hinge may be derived from an IgG2 hinge in which the first two cysteines have been mutated to different amino acids. The assembly of such hybrid constant regions is described in U.S. Patent Application Publication No. 20030044423, the disclosure of which is incorporated herein by reference.

[0169] In one embodiment, the constant region may contain a CH2 and / or a CH3 with one or any combination of the mutations set forth in Table D below.

[0170] [Table 4]

[0171] In certain embodiments, the bifunctional molecule, preferably the binding moiety, comprises a human IgG1 heavy chain constant domain or an IgG1 Fc domain, optionally containing the following amino acids: T250Q / M428L; M252Y / S254T / T256E+H433K / N434F; E233P / L234V / L235A / G236A+A327G / A330S / P331S; E333A; S239D / A330L / I332E; P257I / Q311; K326W / E333S; S239D / I332 E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K322A; and K444A, preferably having a substitution or combination of substitutions selected from the group consisting of N297A, optionally in combination with M252Y / S254T / T256E, and L234A / L235A.

[0172] In another embodiment, the binding moiety comprises a human IgG4 heavy chain constant domain or a human IgG4 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. Even more preferably, the bifunctional molecule, preferably the binding moiety, comprises an IgG4 Fc region with S228P that stabilizes IgG4.

[0173] In certain embodiments, amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. In certain embodiments, the Fc region variant retains some, but not all, effector functions. Such antibodies may be useful, for example, in applications where in vivo antibody half-life is important but certain effector functions are unnecessary or deleterious. Examples of effector functions include complement-dependent cytotoxicity (CDC) and antibody-mediated complement-mediated cytotoxicity (ADCC). Numerous substitutions or deletions that alter effector function are known in the art.

[0174] In one embodiment, the constant region contains a mutation that reduces affinity for an Fc receptor or reduces an Fc effector function. For example, the constant region may contain a mutation that eliminates a glycosylation site in the constant region of an IgG heavy chain. Preferably, the CH2 domain contains a mutation that eliminates a glycosylation site in the CH2 domain.

[0175] In one embodiment, the anti-hPD1 according to the invention has a heavy chain constant domain of SEQ ID NO: 39 or 52 and / or a light chain constant domain of SEQ ID NO: 40, in particular a heavy chain constant domain of SEQ ID NO: 39 or 52 and a light chain constant domain of SEQ ID NO: 40.

[0176] In another embodiment, the anti-hPD1 according to the invention has a heavy chain constant domain of SEQ ID NO: 52 and / or a light chain constant domain of SEQ ID NO: 40, in particular a heavy chain constant domain of SEQ ID NO: 52 and a light chain constant domain of SEQ ID NO: 40.

[0177] [Table 5]

[0178] Amino acid alterations near the junction of the Fc and non-Fc portions can dramatically increase the serum half-life of Fc fusion proteins (WO 01 / 58957). Thus, the junction region of the proteins or polypeptides of the present invention may contain alterations, preferably within about 10 amino acids of the junction point, compared to the naturally occurring sequences of 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 has been replaced. Preferably, the C-terminal lysine of the IgG sequence has been replaced with a non-lysine amino acid, such as alanine or leucine, to further increase serum half-life.

[0179] All subclasses of human IgG have a C-terminal lysine residue (K444) in the antibody heavy chain that is cleaved in circulation. This cleavage in the blood may impair the biological activity of the bifunctional molecule by releasing IL-7. To circumvent this problem, the K444 amino acid in the IgG1 or IgG4 domain can be substituted with alanine to reduce proteolytic cleavage. This mutation is widely used in antibodies. Thus, in one embodiment, the anti-PD1 antibody contains at least one additional amino acid substitution consisting of K444A.

[0180] In one embodiment, the anti-PD1 antibody contains an additional cysteine ​​residue in the C-terminal domain of the IgG to create an additional disulfide bond and potentially limit the flexibility of the bifunctional molecule.

[0181] In certain embodiments, the antibody may be altered to increase, decrease, or eliminate the extent to which it is glycosylated.

[0182] Checkpoint inhibitors The present inventors demonstrate herein that bifunctional molecules of the present invention combine the effect of an IL-7 variant or mutant on the IL-7 receptor with blockade of the inhibitory effect of PD-1, making them suitable for optimizing the effect of checkpoint inhibitors such as anti-PD-1 antibodies. In particular, a synergistic effect on the activation of T cells, particularly exhausted T cells, and more particularly on TCR signaling, is demonstrated. The present inventors demonstrate, in particular, activation on the same immune cells, provided by the binding of the anti-PD-1 antibody and IL-7 contained in the bifunctional molecule on the same cells. This synergistic effect is never observed when the IL-7 antibody and the anti-PD-1 antibody are used as separate compounds. Therefore, it is conceivable that any molecule other than PD-1, particularly an exhaustion factor, expressed on immune cells expressing IL-7R, can be induced by the bifunctional constructs of the present invention. Thus, in embodiments, the bifunctional molecule comprises an antibody or antigen-binding fragment thereof directed against a target other than PD-1 expressed on immune cells. For example, the target may be a receptor expressed on the surface of immune cells, particularly T cells. The receptor may be an inhibitor receptor. Alternatively, the receptor may be an activating receptor.

[0183] As used herein, the term "target" refers to a peptide, polypeptide, protein, antigen, or epitope expressed on the outer surface of an immune cell. With respect to expression of a target on the surface of an immune cell, the term "expressed" refers to a target that is present or displayed on the outer surface of the cell. The term "specifically expressed" means that the target is expressed on the immune cell but is not substantially expressed on other cell types, such as, particularly, tumor cells.

[0184] In one embodiment, the target is specifically expressed by immune cells of a healthy subject or a subject suffering from a disease, particularly cancer. This means that the target shows higher expression levels in immune cells than in other cells, or that the ratio of immune cells expressing the target to all immune cells is higher than the ratio of other cells expressing the target to all other cells. Preferably, the expression level or ratio is 2, 5, 10, 20, 50, or 100 times higher. The expression level or ratio can be determined more specifically for a particular type of immune cell, such as T cells, more specifically CD8+ T cells, effector T cells, or exhausted T cells, or in certain situations for a subject suffering from a disease, such as cancer or an infectious disease.

[0185] In one embodiment, the target is an immune checkpoint. Preferably, the target is selected from the group consisting of PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8. Such targets are described in more detail in Table F, below.

[0186] [Table 6A]

[0187] [Table 6B]

[0188] Thus, in this aspect, the antibody or antigen fragment thereof comprised in the bifunctional molecule according to the invention binds to a target selected from the group consisting of CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8.

[0189] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprised in the bifunctional molecule according to the present invention is selected from the group consisting of CTLA-4, BTLA, TIGIT, LAG3, and TIM3.

[0190] Also known are antibodies against TIM3 and bifunctional or bispecific molecules that target TIM3, such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367, and RG7769 (Roche). In some embodiments, the TFM-3 antibody is as described in WO 2013006490, WO 2016 / 161270, WO 2018 / 085469, WO 2018 / 129553, WO 2011 / 155607, U.S. Pat. No. 8,552,156, EP 2581113, and U.S. Patent Application No. 2014 / 044728.

[0191] In addition, antibodies against CTLA-4 and bifunctional or bispecific molecules targeting CTLA-4 are known, such as ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), and MEDI5752 (AstraZeneca). Anti-CTLA-4 antibodies are disclosed in WO 18025178, WO 19179388, WO 19179391, WO 19174603, WO 19148444, WO 19120232, WO 19056281, WO 19023482, WO 18209701, WO 18165895, WO 18160536, WO 18156250, WO 18106862, WO 18 and also WO 106864, WO 18068182, WO 18035710, WO 18025178, WO 17194265, WO 17106372, WO 17084078, WO 17087588, WO 16196237, WO 16130898, WO 16015675, WO 12120125, WO 09100140, and WO 07008463.

[0192] Antibodies against LAG-3 and bifunctional or bispecific molecules targeting LAG-3 are also known, such as BMS-986016, IMP701, MGD012, or MGD013 (bispecific PD-1 and LAG-3 antibodies). Anti-LAG-3 antibodies are also disclosed in WO 2008132601, EP 2320940, and WO 19152574.

[0193] Antibodies against BTLA are also known in the art, such as hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mab19A7, or hu Mab4C7. Antibody TAB004 against BTLA is currently undergoing clinical trials in subjects with advanced malignancies. Anti-BTLA antibodies are also disclosed in WO 08076560, WO 10106051 (e.g., BTLA8.2), WO 11014438 (e.g., 4C7), WO 17096017, and WO 17144668 (e.g., 629.3).

[0194] The art has disclosed BMS-986207 or AB154, BMS-986207 CPA, as disclosed in WO 19232484. 9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536 .1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5, CHA.9. Antibodies against TIGIT are also known, such as CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8. Anti-TIGIT antibodies are disclosed in WO 16028656, WO 16106302, WO 16191643, WO 17030823, WO 17037707, WO 17053748, WO 17152088, WO 18033798, WO 18102536, WO 18102746, and WO 18160704. , WO 18200430, WO 18204363, WO 19023504, WO 19062832, WO 19129221, WO 19129261, WO 19137548, WO 19152574, WO 19154415, WO 19168382, and WO 19215728.

[0195] Antibodies against CD160 are also known in the art, such as CL1-R2 CNCM I-3204 as disclosed in WO 06015886, or others as disclosed in WO 10006071, WO 10084158, WO 18077926.

[0196] In a specific embodiment, the bifunctional molecule according to the present invention comprises an anti-CTLA-4 antibody or antigen-binding fragment thereof, preferably a human, humanized, or chimeric anti-CTLA-4 antibody or antigen-binding fragment thereof. Preferably, the antibody is a CTLA-4 antagonist. Thus, the bifunctional molecule combines the effect of IL-7wt, a variant or mutant thereof, with blockade of the inhibitory effect of CTLA-4 on the IL-7 receptor, and can exhibit a synergistic effect on T cells, particularly exhausted T cells, and more particularly on the activation of TCR signaling.

