Bifunctional molecules against human PD-1

Abstract

The present invention provides bifunctional molecules comprising a humanized anti-hPD-1 antibody, or an antigen-binding fragment thereof, linked to an immunotherapeutic agent capable of specifically enhancing an immune response, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to the field of immunotherapy. The present invention provides bifunctional molecules comprising a humanized anti-PD1 antibody or antibody fragment thereof linked to an immunotherapeutic agent, and uses thereof. [Background technology]

[0002] Targeting T cell inhibitory checkpoints with therapeutic antibodies to reverse inhibition is an area of ​​intense research (for a 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. Immune checkpoints in innate myeloid cells (macrophages, dendritic cells, MDSCs, PMNs) remain poorly studied, yet these cells are the most abundant immune cell type in many solid tumors and are often associated with poor outcomes. Combination immune checkpoint therapy targeting both the innate immune response (mediated by myeloid cells) and the adaptive immune response (mediated by T cells) has demonstrated 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. In particular, PD-1, which functions as an immune checkpoint, plays an important role in downregulating the immune system by preventing T cell activation, thereby reducing autoimmunity and promoting self-tolerance. Two PD-1 ligands, 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 in 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, resulting in reduced activation and cytokine production.

[0005] Both strategies of disrupting their interaction using anti-PD1 and anti-PDL1 inhibitors have been successful in cancer treatment (Brahmer et al., N Eng J Med, 366(26), 2012; Powles et al., Nature, 515(7528), 2014; Topalian et al., N Eng J Med, 366(26), 2012; Ansell, Curr Opin Hematol, 22(4), 2015). However, the accumulation of immunosuppressive and hypostimulatory myeloid cells within the tumor microenvironment limits the efficiency of T cell responses and the effectiveness of immunotherapies, particularly those targeting immune checkpoints such as PD-1 / PD-L1. At the same time, immunotherapies targeting innate immune checkpoints have had limited efficacy on their own, as T cell responses remain blocked primarily due to a lack of costimulation within the tumor microenvironment and / or by binding of coinhibitory molecules to ligands expressed by tumor cells or antigen-presenting cells. Combination immunotherapies targeting both adaptive (T cell) and innate (myeloid cell) immune checkpoints have shown potent efficacy at the preclinical level. [Prior art documents] [Patent documents]

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[0007] [Non-Patent Document 1] Pardoll, Nat Rev Cancer. 2012; vol. 12: 253-264. [Non-patent document 2] Schildberg et al., Volume 44 (No. 5), Immunity, 2016 [Non-patent document 3] Sharma et al., Cell, Vol. 161(No. 2), 2015 [Non-patent document 4] Pardoll, Nature Reviews Cancer, Vol. 12(4), 2012 [Non-patent document 5] Freeman et al. (2000) J Exp Med 192:1027-34 [Non-patent document 6] Latchman et al. (2001) Nat Immunol 2:261-8 [Non-Patent Document 7] Carter et al. (2002) Eur J Immunol 32:634-43 [Non-patent document 8] Brahmer et al., N Eng J Med, 366(26), 2012 [Non-Patent Document 9] Powles et al., Nature, Vol. 515 (No. 7528), 2014 [Non-Patent Document 10] Topalian et al., N Eng J Med, 366(26), 2012 [Non-licensed Document 11] Ansell, Curr Opin Hematol, Volume 22 (No. 4), 2015 [Non-licensed Document 12] Wahl, 1983, J. Nucl. 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Reprod., Volume 23 (1980), pp. 243-252 [Non-licensed Document 50] Mather, JP, Annals NY Acad. Sci., Volume 383 (1982) Pages 44~68 [Non-licensed Document 51] Urlaub, G., Proc. Natl. Acad. Sci. USA, Volume 77 (1980) Pages 4216~4220 [Non-licensed Document 52] Yazaki, P. and Wu, AM, Methods in Molecular Biology, Volume 248, Lo, BKC (Editor), Humana Press, Tokyo, Japan (2004), pp. 255~268 [Non-licensed Document 53] Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st Edition (2005) [Non-Patent Document 54] Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980) [Non-Patent Document 55] Iwai et al. (2005) Int. Immunol. 17:133-144 [Non-Patent Document 56] Antonia et al., Immuno-oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258–6268, 2014 Summary of the Invention [Problem to be solved by the invention]