[0197] In another specific embodiment, a bifunctional molecule according to the invention comprises an anti-BTLA antibody or antigen-binding fragment thereof, preferably a human, humanized, or chimeric anti-BTLA antibody or antigen-binding fragment thereof. Preferably, the antibody is an antagonist of BTLA. Thus, the bifunctional molecule combines the effect of IL-7wt, a variant or mutant thereof, with blockade of the inhibitory effect of BTLA on the IL-7 receptor, and can exhibit synergistic effects on T cells, particularly exhausted T cells, and more particularly on activation of TCR signaling.

[0198] In another specific embodiment, the bifunctional molecule of the present invention comprises an anti-TIGIT antibody or an antigen-binding fragment thereof, preferably a human, humanized, or chimeric anti-TIGIT antibody or an antigen-binding fragment thereof. Preferably, the antibody is an antagonist of TIGIT. Thus, the bifunctional molecule combines the effect of IL-7wt, its variant, or mutant with the blockade of the inhibitory effect of TIGIT on the IL-7 receptor, and can exhibit a synergistic effect on T cells, particularly exhausted T cells, and more particularly on the activation of TCR signaling.

[0199] In another specific embodiment, the bifunctional molecule according to the present invention comprises an anti-LAG-3 antibody or antigen-binding fragment thereof, preferably a human, humanized, or chimeric anti-LAG-3 antibody or antigen-binding fragment thereof. Preferably, the antibody is an antagonist of LAG-3. Thus, the bifunctional molecule combines the effect of IL-7wt, a variant or mutant thereof, with blocking the inhibitory effect of LAG-3 on the IL-7 receptor, and can have a synergistic effect on T cells, particularly exhausted T cells, and more particularly on the activation of TCR signaling.

[0200] In another specific embodiment, the bifunctional molecule according to the invention comprises an anti-TIM3 antibody or antigen-binding fragment thereof, preferably a human, humanized, or chimeric anti-TIM3 antibody or antigen-binding fragment thereof. Preferably, the antibody is an antagonist of TIM3. Thus, the bifunctional molecule combines the effect of an IL-7 variant or mutant on the IL-7 receptor with blockade of the inhibitory effect of TIM3, which can have a synergistic effect on T cells, particularly exhausted T cells, and more particularly on activation of TCR signaling.

[0201] Peptide Linker The present invention includes bifunctional molecules that may contain a peptide linker between the anti-PD-1 antibody or fragment thereof and IL-7. The peptide linker is typically of sufficient length and flexibility to ensure that the two protein elements connected between them have sufficient spatial freedom to perform their functions and to avoid the effects of α-helix and β-fold formation on the stability of the recombinant bifunctional molecule.

[0202] In an embodiment of the present disclosure, the anti-hPD1 antibody is preferably linked to IL-7 via a peptide linker. In other words, the present invention relates to a bifunctional molecule comprising an anti-PD1 antibody or antigen-binding fragment thereof, as detailed herein, having a chain, e.g., a light chain or a heavy chain or a fragment thereof, preferably a heavy chain or a fragment thereof, linked to IL-7 via a peptide linker. As used herein, the term "linker" refers to a sequence of at least one amino acid linking the IL-7 and anti-PD-1 immunoglobulin sequence portions. Such a linker may be useful for preventing steric hindrance. The linker is typically 3 to 44 amino acid residues in length. Preferably, the linker has 3 to 30 amino acid residues. In some embodiments, the linker has 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.

[0203] In embodiments, the present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody, or antigen-binding fragment thereof, as defined above, and IL-7, wherein a chain of the antibody, e.g., a light chain or a heavy chain, preferably the heavy chain, and even more preferably the C-terminus of the heavy chain or the light chain, is linked to the N-terminus of IL-7, preferably IL-7, by a peptide linker.

[0204] In a particular aspect, the present invention relates to a bifunctional molecule comprising an anti-hPD-1 antibody, or antigen-binding fragment thereof, as defined above, wherein IL-7 is linked to the C-terminus of the heavy chain of the antibody (e.g., the C-terminus of the heavy chain constant domain), preferably by a peptide linker.

[0205] In embodiments, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or antigen-binding fragment thereof, as defined above, wherein IL-7 is linked to the C-terminus of the light chain of the antibody (e.g., the C-terminus of the light chain constant domain), preferably by a peptide linker.

[0206] The linker sequence may be a naturally occurring or non-naturally occurring sequence. 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 are linkers derived from the hinge region of heavy chain antibodies, as described in WO 96 / 34103 and WO 94 / 04678. Another example is a polyalanine linker sequence. Further preferred examples of linker sequences are 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.

[0207] In one embodiment, the linker comprised in the bifunctional molecule is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2Ser, and (Gly3Ser2)3, preferably (Gly4Ser)3. Preferably, the linker is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, and (Gly3Ser2)3. Even more preferably, the linker is (GGGGS)3.

[0208] In embodiments, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or fragment thereof as defined above, wherein the antibody or fragment thereof is linked to IL-7 by a linker sequence preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, even more preferably by (GGGGS)3. Preferably, the linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, and (GGGS)3.

[0209] Preferably, the C-terminus of the heavy chain, preferably the heavy chain of an anti-PD-1 antibody, is genetically fused to the N-terminus of IL-7 via a flexible (Gly4Ser)3 linker. At the fusion junction, the C-terminal lysine residue of the antibody heavy chain can be mutated to alanine to reduce proteolytic cleavage.

[0210] Preferably, the C-terminus of the heavy chain, preferably the light chain, of the anti-PD-1 antibody is genetically fused to the N-terminus of IL-7 via a flexible (Gly4Ser)3 linker. At the fusion junction, the C-terminal lysine residue of the antibody light chain can be mutated to alanine to reduce proteolytic cleavage.

[0211] IL-7 The bifunctional molecule according to the present invention comprises an additional or second entity which comprises interleukin-7, or a variant or fragment thereof.

[0212] Preferably, the IL-7 protein is human IL-7 or a variant thereof. Thus, the IL-7 or variant thereof has an amino acid sequence that has at least 75% identity with wild-type IL-7, in particular with the protein of SEQ ID NO: 51.

[0213] In one embodiment, the bifunctional molecule comprises the 152 amino acid typical wild-type IL-7 human protein (SEQ ID NO: 51). Preferably, the IL-7 protein is that of SEQ ID NO: 51. The IL-7 protein may include or lack its peptide signal.

[0214] A "variant" of an IL-7 protein is defined as an amino acid sequence in which one or more amino acids have been altered. Variants may have "conservative" or "non-conservative" modifications. Such modifications can include amino acid substitutions, deletions, and / or insertions. Guidance for determining which and how many amino acid residues can be substituted, inserted, or deleted without impairing biological properties (e.g., activity, binding ability, and / or structure) can be found using computer programs well known in the art, such as molecular modeling or alignment software. In certain embodiments, variant IL-7 proteins encompassed within the present invention include, in particular, IL-7 proteins that retain substantially equivalent biological properties compared to wild-type IL-7. In alternative embodiments, variant IL-7 proteins encompassed within the present invention include, in particular, 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. Variants of IL-7 also include altered polypeptide sequences of IL-7 (e.g., oxidized, reduced, deaminated, or truncated forms). In particular, truncated forms or fragments of IL-7 that retain biological properties equivalent to the full-length IL-7 protein are included within the scope of the present invention. In one embodiment, interleukin-7 is any biologically active fragment thereof. Variants of IL-7 more preferably include naturally occurring allelic variants resulting from natural genetic polymorphisms, including SNPs, splicing variants, etc.

[0215] The biological activity of the IL-7 protein can be measured using an in vitro cell proliferation assay. Preferably, the IL-7 variants according to the invention retain at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 80%, 90%, 95%, and even more preferably 99% of the biological activity of wild-type human IL-7.

[0216] Variant IL-7 proteins also include polypeptides having at least about 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more sequence identity to wild-type IL-7, particularly the protein of SEQ ID NO:51.

[0217] A preferred IL-7 according to the present invention is a human IL-7 polypeptide comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 51, EP 314415, or WO 2004 / 018681 A2, and any naturally occurring variants and homologues thereof.

[0218] In one aspect, the IL-7 polypeptide 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 the culture of a host cell (e.g., a microorganism, or an insect, or a plant, or a mammal), or from a transgenic plant or animal that has been genetically engineered to contain a nucleic acid molecule encoding an IL-7 polypeptide. Preferably, the recombinant IL-7 is human recombinant IL-7 (e.g., human IL-7 produced in a recombinant expression system).

[0219] The present invention also provides bifunctional molecules containing IL-7 proteins with enhanced biological activity compared to wild-type IL-7 proteins. For example, as described in U.S. Patent No. 7,960,514, IL-7 proteins with disulfide bond patterns of Cys2-Cys92, Cys34-Cys129, and Cys47-141 are more active in vivo than wild-type recombinant IL-7 proteins. As described in European Patent No. 1,904,635, etc., hyperglycosylation of IL-7, such as an IL-7 protein in which Asn116 is non-glycosylated but Asn70 and Asn91 are glycosylated, enhances IL-7 biological activity.

[0220] Alternatively, the present invention provides bifunctional molecules comprising IL-7 proteins that have reduced immunogenicity compared to wild-type IL-7 proteins, particularly by removing T cell epitopes within IL-7 that can stimulate an immune response. Examples of such IL-7 proteins are described in WO2006061219.

[0221] In certain aspects, the present disclosure also provides bifunctional molecules comprising an IL-7 variant or mutant. The terms "interleukin-7 variant," "mutant IL-7," "IL-7 variant," "IL-7 variant," "IL-7m," or "IL-7v" are used interchangeably herein.

[0222] In this situation, the IL-7 variant or mutant does not retain substantially equivalent biological properties (e.g., activity, binding ability, and / or structure) compared to wild-type IL-7. The IL-7 variant or mutant contains at least one mutation. In particular, the at least one mutation reduces the affinity of the IL-7 variant or mutant for the IL-7 receptor (IL-7R) but does not result in a loss of IL-7R recognition. Thus, the IL-7 variant or mutant retains the ability to activate IL-7R, e.g., as measured by pStat5 signaling, as disclosed, e.g., in Bitar et al., Front. Immunol., 2019, Vol. 10. The biological activity of the IL-7 protein can be measured using an in vitro cell proliferation assay or by measuring p-Stat5 in T cells by ELISA or FACS. Preferably, IL-7 variants according to the present invention have at least a 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 250-fold, 500-fold, 750-fold, 1000-fold, 2500-fold, 5000-fold, or 8000-fold reduced biological property (e.g., activity, binding ability, and / or structure) compared to wild-type IL-7, preferably wth-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 1%, 5%, 10%, 20%, 30%, 40%, 50%, 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.

[0223] In one aspect, the IL-7 variant or mutant differs from wt-IL-7 by at least one amino acid mutation that i) reduces 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) improves the pharmacokinetics of the IL-7 variant compared to wt-IL-7. More particularly, the IL-7 variant or mutant further retains the ability to activate IL-7R, particularly via pStat5 signaling.

[0224] In another aspect, bifunctional molecules comprising an IL-7 variant or mutant differ from wt-IL-7 by at least one amino acid mutation that i) reduces the affinity of the bifunctional molecule for the IL-7 receptor (IL-7R) compared to the affinity of a bifunctional molecule comprising wt-IL-7 for IL-7R, and ii) improves the pharmacokinetics of the bifunctional molecule comprising an IL-7 variant or mutant compared to a bifunctional molecule comprising wt-IL-7. More particularly, bifunctional molecules comprising an IL-7 variant or mutant further retain the ability to activate IL-7R, particularly via pStat5 signaling. For example, the binding of a bifunctional molecule comprising 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 comprising 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 comprising wild-type IL-7.

[0225] In certain aspects, the IL-7 variant or mutant exhibits reduced affinity for the IL-7 receptor (IL-7R) compared to the affinity of with-IL-7 for IL-7R. In particular, the IL-7 variant or mutant exhibits reduced affinity for CD127 and / or CD132 compared to the affinity of with-IL-7 for CD127 and / or CD132, respectively. Preferably, the IL-7 variant or mutant exhibits reduced affinity for CD127 compared to the affinity of with-IL-7 for CD127.

[0226] Preferably, the at least one amino acid mutation reduces the affinity of the IL-7 variant or mutant for IL-7R, in particular for CD132 or CD127, by at least 10-fold, 100-fold, 10000-fold, 10000-fold, or 100000-fold compared to the affinity of wt-IL-7 for IL-7R. Such affinity comparisons can be performed by any method known to those skilled in the art, such as ELISA or Biacore.

[0227] Preferably, the at least one amino acid mutation reduces the affinity of the IL-7 variant or mutant for IL-7R but does not reduce the biological activity of the IL-7 variant or mutant compared to IL-7 wt, particularly as measured by pStat5 signaling.

[0228] Alternatively, the at least one amino acid mutation reduces the affinity of the IL-7 variant or mutant for IL-7R but does not significantly reduce the biological activity of IL-7m compared to IL-7 wt, particularly as measured by pStat5 signaling.

[0229] Additionally or alternatively, the IL-7 variant or mutant improves the pharmacokinetics of a bifunctional molecule comprising an IL-7 variant or mutant compared to a bifunctional molecule comprising wild-type IL-7. In particular, the IL-7 variant or mutant of the present invention improves the pharmacokinetics of a bifunctional molecule comprising an IL-7 variant or mutant by at least 10-fold, 100-fold, or 1000-fold compared to a bifunctional molecule comprising wt-IL-7. Pharmacokinetic profile comparison can be performed by any method known to those skilled in the art, such as in vivo drug injection and dosage ELISA of drug in serum at multiple time points, as shown in Example 9.

[0230] As used herein, the terms "pharmacokinetics" and "PK" are used interchangeably and refer to the fate of a compound, substance, or drug administered to a living organism. Pharmacokinetics specifically includes the ADME or LADME scheme, which describes release (i.e., release of a substance from a composition), absorption (i.e., entry of a substance into the blood circulation), distribution (i.e., dispersion or diffusion of a substance throughout the body), metabolism (i.e., transformation or breakdown of a substance), and excretion (i.e., removal or clearance of a substance from an 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, such as elimination half-life, elimination constant rate, clearance (i.e., the volume of plasma from which a drug is cleared per unit time), Cmax (maximum serum concentration), and drug exposure (determined by the area under the curve) (Scheff et al., Pharm Res., 2011, 28, 1081-9).

[0231] Thus, improved pharmacokinetics through the use of an IL-7 variant or mutant 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.

[0232] In certain embodiments, at least one mutation in the IL-7 variant or mutant improves the terminal half-life of the bifunctional molecule comprising the IL-7 variant or mutant compared to a bifunctional molecule comprising IL-7 wt.

[0233] In one embodiment, the IL-7 variant or mutant 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 to the 152 amino acid wild-type human IL-7 (wth-IL-7) protein, such as that disclosed in SEQ ID NO: 51. Preferably, the IL-7 variant or mutant 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 to SEQ ID NO: 51.

[0234] In particular, at least one mutation occurs at IL-7 amino acid positions 74 and / or 142. Additionally or alternatively, at least one mutation occurs at amino acid positions 2 and 141, 34 and 129, and / or 47 and 92. These positions refer to the amino acid positions set forth in SEQ ID NO:51.

[0235] 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 set forth in SEQ ID NO: 51. Thus, for example, the mutation W142H represents a substitution of tryptophan with histidine in wth-IL7 to obtain IL-7m with a histidine at amino acid position 142. Such mutants are set forth, for example, in SEQ ID NO: 56.

[0236] In one embodiment, an IL-7 variant or mutant contains a set of substitutions to disrupt disulfide bonds between C2 and C141, between C47 and C92, and between C34-C129. Specifically, the IL-7 variant or mutant contains two sets of substitutions to disrupt 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. Thus, the 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 set forth in SEQ ID NO: 51. Such IL-7 variants or mutants are specifically set forth in SEQ ID NOs: 53-55 (SS1, SS2, and SS3, respectively). Preferably, the IL-7 variant or mutant comprises the amino acid substitutions C2S-C141S and C47S-C92S. Even more preferably, the IL-7 variant or mutant has the sequence set forth in SEQ ID NO: 54.

[0237] In another embodiment, the IL-7 variant or mutant comprises at least one mutation selected from the group consisting of W142H, W142F, and W142Y. Such IL-7 variants or mutants are specifically set forth in the sequences set forth in SEQ ID NOs: 57-58, respectively. Preferably, the IL-7 variant or mutant comprises the mutation W142H. Even more preferably, the IL-7 variant or mutant comprises the sequence set forth in SEQ ID NO: 56.

[0238] In another embodiment, the IL-7 variant or mutant comprises at least one mutation selected from the group consisting of D74E, D74Q, and D74N, preferably D74E and D74Q. Such IL-7 variants or mutants are specifically set forth in the sequences set forth in SEQ ID NOs: 63 to 65, respectively. Preferably, the IL-7 variant or mutant comprises the mutation D74E. Even more preferably, the IL-7 variant or mutant comprises the sequence set forth in SEQ ID NO: 63.

[0239] In another embodiment, the IL-7 variant or mutant comprises at least one mutation selected from the group consisting of Q11E, Y12F, M17L, Q22E, and / or K81R. These mutations refer to the amino acid positions set forth in SEQ ID NO: 51. Such IL-7 variants or mutants are specifically set forth in the sequences set forth in SEQ ID NOs: 59, 60, 61, 62, and 66, respectively.

[0240] In one embodiment, the IL-7 variant or mutant comprises at least one mutation consisting of i) W142H, W142F, or W142Y, and / or ii) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or iii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0241] In one embodiment, the IL-7 variant or mutant comprises a W142H substitution and at least one mutation consisting of i) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0242] In one embodiment, the IL-7 variant or mutant comprises a D74E substitution and at least one mutation consisting of i) W142H, W142F, or W142Y, and / or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0243] In one embodiment, the IL-7 variant or mutant comprises the 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.

[0244] In one embodiment, the IL-7 variant or mutant comprises i) D74E and W142H substitutions, and ii) mutations C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0245] An IL-7 variant or mutant may include or lack the peptide signal.

[0246] In one embodiment, a bifunctional molecule according to the present invention comprises an IL-7 variant comprising or consisting of the amino acid sequence set forth in SEQ ID NOs: 53 to 58 or 63 to 65. Even more preferably, a bifunctional molecule according to the present invention comprises an IL-7 variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 54, 56, or 63.

[0247] Bifunctional molecules or "Bicki" The present invention particularly provides a bifunctional molecule comprising or consisting of an anti-hPD1 antibody or antibody fragment thereof and IL-7, as disclosed herein above, wherein the anti-hPD1 antibody or antibody fragment thereof is covalently linked to IL-7, preferably by a peptide linker as disclosed herein above, in particular as a fusion protein.

[0248] In particular, a bifunctional molecule according to the invention comprises two entities: a first entity comprising or consisting essentially of an anti-hPD1 antibody or a fragment thereof; and a second entity comprising or consisting essentially of interleukin-7 (IL-7), preferably human IL-7, optionally linked by a peptide linker.

[0249] In particular, bifunctional molecules according to the present invention comprise one, two, three, or four IL-7 molecules. In particular, a bifunctional molecule may comprise only one IL-7 molecule linked to only one of the light or heavy chains of an anti-PD-1 antibody. Alternatively, a bifunctional molecule may comprise two IL-7 molecules linked to either the light or heavy chain of an anti-PD-1 antibody. Alternatively, a bifunctional molecule may comprise two IL-7 molecules, one linked to the light chain of an anti-PD-1 antibody and the second linked to the heavy chain of an anti-PD-1 antibody. Alternatively, a bifunctional molecule may comprise three IL-7 molecules, two linked to either the light or heavy chain of an anti-PD-1 antibody and the last linked to the other chain of an anti-PD-1 antibody. Finally, the bifunctional molecule may also comprise four IL-7 molecules, two linked to the light chain of an anti-PD-1 antibody and two linked to the heavy chain of an anti-PD-1 antibody, and thus the bifunctional molecule comprises one to four immunotherapeutic molecules as disclosed herein.