[0008] However, the validation and development of combinatorial 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 innate myeloid immune cells and have effective and positive effects on adaptive immune responses, particularly T-cell immune responses. The inventors have achieved a significant advance with the invention disclosed herein. [Means for solving the problem]

[0009] The present inventors provide a bifunctional molecule comprising a humanized anti-hPD-1 antibody and an immunotherapeutic agent, which is promising for numerous therapeutic applications, particularly the treatment of cancer. The present invention is based on the development of a humanized antibody that specifically targets human PD-1, exhibiting high binding affinity to PD-1 and potently competing with its ligands, PDL-1 and PD-L2. Surprisingly, this humanized antibody exhibits a high humanness score, enabling high production yields even when produced as a bifunctional molecule linked to an immunotherapeutic agent. Indeed, this humanized antibody has been engineered to be highly manufacturable in mammalian cell-based production systems, particularly when an active protein domain is fused to the C-terminus of its heavy or light chain, a situation typically associated with poor manufacturability.

[0010] In a first embodiment, the bifunctional molecule comprises: (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; 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, 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; Humanized PD-1 antibody or antigen-binding fragment thereof and (b) an immunotherapeutic agent or a fragment thereof It consists of The C-terminus of the heavy and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked, preferably by a peptide linker, to the N-terminus of the immunotherapeutic agent as a fusion protein.

[0011] In particular, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprises: (a) a VH comprising 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.

[0012] Preferably, the antibody or antigen-binding fragment thereof is an antagonist of human PD-L1 and / or PD-L2 binding to human PD-1.

[0013] Preferably, the immunotherapeutic agent or fragment thereof is selected from the group consisting of tumor targeting peptides, cytokines, cytokine receptors, chemokines, chemokine receptors, costimulatory molecules, inhibitory or co-inhibitory molecules, enzymes, molecular chaperone inhibitors, and type I or type II human transmembrane immunity proteins, preferably their extracellular domains.

[0014] In particular, the immunotherapeutic agent or a fragment thereof has a size comprised between 10 kDa and 50 kDa.

[0015] In certain embodiments, the immunotherapeutic agent is preferably selected from the group consisting of ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, nectin-1, nectin-2, nectin-3, NELC-5, TLT-2, LFA-3, TIM3, TIM4, LAIR1, SIRPG, IL10R, IL6RA, IL-1R1, IL-1RAcP, IL-1R2, IL-1R3, IL-1R4, IL-1R5, IL-1R6, IL-1R7, IL-1R8, IL-1R9, IL-1R10, IL-1R11, IL-1R12, IL-1R13, IL-1R14, IL-1R15, IL-1R16, IL-1R17, IL-1R18, IL-1R19 ... The human type I transmembrane immune protein or a fragment thereof is selected from the group consisting of L6RB, TGFBRII, CSF1R, IL22R, VEGFR1, VEGFR2, VEGFR3, CD111, CD112, CD155, CD113, VISTA, CD244, OX40, SIRP alpha, CD80, CD24, Siglec-10, Fas, IL15RA, SIRB1, SIRB2, LTBR, ​​IL21R, and GITR.

[0016] In another aspect, the immunotherapeutic agent is a type II human transmembrane immunity protein or a fragment thereof, preferably selected from the group consisting of CD40L, OX40L, FasL, TRAIL, TNF, LIGHT, APRIL, GITRL, CD30, CD70, CD40, CD27, CD30, CD153, RANK, CD96, CLEC1, CLEC2 / CLE1B, CLEC3A, CLEC4A, CLEC4E, CLEC4L, CLEC51, CLEC6, CLEC7A, NKG2D, BTL-II, TGFRII, DECTIN-1, DC-SIGN, LT-alpha, LT-beta, 4-1BBL, and MINCLE, and preferably a member of the TNF family.

[0017] In another aspect, the immunotherapeutic agent is a cytokine or fragment thereof selected from the group consisting of TGFβ, IL-1, IL-2, IL-6, IL-7, IL-10, IL-12A, IL12B, IL15, IL-21, IL-4, and IL-18.

[0018] In very particular embodiments, the immunotherapeutic agent is human IL-2 or a variant thereof, in particular an IL-2 variant having the sequence shown in SEQ ID NO: 58 and having the substitutions F42A, Y45A, and L72G, preferably T3A, F42A, Y45A, L72G, and C125A.