[0250] In one embodiment, only one of the light chains comprises one IL7 molecule (e.g., the bifunctional molecule comprises one IL7 molecule), only one of the heavy chains comprises one IL7 molecule (e.g., the bifunctional molecule comprises one IL7 molecule), each light chain comprises one IL-7 molecule (e.g., the bifunctional molecule comprises two IL7 molecules), each heavy chain comprises one IL-7 molecule (e.g., the bifunctional molecule comprises two IL7 molecules), or only one of the light chains and only one of the heavy chains comprises one immunotherapy. Each light chain contains one IL7 molecule and only one of the heavy chains contains one IL7 molecule (e.g., a bifunctional molecule contains two IL7 molecules), each heavy chain contains one IL7 molecule and only one of the light chains contains one IL7 molecule (e.g., a bifunctional molecule contains three IL7 molecules), or both the light and heavy chains contain one IL7 molecule (e.g., a bifunctional molecule contains four IL7 molecules).

[0251] In one embodiment, the bifunctional molecule according to the present invention comprises: (a) an anti-human PD-1 antibody or an antigen-binding fragment thereof comprising (i) a heavy chain and (ii) a light chain; and (b) human interleukin-7 (IL-7) or a fragment or variant thereof comprising or consisting of The antibody heavy and / or light chains or fragments thereof are covalently linked to IL-7 by a peptide linker, preferably as a fusion protein.

[0252] Preferably, the bifunctional molecule according to the present invention comprises: (a) a humanized anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising (i) a heavy chain and (ii) a light chain; and (b) human interleukin 7 (IL-7) or a variant or fragment thereof comprising or consisting of The antibody heavy or light chain or fragment thereof is covalently linked to IL-7 by a peptide linker, preferably as a fusion protein.

[0253] Preferably, such bifunctional molecules comprise at least one peptide linker connecting the N-terminus of IL-7 to the C-terminus of the heavy chain or light chain or both of the anti-human PD-1 antibody, the peptide linker preferably being selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably (GGGGS)3.

[0254] Preferably, the N-terminus of IL-7 is connected to the C-terminus of the heavy chain, the light chain, or both of the anti-human PD-1 antibody via at least one peptide linker, or the C-terminus of IL-7 is connected to the N-terminus of the heavy chain, the light chain, or both of the anti-human PD-1 antibody via at least one peptide linker.

[0255] In one embodiment, the bifunctional molecule according to the present invention comprises: (a) an anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising (i) a heavy chain and (ii) a light chain; (b) human interleukin 7 (IL-7) or a variant or fragment thereof, and (c) a peptide linker connecting the N-terminus of IL-7 to the C-terminus of the heavy chain or light chain, or both, of the anti-human PD-1 antibody, preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably (GGGGS)3. comprising or consisting of

[0256] In certain embodiments, the bifunctional molecule according to the present invention comprises: (a) (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 Including, - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with 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: 1; - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with 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: 2; - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, in which X1 is either D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N and E, and optionally with 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: 3; - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, wherein 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: 12; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with 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; an anti-human PD-1 antibody or an antigen-binding fragment thereof; and (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof comprising or consisting of The antibody heavy and / or light chains or fragments thereof are covalently linked to IL-7 as a fusion protein, preferably by a peptide linker.

[0257] In another embodiment, the bifunctional molecule according to the present invention comprises: (a) (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 Including, - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with 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: 1; - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with 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: 2; - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, wherein either Xi is D and X2 is selected from the group consisting of T, H, A, Y, N, E, preferably in the group consisting of H, A, Y, N, E, or Xi is E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N, E and S, and optionally with 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: 3, - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, wherein 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: 12; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with 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; an anti-human PD-1 antibody or an antigen-binding fragment thereof; and (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof comprising or consisting of The antibody heavy or light chain or both or fragments thereof are covalently linked to IL-7 as a fusion protein, preferably by a peptide linker.

[0258] In another embodiment, the bifunctional molecule according to the present invention comprises: (a) - a heavy chain CDR1 (HCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 1, optionally with 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: 1; - a heavy chain CDR2 (HCDR2) comprising or consisting of the amino acid sequence of SEQ ID NO: 2, optionally with 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: 2; - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11, optionally with 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: 4, 5, 6, 7, 8, 9, 10, or 11; - a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, optionally with 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: 13 or SEQ ID NO: 14, - a light chain CDR2 (LCDR2) comprising or consisting of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - a light chain CDR3 (LCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 16, optionally with 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; a humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprising: (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof comprising or consisting of The antibody heavy or light chain or fragment thereof is covalently linked to IL-7 as a fusion protein, preferably by a peptide linker.

[0259] Preferably, the peptide linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably is (GGGGS)3.

[0260] In another embodiment, the present invention provides (a) (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein Xi is D or E and X is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and optionally with 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; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein 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. a humanized anti-hPD1 antibody comprising: (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof; (c) a peptide linker between the light chain and / or heavy chain of the anti-hPD1 antibody and human IL-7 or a variant or fragment thereof selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably (GGGGS)3. The present invention relates to a bifunctional molecule comprising:

[0261] Preferably, the N-terminus of IL-7 is connected to the C-terminus of the heavy chain, the light chain, or both of the anti-human PD-1 antibody via at least one peptide linker, or the C-terminus of IL-7 is connected to the N-terminus of the heavy chain, the light chain, or both of the anti-human PD-1 antibody via at least one peptide linker.

[0262] In another embodiment, the present invention provides a) (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein Xi is D or E and X is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and optionally with 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; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein 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. a humanized anti-hPD1 antibody comprising: (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof A bifunctional molecule comprising or consisting of: The C-terminus of the heavy and / or light chain of the antibody or antigen-binding fragment thereof is preferably covalently linked to the N-terminus of IL-7 by a (GGGGS)3 peptide linker, in relation to the bifunctional molecule.

[0263] In another embodiment, the present invention provides (a) (i) 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, optionally with 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: 18, 19, 20, 21, 22, 23, 24, or 25, respectively; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28, optionally with 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. a humanized anti-hPD1 antibody comprising: (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof A bifunctional molecule comprising or consisting of: For bifunctional molecules, the C-terminus of the heavy and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of IL7, preferably by a (GGGGS)3 peptide linker, to form a fusion protein.

[0264] In a preferred embodiment, the C-terminus of the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of IL-7 to form a fusion protein. Preferably, only the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to IL-7.

[0265] In another embodiment, the present invention provides a) (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein Xi is D or E and X is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and optionally with 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; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein 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. a humanized anti-hPD1 antibody comprising: (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof A bifunctional molecule comprising or consisting of: For bifunctional molecules, the C-terminus of the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of IL7, preferably by a (GGGGS)3 peptide linker, to form a fusion protein.

[0266] In another embodiment, the present invention provides a) (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 24; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28 a humanized anti-hPD1 antibody comprising: (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof A bifunctional molecule comprising or consisting of: For bifunctional molecules, the C-terminus of the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of IL7, preferably by a (GGGGS)3 peptide linker, to form a fusion protein.

[0267] Preferably, the antibody or antibody fragment thereof has an IgG1 domain or an IgG4 Fc domain.

[0268] In one aspect, the antibody or antibody fragment thereof optionally comprises: T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S239D / I and K444A, preferably selected from the group consisting of N297A, optionally in combination with M252Y / S254T / T256E, and L234A / L235; 332E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K322A; and K444A, and even more preferably the IgG1 Fc domain has the mutation N297A, such as those described above.

[0269] In another embodiment, the antibody or antibody fragment thereof comprises an IgG4 Fc domain, optionally with a substitution or combination of substitutions selected from the group consisting of S228P; L234A / L235A, S228P+M252Y / S254T / T256E, and K444A, and even more preferably the IgG4 Fc domain has the mutation S228P, such as those described above.

[0270] Optionally, in any of the above-specified embodiments, the IL-7 is an IL-7 variant or mutant.

[0271] More particularly, an IL-7 variant or mutant exhibits at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 51, and such an IL-7 variant comprises at least one amino acid mutation that i) reduces the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R, ii) retains the ability to activate IL-7R, and iii) improves the pharmacokinetics of a bifunctional molecule comprising the IL-7 variant compared to a bifunctional molecule comprising wth-IL-7. More preferably, such a mutant i) reduces the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R, and ii) improves the pharmacokinetics of a bifunctional molecule comprising the IL-7 variant compared to a bifunctional molecule comprising wth-IL-7.

[0272] More specifically, an IL-7 variant or mutant may exhibit at least 75% identity to wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 51, and such IL-7 variants contain at least one mutation selected from the group consisting of: (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S; (ii) W142H, W142F, or W142Y; (iii) D74E, D74Q, or D74N, preferably D74E or D74Q; iv) Q11E, Y12F, M17L, Q22E, and / or K81R, or any combination thereof.

[0273] An IL-7 variant or mutant may comprise at least one set of substitutions 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 set forth in SEQ ID NO: 51. Such IL-7 variants or mutants are specifically set forth in SEQ ID NOs: 53-55 (SS1, SS2, and SS3, respectively). Preferably, the IL-7 variant or mutant comprises the amino acid substitutions C2S-C141S and C47S-C92S. Even more preferably, the IL-7 variant or mutant has the sequence set forth in SEQ ID NO: 54.

[0274] The IL-7 variant or mutant may contain at least one mutation selected from the group consisting of W142H, W142F, and W142Y. Such IL-7 variants or mutants are specifically set forth in the sequences set forth in SEQ ID NOs: 57-58, respectively. Preferably, the IL-7 variant or mutant contains the mutation W142H. Even more preferably, the IL-7 variant or mutant has the sequence set forth in SEQ ID NO: 56.

[0275] The IL-7 variant or mutant may contain at least one mutation selected from the group consisting of D74E, D74Q, and D74N, preferably D74E or D74Q. Such IL-7 variants or mutants are specifically set forth in the sequences set forth in SEQ ID NOs: 63 to 65, respectively. Preferably, the IL-7 variant or mutant contains the mutation D74E. Even more preferably, the IL-7 variant or mutant has the sequence set forth in SEQ ID NO: 63.

[0276] IL-7 variants or mutants may contain at least one mutation selected from the group consisting of Q11E, Y12F, M17L, Q22E, and / or K81R. These mutations refer to the amino acid positions set forth in SEQ ID NO: 51. Such IL-7 variants or mutants are specifically set forth in the sequences set forth in SEQ ID NOs: 59, 60, 61, 62, and 66, respectively.

[0277] The IL-7 variant or mutant may comprise at least one mutation consisting of i) W142H, W142F, or W142Y, and / or ii) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or iii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0278] The IL-7 variant or mutant may comprise a W142H substitution and at least one mutation consisting of i) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0279] The IL-7 variant or mutant may comprise a D74E substitution and at least one mutation consisting of i) W142H, W142F, or W142Y, and / or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0280] The IL-7 variant or mutant may comprise the 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.

[0281] IL-7 variants or mutants may comprise i) D74E and W142H substitutions, and ii) mutations C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0282] An IL-7 variant or mutant may comprise or consist of the amino acid sequence shown in SEQ ID NO: 53, 54, 55, 56, 57, 58, 63, 64, or 65.

[0283] In a particular embodiment, the IL-7 is an IL-7 variant according to the invention, and the antibody or antibody fragment thereof optionally comprises the following amino acid sequence: T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / and K444A, preferably N297A, optionally in combination with M252Y / S254T / T256E, and L234A / L235; even more preferably, the IgG1 Fc domain has the mutation N297A, such as those described above. Preferably, the antibody or fragment thereof is linked to the IL-7 or variant thereof by a linker selected from the group consisting of (GGGGS)3, (GGGGS)4, and (GGGS)3, more preferably by (GGGGS)3. Preferably, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N. More preferably, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N. Even more preferably, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, and D74E.

[0284] In a particular embodiment, the bifunctional molecule according to the invention comprises: (a) an antibody or antibody fragment thereof, such as those described herein above, which specifically binds to a target expressed on the surface of an immune cell, preferably on a T cell, and more preferably the target is selected from the group consisting of PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8, preferably the group consisting of PD-1, TIM3, CD244, LAG-3, BTLA, TIGIT, and CD160; (b) human interleukin-7 of SEQ ID NO: 51 or a variant or fragment thereof, and (c) optionally a peptide linker selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGS, GGG, GGS, and (GGGS)3, preferably (GGGGS)3; It is a fusion protein comprising or consisting of:

[0285] Any and all of the specific aspects and embodiments detailed above disclosed with respect to the bifunctional anti-PD-1 molecules may be applied to such alternative bifunctional molecules.

[0286] In certain aspects, the antibodies or antibody fragments thereof, such as those described herein above, specifically bind to a target expressed on the surface of immune cells, preferably on T cells, more preferably the target is selected from the group consisting of PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8, preferably the group consisting of PD-1, TIM3, CD244, LAG-3, BTLA, TIGIT, and CD160; IL-7 is a target of interest in the present invention. and the antibody or antibody fragment thereof is optionally an IL-7 variant according to the invention, wherein the antibody or antibody fragment thereof is optionally an IL-7 variant according to the invention, wherein the antibody or antibody fragment thereof is optionally an IL-7 variant according to the invention, wherein the antibody or antibody fragment thereof is optionally an IL-7 variant according to the invention, wherein the antibody or antibody fragment thereof is optionally an IL-7 variant according to the invention, wherein the antibody or antibody fragment and K444A, preferably N297A, optionally in combination with M252Y / S254T / T256E, and L234A / L235; and even more preferably, the IgG1 Fc domain has the mutation N297A, such as those described above. Preferably, the antibody or fragment thereof is linked to the IL-7 or variant thereof by a linker selected from the group consisting of (GGGGS)3, (GGGGS)4, and (GGGS)3, more preferably by (GGGGS)3. Preferably, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N.More preferably, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N. Even more preferably, the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, and D74E.

[0287] Binding of bifunctional molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, anti-hPD-1 antibody / IL-7 complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to IL-7 or the receptor for IL-7.

[0288] In some examples, the bifunctional molecules described herein inhibit the PD-1 signaling pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

[0289] Preferably, such bifunctional molecules are capable of blocking or inhibiting the interaction of PD-1 with its ligands (e.g., PD-L1 and / or PD-L2). In certain embodiments, the bifunctional molecules inhibit the binding interaction of PD-1 with its ligands (e.g., PD-L1 and / or PD-L2) by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0290] In some examples, the bifunctional molecules described herein inhibit the PD-1 signaling pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

[0291] In some instances, the bifunctional molecules described herein stimulate IFN gamma secretion and / or alpha 4 and beta 7.

[0292] In another example, the bifunctional molecules described herein promote T cell infiltration in tumors.

[0293] In some examples, the bifunctional molecules described herein stimulate the IL-7R signaling pathway by at least 10%, at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

[0294] In other embodiments, the bifunctional molecules described herein retain substantially equivalent biological IL-7 properties compared to wild-type IL-7. For example, they retain biological properties comparable to those of the full-length IL-7 protein. The biological activity of the IL-7 protein can be measured using an in vitro cell proliferation assay or by measuring P-Stat5 in T cells by ELISA or FACS. Preferably, the IL-7 bifunctional molecules described herein retain at least 10%, 20%, 30%, 40%, 50%, or 60% of the biological activity compared to wild-type human IL-7, preferably at least 80%, 90%, 95%, and even more preferably 99% of the biological activity compared to wild-type IL-7. For example, biological activity can be assessed by measuring the ability of the bifunctional molecules described herein to bind to the IL-7R and / or compete with wild-type IL-7 for binding to the IL-7R.

[0295] In another example, the bifunctional molecules described herein induce cytokine secretion and / or proliferation of naive, partially exhausted and / or fully exhausted T cell subsets.

[0296] Preparation of bifunctional molecules - Nucleic acid molecules encoding bifunctional molecules, recombinant expression vectors and host cells containing them To generate a bifunctional molecule of the invention, an anti-hPD1 antibody of the invention is operably linked to IL-7 or a variant thereof.

[0297] Both entities of the bifunctional molecule are encoded in the same vector and produced as a fusion protein. Accordingly, also disclosed herein are nucleic acids encoding any of the bifunctional molecules described herein, vectors such as expression vectors or recombinant viruses that contain such nucleic acids, and host cells that contain the nucleic acids and / or vectors.

[0298] To produce a bifunctional fusion protein of the invention that is secreted in a stable form by mammalian cells, the nucleic acid sequence encoding the bifunctional molecule is subcloned into an expression vector commonly used to transfect mammalian cells. Basic techniques for producing molecules containing antibody sequences are described in Coligan et al. (eds.), Current Protocols in Immunology, pp. 10.19.1-10.19.11 (Wiley Interscience, 1992), the contents of which are incorporated herein by reference, and in W. H. Freeman and Company's "Antibody Engineering: A Practical Guide" (1992), whose relevant reviews of molecule production are interspersed throughout the text.

[0299] In general, such methods include: (1) transfecting or transforming a suitable host cell with a polynucleotide encoding a recombinant bifunctional molecule of the invention or a variant thereof, or a vector containing said polynucleotide; (2) culturing the host cells in an appropriate medium; and (3) Optionally, isolating or purifying the protein from the medium or the host cell. Includes.

[0300] The present invention further relates to nucleic acids encoding the bifunctional molecules as disclosed above, vectors, preferably expression vectors, comprising the nucleic acids of the invention, genetically engineered host cells transformed with the vectors of the invention or directly with sequences encoding the recombinant bifunctional molecules, and methods for producing the proteins of the invention by recombinant techniques.

[0301] Nucleic acids, vectors, and host cells are described in more detail herein below.

[0302] Nucleic acid sequence The present invention also relates to a nucleic acid molecule encoding a bifunctional molecule as defined above, or to a group of nucleic acid molecules encoding a bifunctional molecule as defined above.

[0303] Antibody DNA sequences may be, for example, amplified from RNA of cells that synthesize immunoglobulins, or synthesized using PCR with cloned immunoglobulins, or synthesized with oligonucleotides encoding known signal peptide amino acid sequences.

[0304] Preferably, the peptide signal comprises or consists of the amino acid sequence of SEQ ID NO: 49 for VH and / or CH and / or the amino acid sequence of SEQ ID NO: 50 for VL and / or CL. In particular, the peptide signal is present at the N-terminus of CH, VH, CL, and / or VL.

[0305] Such nucleic acids can encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of an antibody (e.g., the light and / or heavy chains of the antibody). Such nucleic acids can be readily isolated and sequenced using conventional procedures.

[0306] In particular, a nucleic acid molecule encoding a bifunctional molecule as defined above is - a first nucleic acid molecule encoding the variable heavy chain domain of an anti-hPD-1 antibody as disclosed herein, optionally having a peptide signal of SEQ ID NO: 49; and - a second nucleic acid molecule encoding the variable light chain domain of an anti-hPD-1 antibody as disclosed herein, optionally having a peptide signal of SEQ ID NO: 50; and - a third nucleic acid encoding IL-7 or a variant thereof, preferably human IL-7 or a variant thereof, operably linked to either the first nucleic acid or the second nucleic acid, or both, optionally via a nucleic acid encoding a peptide linker; Includes.

[0307] Preferably, the nucleic acid molecule encoding the bifunctional molecule as defined above comprises: - a first nucleic acid molecule encoding a variable heavy chain domain according to SEQ ID NO: 17, in which X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N and E, and optionally having a peptide signal according to SEQ ID NO: 49, and - a second nucleic acid molecule encoding the variable light chain domain of SEQ ID NO: 26, wherein X is G or T, and optionally having a peptide signal of SEQ ID NO: 50, and - a third nucleic acid molecule encoding human IL-7 of SEQ ID NO: 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63, or a variant or fragment thereof, operably linked to either the first nucleic acid or the second nucleic acid, or both, optionally via a nucleic acid encoding a peptide linker. Includes.