[0019] 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.

[0020] 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 The heavy chain constant domain is derived from a human IgG1 heavy chain constant domain with a substitution or combination of substitutions selected from the group consisting of: 9D / I332E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K444A, and K322A, preferably N297A, and L234A / L235A, optionally in combination with M252Y / S254T / T256E.

[0021] 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.

[0022] The present invention also relates to an isolated nucleic acid sequence or group of isolated nucleic acid molecules encoding a bifunctional molecule as disclosed herein, a vector comprising the nucleic acid or group of nucleic acid molecules, and a host cell comprising the vector or nucleic acid or group of nucleic acid molecules disclosed herein.

[0023] In one aspect, the 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.

[0024] In another aspect, the present invention relates to a pharmaceutical composition comprising a bifunctional molecule, a nucleic acid or a group of nucleic acid molecules, a vector as disclosed herein, and a pharmaceutically acceptable carrier.

[0025] 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.

[0026] Finally, the present invention relates to a pharmaceutical composition, a bifunctional molecule, a nucleic acid or group of nucleic acid molecules, a vector, or a host cell as disclosed herein for use as a medicament. In a particular embodiment, the pharmaceutical composition, the bifunctional molecule, the nucleic acid or group of nucleic acid molecules, the vector, or the host cell as disclosed herein is for use in the treatment of cancer. Preferably, the cancer is a hematological malignancy or solid tumor with expression of PD-1 and / or PD-L1, e.g., a cancer selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndrome, and acute myeloid leukemia; a cancer induced by a virus or associated with an immune deficiency, e.g., 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 (NHL), including diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classical cancers selected from the group consisting of 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.

[0027] In another particular embodiment, the pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell as disclosed herein is for use in the treatment of an infectious disease, preferably a chronic infectious disease, even more preferably a chronic viral infection 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.

[0028] 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. [Brief explanation of the drawings]