[0308] Preferably, the nucleic acid molecule encoding the bifunctional molecule as defined above comprises: - a first nucleic acid molecule encoding a variable heavy chain domain of the amino acid sequence shown in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25, optionally having a peptide signal of SEQ ID NO: 49, and - a second nucleic acid molecule encoding a variable light chain domain of the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 28, optionally with a peptide signal of SEQ ID NO: 50, and - a third nucleic acid molecule encoding human IL-7 of SEQ ID NO: 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 or a variant thereof, operably linked to either the first nucleic acid or the second nucleic acid, or both, optionally via a nucleic acid encoding a peptide linker. Includes.

[0309] In very particular embodiments, the nucleic acid molecule encoding the variable heavy chain domain has the sequence set forth in SEQ ID NO: 73 and / or the nucleic acid molecule encoding the variable light chain domain has the sequence set forth in SEQ ID NO: 74.

[0310] By "operably linked" is intended that the nucleic acid encodes a protein fusion comprising a variable heavy or light chain domain, optionally a peptide linker, and IL-7. Preferably, the linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably (GGGGS)3.

[0311] In one embodiment, the nucleic acid molecule is an isolated, particularly a non-naturally occurring, nucleic acid molecule.

[0312] The nucleic acid molecule or group of nucleic acid molecules encoding the bifunctional molecule according to the invention is preferably comprised in a vector or group of vectors.

[0313] vector In another aspect, the present invention relates to a vector comprising a nucleic acid molecule or a group of nucleic acid molecules as defined above.

[0314] As used herein, a "vector" is a nucleic acid molecule used as a vehicle for transferring genetic material into cells. The term "vector" encompasses plasmids, viruses, cosmids, and artificial chromosomes. Genetically engineered vectors generally contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is generally a nucleotide sequence, typically a DNA sequence, that contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may include additional features in addition to the transgene insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically intended for expressing a transgene in target cells and generally contain regulatory sequences.

[0315] In one embodiment, both the heavy chain coding sequence and the light chain coding sequence, and / or the constant region of the anti-PD1 antibody, are contained in a single expression vector. The heavy chain coding sequence and the light chain coding sequence may each be operably linked to a suitable promoter, and the heavy chain and / or the light chain may be operably linked to an immunotherapeutic agent according to the invention. Alternatively, expression of both the heavy chain and the light chain may be driven by the same promoter. In another embodiment, the heavy chain and the light chain of the antibody are each cloned into individual vectors, and one or both of the heavy chain and the light chain, the heavy chain and / or the light chain, are operably linked to an immunotherapeutic agent according to the invention. In the latter case, expression vectors encoding the heavy chain and the light chain can be co-transfected into a single host cell for expression of both chains, and the two chains can assemble to form an intact antibody either in vivo or in vitro. Alternatively, the heavy chain-encoding expression vector and the light chain-encoding expression vector can be introduced into different host cells to express each of the heavy chain and the light chain, which can then be purified and assembled to form an intact antibody in vitro.

[0316] Those skilled in the art can clone a nucleic acid molecule encoding a humanized anti-PD-1 antibody or antibody fragment thereof into a vector and then transform it into a host cell. Thus, the present invention also provides a recombinant vector comprising a nucleic acid molecule encoding an anti-PD-1 antibody of the present invention or a fragment thereof. In a 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.

[0317] Suitable expression vectors typically contain: (1) prokaryotic DNA elements encoding a bacterial origin of replication and an antibiotic resistance marker to provide for growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control transcription initiation, such as a promoter; and (3) DNA elements that control processing of the transcript, such as transcription termination / polyadenylation sequences.

[0318] Methods known to those skilled in the art can be used to construct expression vectors containing the nucleic acid sequences of the bifunctional molecules described herein and appropriate regulatory elements for transcription / translation. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence is operatively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector may further contain a ribosome binding site for initiating translation, a transcription terminator, etc.

[0319] Expression vectors can be introduced into host cells using a variety of techniques, including calcium phosphate transfection, liposome-mediated transfection, and electroporation. Preferably, transfected cells in which the expression vector has stably integrated into the host cell genome are selected and propagated to produce stable transformants. Techniques for introducing vectors into eukaryotic cells and for selecting stable transformants using dominant selectable markers are described in Sambrook, Ausubel, and Bebbington, "Expression of Antibody Genes in Nonlymphoid Mammalian Cells," in 2 METHODS: A companion to methods in enzymology, Vol. 136 (1991), and Murray (ed.), Gene transfer and expression protocols (Humana Press, 1991). Suitable cloning vectors are described in Sambrook et al. (eds.), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition (Cold Spring Harbor Press, 1989) (hereinafter "Sambrook"); Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience, 1987) (hereinafter "Ausubel"); and Brown (eds.), MOLECULAR BIOLOGY LABFAX (Academic Press, 1991).

[0320] host cell In another aspect, the present invention relates to a host cell comprising a vector or a nucleic acid molecule or a group of nucleic acid molecules as defined above, for example for the purpose of producing a bifunctional molecule.

[0321] As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be or has been a recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antibody constructs of the present invention; and / or the antibody construct or bifunctional molecule itself. Introduction of each substance into a cell can be accomplished by transformation, transfection, and the like. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, including, but not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., mouse, rat, rabbit, macaque, or human cells.

[0322] In one embodiment, the host cell contains (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and / or an amino acid sequence comprising the VH of an antibody and / or a constant region of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody.

[0323] In another embodiment, the host cell contains (e.g., is transformed with) a vector that comprises both entities of the bifunctional molecule. Preferably, the host cell contains (e.g., is transformed with) a vector that comprises a first nucleic acid molecule encoding the variable heavy chain domain of an anti-hPD-1 antibody as disclosed herein and a second nucleic acid molecule encoding the variable light chain domain of an anti-hPD-1 antibody as disclosed herein operably linked to a third nucleic acid encoding IL-7 or a variant or mutant thereof, preferably human IL-7 or a variant thereof.

[0324] Also provided herein are methods for producing humanized anti-PD1 antibodies. The methods include culturing a host cell containing nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). In particular, for recombinant production of a humanized anti-PD1 antibody, for example, nucleic acid encoding the antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell.

[0325] The bifunctional molecules of the present invention are preferably expressed in eukaryotic cells, such as mammalian cells, plant cells, insect cells, or yeast cells. Mammalian cells are particularly preferred eukaryotic hosts because they provide suitable post-translational modifications, such as glycosylation. Preferably, such suitable eukaryotic host cells may be fungi, such as Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe; insect cells, such as Mythimna separata; plant cells, such as tobacco; and mammalian cells, such as BHK cells, 293 cells, CHO cells, NSO cells, and COS cells. Other examples of useful mammalian host cell lines include CV-1 in origin with SV40 genes cells (COS cells), monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham, FL et al., J. Gen Virol., 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., Mather, JP, Biol. Reprod., 23 (1980) pp. 243-252); human epithelial kidney cells (HEK cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TRI cells, as described, for example, in Mather, J.P. et al., Annals NY Acad. Sci., 383 (1982) pp. 44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA, 77 (1980) pp. 4216-4220); and myeloma cell lines such as Y0, NSO, and Sp2 / 0.For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, NJ, USA (2004), pp. 255-268. For example, mammalian cell lines adapted for growth in suspension may be useful.

[0326] In particular, the host cells of the present invention are selected from the group consisting of CHO cells, COS cells, NSO cells, and HEK cells.

[0327] For mammalian hosts, the transcriptional and translational control signals of the expression vectors may be derived from viral sources such as adenovirus, bovine papillomavirus, or simian virus, with the control signals associated with particular genes exhibiting high levels of expression. Suitable transcriptional and translational control sequences can also be obtained from mammalian genes such as actin, collagen, myosin, and metallothionein genes.

[0328] Stable transformants producing the bifunctional molecules of the present invention can be identified using a variety of methods. After molecule-producing cells are identified, the host cells are cultured under conditions (e.g., temperature, medium) suitable for their growth and expression of the bifunctional molecules. The bifunctional molecules are then isolated and / or purified by any method known in the art. These methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (such as salt precipitation), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and combinations thereof. As described, for example, by Coligan, bifunctional molecule isolation techniques can include affinity chromatography with Protein A Sepharose, size exclusion chromatography, and ion exchange chromatography, among others. Protein A is preferably used to isolate the bifunctional molecules of the present invention.

[0329] Pharmaceutical compositions and methods of administration thereof The present invention also relates to pharmaceutical compositions comprising, preferably as an active ingredient or compound, any of the bifunctional molecules described herein, nucleic acid molecules, groups of nucleic acid molecules, vectors, and / or host cells as disclosed hereinabove. The formulations can be sterilized and, if desired, mixed with auxiliary agents such as pharmaceutically acceptable carriers and excipients that do not adversely interact with the bifunctional molecules of the invention, nucleic acids of the invention, vectors, and / or host cells. Optionally, the pharmaceutical compositions may further comprise additional therapeutic agents, as described in more detail below.

[0330] Preferably, pharmaceutical compositions of the present invention may comprise a bifunctional molecule as described herein, a nucleic acid molecule, a group of nucleic acid molecules, a vector, and / or a host cell as described hereinabove, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, excipients, salts, and antioxidants as described hereinbelow. Desirably, a pharmaceutically acceptable form is used that does not adversely affect the desired immunopotentiating effect of the bifunctional molecule of the present invention. For ease of administration, the bifunctional molecules as described herein can be formulated into pharmaceutical compositions for in vivo administration. Means for formulating such compositions are described in the art (see, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st Edition (2005)).

[0331] In particular, pharmaceutical compositions according to the present invention can be formulated for any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration. Preferably, pharmaceutical compositions according to the present invention are formulated for enteral or parenteral administration. Compositions and formulations for parenteral administration may comprise sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carder compounds, and other pharmaceutically acceptable carriers or excipients.

[0332] Pharmaceutical compositions can be prepared in the form of a lyophilized formulation or aqueous solution by mixing a substance having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A., ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used and include: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0333] Solid pharmaceutically acceptable vehicles may contain one or more substances that can also act as flavoring agents, lubricants, solubilizers, suspending agents, pigments, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, pigments, coatings, or tablet disintegrating agents. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low-melting waxes, and ion exchange resins. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Except insofar as any conventional media or substances are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated.

[0334] The bifunctional molecules of the present invention can be dissolved or suspended in pharmaceutically acceptable liquid vehicles such as water, organic solvents, ethanol, and polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), pharmaceutically acceptable oils or fats, or mixtures of both, as well as suitable mixtures thereof. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, humectants, thickeners, colorants, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid vehicles for oral and enteral administration include water (partially containing additives such as those described above, e.g., cellulose derivatives, preferably carboxymethylcellulose sodium solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for enteral administration. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0335] The pharmaceutical compositions of the present invention may further comprise one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkali or alkaline earth metals such as sodium, potassium, magnesium, and calcium, and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0336] The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant, examples of which include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0337] To facilitate delivery, either the bifunctional molecule or its encoding nucleic acid may be conjugated to a chaperone agent. The chaperone agent 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. The chaperone agent may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, and polyphosphazine. In one example, the chaperone agent is a micelle, liposome, nanoparticle, or microsphere. Methods for preparing such micelles, liposomes, nanoparticles, or microspheres are well known in the art. See, for example, U.S. Patent Nos. 5,108,921; 5,354,844; 5,416,016; and 5,527,5285.

[0338] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations and / or suitable for injection. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0339] In one embodiment, the pharmaceutical composition is an injectable composition that may contain various carriers, such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol). For intravenous injection, water-soluble antibodies can be administered by infusion, where a formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular formulations, e.g., a sterile formulation of a suitable soluble salt form of the antibody can be dissolved and administered in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0340] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the ingredients listed above, followed by sterile microfiltration as needed.Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above.For sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying (lyophilization) to obtain a powder of active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate salts and gelatin.

[0341] Prevention of the presence of microorganisms can be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0342] Those skilled in the art will understand that the formulations of the present invention may be isotonic with human blood, i.e., 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, using a vapor pressure or ice-freezing osmometer. The tonicity of the formulation is adjusted using a tonicity adjuster. A "tonicity adjuster" is a pharmaceutically acceptable inert substance that can be added to a formulation to provide isotonicity to the formulation. Tonicity adjusters suitable for the present invention include, but are not limited to, saccharides, salts, and amino acids.

[0343] Pharmaceutical compositions according to the present invention may be formulated to release the active ingredient (e.g., a bifunctional molecule of the present invention) substantially immediately after administration or at any predetermined time or period after administration. In some embodiments, pharmaceutical compositions may employ time-release, delayed-release, and sustained-release delivery systems so that delivery of the composition occurs prior to and in sufficient time to cause sensitization of the area to be treated. Means known in the art may be used to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed release of the composition. Such systems may avoid repeated administration of the composition, thereby increasing convenience for the subject and the physician.

[0344] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.

[0345] Subjects, Regimen, and Administration The present invention relates to a bifunctional molecule as disclosed herein for use as a medicament, or for use in treating a disease, or for administration to a subject, or for use as a medicament; a nucleic acid or vector encoding the same, a host cell, or a pharmaceutical composition, nucleic acid, vector, or host cell. The present invention also relates to the use of a pharmaceutical composition, nucleic acid, vector, or host cell of the present invention, or a bifunctional molecule comprising an anti-PD1 antibody or antibody fragment thereof and IL-7 or a variant thereof, in the manufacture of a medicament for treating a disease in a subject. Finally, the present invention relates to a method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or a bifunctional molecule comprising an anti-PD1 antibody or antibody fragment thereof and IL-7 or a variant thereof. Exemplary treatments are described in more detail herein below in the "Methods and Uses" section.

[0346] The subject to be treated may be a human, in particular a human in the prenatal stage, a newborn, a child, an infant, an adolescent, or an adult, in particular an adult at least 30, 40 years of age, preferably an adult at least 50 years of age, even more preferably an adult at least 60 years of age, and even more preferably an adult at least 70 years of age.

[0347] In particular, the subject suffers from a disease in which the PD-1 / PD-L1 pathway may be involved, particularly a disease in which at least one of the ligands of PD-1 (e.g., PD-L1 and / or PD-L2) or PD-1 is expressed, particularly overexpressed. Preferably, the subject suffers from cancer, even more preferably a PD1-, PD-L1-, and / or PD-L2-positive cancer, or a PD-1-positive cancer. Exemplary diseases and cancers are described in more detail herein below in the "Methods and Uses" section.

[0348] In certain embodiments, the subject has already received at least one treatment, preferably several courses of treatment, prior to administration of the bifunctional molecule according to the invention comprising an anti-PD1 antibody or antibody fragment thereof and IL-7 or a variant thereof, or the pharmaceutical composition according to the invention.

[0349] The bifunctional molecules or pharmaceutical compositions disclosed herein can be administered to a subject using conventional methods known to those skilled in the medical field, depending on the type of disease or site of disease to be treated. The compositions can be administered by conventional routes, for example, orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intratumoral, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. When administered parenterally, the pharmaceutical compositions of the present invention are preferably administered via the intravenous route. When administered enterally, the pharmaceutical compositions of the present invention are preferably administered via the oral route. The compositions can also be administered topically.

[0350] The form of the pharmaceutical composition, the route of administration and the dosage of the pharmaceutical composition or bifunctional molecule according to the invention can be adjusted by those skilled in the art depending on the type and severity of the infection, the age, weight, sex and general health of the patient, in particular the patient. The compositions of the invention can be administered in several ways depending on whether a local or systemic treatment is desired.

[0351] Preferably, treatment with a bifunctional molecule or with a pharmaceutical composition according to the invention is administered periodically, preferably daily, weekly, or monthly, more preferably daily to every 1, 2, 3, or 4 weeks. In certain embodiments, treatment is administered several times daily, preferably two or three times daily.

[0352] The duration of treatment with the bifunctional molecules or pharmaceutical compositions according to the present invention is preferably comprised between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, even more preferably between 1 day and 4 weeks, and even more preferably between 1 day and 2 weeks. Alternatively, treatment may be continued for as long as the disease persists.

[0353] The bifunctional molecules disclosed herein can be provided at an effective dose range of about 1 ng / kg body weight to about 30 mg / kg body weight, 1 μg / kg to about 20 mg / kg, 10 μg / kg to about 10 mg / kg, or 100 μg / kg to 5 mg / kg, optionally every 1, 2, 3, or 4 weeks, preferably by parenteral or oral administration, particularly by intravenous or subcutaneous administration.

[0354] In particular, the bifunctional molecules according to the invention may be administered at sub-therapeutic doses. The term "sub-therapeutic dose," as used herein, refers to a dose below effective monotherapy dosage levels commonly used to treat a disease or a dose that is not currently typically used for effective monotherapy with anti-hPD1 antibodies.

[0355] Methods and Uses Use in the treatment of disease The bifunctional molecules, nucleic acids, vectors, host cells, compositions, and methods of the present invention have numerous in vitro and in vivo utilities and applications. For example, the bifunctional molecules, nucleic acids, vectors, host cells, and / or pharmaceutical compositions described herein can be used as therapeutic agents, diagnostic agents, and pharmaceutical research agents. 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 treatment methods and / or for therapeutic purposes. In particular, the bifunctional molecules, nucleic acids, vectors, or pharmaceutical compositions provided herein may be useful for treating any disease or condition, preferably any disease or condition involving PD-1, such as cancer, autoimmune diseases, and other diseases associated with immune deficiencies, such as infectious diseases or T-cell dysfunction. Even more preferably, 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 in need thereof, comprising administering to the subject an effective amount of a bifunctional molecule or pharmaceutical composition as defined above. Examples of such diseases are described in more detail herein below.

[0356] In particular, the bifunctional molecules according to the present invention are referred to as "bifunctional checkpoint inhibitors" because they target both the PD-1 / PD-L1 / PD-L2 pathway and the IL7 pathway.

[0357] The present invention particularly relates to bifunctional molecules, nucleic acids, groups of nucleic acids or vectors encoding same, or pharmaceutical compositions comprising same, for use in the treatment of pathologies, diseases and / or disorders that can be prevented or treated by inhibiting the binding of PD-L1 and / or PD-L2 to PD-1.

[0358] The bifunctional molecules of the present invention target CD127+ immune cells, particularly CD127+ T cells. Such cells can be found in the following specific target areas: resident lymphoid cells in lymph nodes (mainly in the paracortex, with occasional cells in the follicles), tonsils (interfollicular area), spleen (mainly in the periarteriolar lymphoid sheaths (PALS) of the white pulp and some scattered cells in the red pulp), thymus (mainly in the medulla or cortex), bone marrow (scattered distribution), GALT (gut-associated lymphoid tissue, mainly in the interfollicular area and lamina propria) in the gastrointestinal tract (stomach, duodenum, jejunum, ileum, cecum, rectum), and MALT (mucosa-associated lymphoid tissue) in the gallbladder. Therefore, the bifunctional molecules of the present invention are particularly interesting for treating diseases located in or involving these areas, especially cancer.

[0359] Thus, disclosed herein are methods for treating diseases, particularly diseases associated with the PD-1 and / or PD-1 / PD-L1 and / or PD-1 / PD-L2 signaling pathways, comprising administering to a subject in need thereof an effective amount of any of the bifunctional molecules or pharmaceutical compositions described herein. The patient's physiological data (e.g., age, size, and weight) and the route of administration must also be taken into consideration to determine the appropriate dosage, so that a therapeutically effective amount is administered to the patient.

[0360] In another aspect, the bifunctional molecules disclosed herein can be administered to a subject, e.g., in vivo, to enhance immunity, preferably to treat a disorder and / or disease. Accordingly, in one aspect, the present invention provides a method of modifying an immune response in a subject, comprising administering to the subject a bifunctional molecule, nucleic acid, vector, or pharmaceutical composition of the present invention, such that the immune response in the subject is modified. Preferably, the immune response is enhanced, augmented, stimulated, or upregulated. The bifunctional molecule or pharmaceutical composition can be used to enhance an immune response, such as T cell activation, in a subject in need of treatment. Enhancement of the immune response can result in inhibition of binding of PD-L1 and / or PD-L2 to PD-1, thereby reducing the immunosuppressive environment and stimulating human T cell proliferation and / or activation and / or IFNγ secretion by human PBMCs.

[0361] The present invention particularly provides a method of enhancing an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a bifunctional molecule, nucleic acid, vector or pharmaceutical composition comprising same as described herein, such that the immune response in the subject is enhanced.

[0362] In some embodiments, the amount of a bifunctional molecule described herein is effective to inhibit PD-1 signaling (e.g., to reduce PD-1 signaling by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control). In other embodiments, the amount of a bifunctional molecule described herein is effective to activate an immune response (e.g., by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control).

[0363] In some embodiments, the amount of bif...

Claims

1. (a)(i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain (VL) comprising LCDR1, LCDR2, and LCDR3; an antagonist anti-human PD-1 antibody or antigen-binding fragment thereof comprising: (b) human interleukin-7 (IL-7) or a variant thereof consisting of the amino acid sequence set forth in SEQ ID NO: 51, wherein the IL-7 variant comprises the amino acid sequence set forth in SEQ ID NO: 51 or has at least 90% sequence identity with wild-type human IL-7 (wth-IL-7) consisting of the amino acid sequence set forth in SEQ ID NO: 51; A bifunctional molecule comprising: the antibody or fragment thereof is covalently linked to the human IL-7 or variant thereof as a fusion protein; the N-terminus of the human IL-7 or variant thereof is connected to the C-terminus of the heavy chain, the light chain, or both of the anti-human PD-1 antibody or antigen-binding fragment thereof; Bifunctional molecules.

2. The bifunctional molecule of claim 1, wherein the N-terminus of the human IL-7 or variant thereof is connected to the C-terminus of the heavy chain, the light chain, or both, of the anti-human PD-1 antibody or antigen-binding fragment thereof by a peptide linker.

3. The bifunctional molecule of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a human antibody.

4. The anti-human PD-1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain (VL) comprising LCDR1, LCDR2, and LCDR3; Including, - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1; - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, wherein X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S; - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, in which X is G or T; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16; A bifunctional molecule according to any one of claims 1 to 3.

5. 5. The bifunctional molecule of claim 1 , wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: (a) a 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; and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T.

6. 6. The bifunctional molecule of claim 1 , wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises or consists of: (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 24; and (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:

28.

7. The anti-PD-1 antibody includes pembrolizumab, nivolumab, pidilizumab, cemiplimab, PDR001, AUNP12, AMP-224, AGEN-2034, BGB-A317, MK-3477, SCH-900475, PF-06801591, JNJ-63723283, genolimuzumab, LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103, MEDI-0680, MEDI0608, JS001, and BI-754091.

4. The bifunctional molecule of claim 1, wherein the bifunctional molecule is selected from the group consisting of: CBT-501, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, AGEN2034, MGA012, IBI308, RG7769, XmAb20717, MEDI5752, FS118, SL-279252, XmAb23104, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4.

8. The bifunctional molecule of any one of claims 1 to 7, wherein the IL-7 or variant thereof comprises or consists of an amino acid sequence having at least 95% identity to wild-type human IL-7 (wth-IL-7) consisting of the amino acid sequence set forth in SEQ ID NO:

51.

9. 9. The bifunctional molecule of claim 1, wherein the IL-7 is an IL-7 variant that exhibits at least 90% identity to wild-type human IL-7 (with IL-7) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 51, and the variant contains at least one amino acid mutation that i) reduces the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of with IL-7 for IL-7R, and ii) improves the pharmacokinetics of the bifunctional molecule comprising the IL-7 variant compared to the bifunctional molecule comprising with IL-7.

10. 10. The bifunctional molecule of claim 9, wherein the at least one mutation is an amino acid substitution or group of amino acid substitutions selected from the group consisting of: (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S; (ii) W142H, W142F, or W142Y; (iii) D74E, D74Q, or D74N; iv) Q11E, Y12F, M17L, Q22E, and / or K81R, or any combination thereof.

11. 10. The bifunctional molecule of claim 9, wherein the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, and C47S-C92S and C34S-C129S.

12. 10. The bifunctional molecule of claim 9, wherein the IL-7 variant comprises an amino acid substitution selected from the group consisting of W142H, W142F, and W142Y.

13. 10. The bifunctional molecule of claim 9, wherein the IL-7 variant comprises an amino acid substitution selected from the group consisting of D74E, D74Q, and D74N.

14. The bifunctional molecule of any one of claims 1 to 9, wherein the IL-7 variant comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 53 to 66.

15. The bifunctional molecule of any one of claims 1 to 9, wherein the IL-7 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, 56, or 63.

16. 16. The bifunctional molecule of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain.

17. The antibody or antigen-binding fragment thereof may comprise a light chain constant domain derived from a human kappa light chain constant domain, and a light chain constant domain derived from a human kappa light chain constant domain. The light chain constant domains are: T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K 17. The bifunctional molecule of any one of claims 1 to 16, comprising a heavy chain constant domain derived from a human IgG1 heavy chain constant domain with substitutions or combinations of substitutions selected from the group consisting of: 326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K322A; and K444A.

18. 17. The bifunctional molecule of any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain with a substitution or combination of substitutions selected from the group consisting of S228P, L234A / L235A, S228P+M252Y / S254T / T256E, and K444A.

19. The peptide linker sequence is (GGGGS) 3 , (GGGGS) 4 , (GGGGS) 2 , GGGGS, GGGS, GGG, GGS, and (GGGS) 3 19. The bifunctional molecule of any one of claims 2 to 18, selected from the group consisting of:

20. The antibody or antigen-binding fragment thereof may comprise a light chain constant domain derived from a human kappa light chain constant domain, and a light chain constant domain derived from a human kappa light chain constant domain. The light chain constant domains are: T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311; and K444A, wherein the antibody or fragment thereof comprises a heavy chain constant domain derived from a human IgG1 heavy chain constant domain having a substitution or combination of substitutions selected from the group consisting of K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K322A; and K444A, wherein the antibody or fragment thereof comprises a linker (GGGGS). 3 16. The bifunctional molecule of any one of claims 11 to 15, wherein the bifunctional molecule is linked to the IL-7 variant by

21. 21. An isolated nucleic acid molecule or group of isolated nucleic acid molecules encoding the bifunctional molecule of any one of claims 1 to 20.

22. 22. A vector comprising the nucleic acid molecule or group of nucleic acid molecules of claim 21.

23. 23. A host cell comprising a vector according to claim 22, or a nucleic acid molecule or group of nucleic acid molecules according to claim 21.

24. 21. A method for producing a bifunctional molecule according to any one of claims 1 to 20, comprising culturing a host cell according to claim 23, and isolating the bifunctional molecule.

25. 24. A pharmaceutical composition comprising a bifunctional molecule according to any one of claims 1 to 20, a nucleic acid molecule or a group of nucleic acid molecules according to claim 21, a vector according to claim 22, or a host cell according to claim 23, and a pharmaceutically acceptable carrier.

26. Alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antivirals, Aurora kinase inhibitors, proapoptotic agents, Bcl-2 family inhibitors, activators of death receptor pathways, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody-drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunological agents, inhibitors of inhibitor of apoptosis proteins (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, rapamycin 26. The pharmaceutical composition of claim 25, further comprising an additional therapeutic agent selected from the group consisting of mammalian target of action inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent 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 inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, epitopes or neoepitopes derived from tumor antigens, and combinations of one or more of these substances.

27. 27. A pharmaceutical composition according to claim 25 or 26, a bifunctional molecule according to any one of claims 1 to 20, a nucleic acid molecule or group of nucleic acid molecules according to claim 21, a vector according to claim 22 or a host cell according to claim 23 for use as a medicament.

28. 28. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 27 for use in the treatment of a disease selected from the group consisting of cancer.

29. The cancers include hematological malignancies or solid tumors with PD-1 and / or PD-L1 expression, hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndromes, and acute myeloid leukemia, cancers induced by viruses or associated with immunodeficiency, Kaposi's sarcoma; cervical cancer, anal cancer, penile cancer, and vulvar squamous cell carcinoma, and oropharyngeal cancer; 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, classical Hodgkin's lymphoma, and leukemia.

29. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 28, wherein the cancer is selected from the group consisting of: Hodgkin's lymphoma, and lymphoproliferative disorders; hepatocellular carcinoma; Merkel cell carcinoma; and cancers associated with human immunodeficiency virus (HIV) infection, and cancers selected from the group consisting of 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, and cervical cancer.

30. 30. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of any one of claims 27 to 29 for use in combination with radiation therapy or an additional therapeutic agent.

31. Additional therapeutic agents include alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antivirals, Aurora kinase inhibitors, proapoptotic agents, Bcl-2 family inhibitors, activators of the death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunological agents, inhibitors of inhibitor of apoptosis proteins (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian rapamycin inhibitors 31. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 30, wherein the bifunctional molecule or group of nucleic acid molecules, vector, or host cell is selected from the group consisting of a target, a microRNA, a mitogen-activated extracellular signal-regulated kinase inhibitor, a multivalent binding protein, a nonsteroidal anti-inflammatory drug (NSAID), a poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitor, a platinum chemotherapeutic agent, a polo-like kinase (Plk) inhibitor, a phosphoinositide-3 kinase (PI3K) inhibitor, a proteasome inhibitor, a purine analog, a pyrimidine analog, a receptor tyrosine kinase inhibitor, a retinoid / deltoid plant alkaloid, a small inhibitory ribonucleic acid (siRNA), a topoisomerase inhibitor, a ubiquitin ligase inhibitor, a hypomethylating agent, a checkpoint inhibitor, a peptide vaccine, an epitope or neoepitope derived from a tumor antigen, and a combination of one or more of these substances.

32. 28. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector or host cell of claim 27 for use in the treatment of an infectious disease.

33. 33. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 32, wherein the infectious disease is caused by a virus selected from the group consisting of HIV, hepatitis virus, herpes 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.

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