[0029] [Figure 1]This figure shows the productivity of chimeric and humanized anti-PD1 bifunctional antibodies fused to type I Ig-like proteins, type II TNF family proteins, or cytokine proteins at the C-terminus of the heavy chain (A), light chain (B), or both heavy and light chains (C). HEK freestyle mice were transiently transfected with lipofectamine and DNA plasmids encoding the humanized or chimeric bifunctional antibodies. Three days after transfection, antibody-containing supernatants were collected, and concentrations were measured by sandwich ELISA using anti-human IgG Fc as the capture antibody and anti-human IgK for detection. Statistical analysis was performed using the Mann-Whitney test (**p<0.05). As an example, in this figure, the cytokine fused to the anti-PD-1 antibody was IL-7. Type I protein fusion protein A corresponds to CD86, protein B corresponds to CD80, and protein C corresponds to SIRPa (i.e., SIRP alpha). Type II protein A corresponds to OX40L, and protein B corresponds to 4-1BBL. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO: 19 and a light chain variable domain as disclosed in SEQ ID NO: 28. [Figure 2]Figure 2 shows the productivity of bifunctional proteins with chimeric, humanized, or other anti-PD-1 scaffolds. HEK freestyle or adherent CHO cells were transiently transfected with DNA plasmids encoding the humanized or chimeric anti-PD-1 antibodies of the present invention, or the nivol2mab or pembrolizumab anti-PD-1 sequences, in which cytokines or type I proteins are fused to the C-terminus of the heavy chain. Shaker flasks or 12-well plates were used for HEK or CHO cell production, respectively. Three days after transfection, antibody-containing supernatants were collected, and concentrations were measured by sandwich ELISA using anti-human IgG Fc as a capture antibody and anti-human IgK for detection. Figure 2A: Productivity of bifunctional anti-PD-1 fused with IL-7 cytokine. Figure 2B: Productivity of bifunctional anti-PD-1 fused with CD80 protein I fusion protein. Figure 2C: Productivity of bifunctional anti-PD-1 fused with SIRPa protein I fusion protein. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:24 and a light chain variable domain as disclosed in SEQ ID NO:28. [Figure 3] Figure 1 shows the productivity of bifunctional proteins with a humanized anti-PD-1 backbone compared with other bifunctional proteins with non-anti-PD-1 backbones. HEK freestyle mice were transiently transfected with DNA plasmids encoding multiple bifunctional proteins with a humanized anti-PD-1 backbone or a non-anti-PD-1 backbone. Three days after transfection, antibody-containing supernatants were collected and concentrations were measured by sandwich ELISA using anti-human IgG Fc as a capture antibody and anti-human IgK for detection. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody with a heavy chain variable domain as disclosed in SEQ ID NO: 19, 22, or 24 and a light chain variable domain as disclosed in SEQ ID NO: 28. [Figure 4]Figure 1 shows a PD-1 binding ELISA assay. Human recombinant PD-1 protein was immobilized and anti-PD-1 bifunctional antibodies were added at various concentrations. Color development was performed using a peroxidase-coupled anti-human Fc antibody. Colorimetric analysis was determined at 450 nm using TMB substrate. (A) Data for anti-PD-1 chimeric antibodies unfused (■) or with three type I Ig-like family proteins A, B, or C fused to the VL domain (◯) or VH domain (●). (B) Data for anti-PD-1 chimeric antibodies unfused (■) or with two type II TNF family proteins A, B, or C fused to the VL domain (◯) or VH domain (●). (C) Data for anti-PD-1 chimeric antibodies unfused (■) or with cytokine family protein A fused to the VL domain (◯) or VH domain (●). In this figure, the cytokine fused to the anti-PD-1 antibody is IL-7. Type I protein fusion protein A corresponds to CD86, protein B corresponds to CD80, and protein C corresponds to SIRPa. Type II protein A corresponds to OX40L, and protein B corresponds to 4-1BBL. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as set forth in SEQ ID NO:19 and a light chain as set forth in SEQ ID NO:28. [Figure 5]Figure 1 shows PD-1 binding ELISA assays of chimeric and humanized bifunctional anti-PD-1 antibodies fused to type I protein (A), type II protein (B), and cytokine protein (C) on the heavy chain. Human recombinant PD-1 protein was immobilized, and anti-PD-1 bifunctional antibodies were added at various concentrations. Color development was performed using a peroxidase-coupled anti-human Fc antibody. Colorimetric determination was performed at 450 nm using TMB substrate. (A) Data for chimeric (●) and humanized (■) anti-PD-1 antibodies fused to three type I Ig-like family proteins A, B, or C. (B) Data for chimeric (●) and humanized (■) anti-PD-1 antibodies fused to type II TNF family protein A or B. (C) Data for chimeric (●) and humanized (■) anti-PD-1 antibodies fused to cytokine family protein A. In this figure, the cytokine fused to the anti-PD-1 antibody was IL-7. Type I protein fusion protein A corresponds to CD86, protein B corresponds to CD80, and protein C corresponds to SIRPa. Type II protein A corresponds to OX40L, and protein B corresponds to 4-1BBL. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as set forth in SEQ ID NO: 19 and a light chain variable domain as set forth in SEQ ID NO: 28. [Figure 6]Figure 1 shows PD-1 binding ELISA assays of chimeric and humanized bifunctional anti-PD-1 antibodies fused to the light chains of type I protein (A), type II protein (B), and cytokine protein (C). Human recombinant PD-1 protein was immobilized, and anti-PD-1 bifunctional antibodies were added at various concentrations. Color development was performed using a peroxidase-coupled anti-human Fc antibody. Colorimetric determination was performed at 450 nm using TMB substrate. (A) Data for anti-PD-1 chimeric (●) antibodies fused to three type I Ig-like family proteins A, B, or C versus humanized (■) antibodies. (B) Data for anti-PD-1 chimeric (●) and humanized (■) antibodies fused to type II TNF family protein A or B. (C) Data for anti-PD-1 chimeric (●) and humanized (■) antibodies fused to cytokine family protein A. In this figure, the cytokine fused to the anti-PD-1 antibody is IL-7. Type I protein fusion protein A corresponds to CD86, protein B corresponds to CD80, and protein C corresponds to SIRPa. Type II protein A corresponds to OX40L, and protein B corresponds to 4-1BBL. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as set forth in SEQ ID NO: 19 and a light chain variable domain as set forth in SEQ ID NO: 28. [Figure 7] Figure 1 shows a PD-1 binding ELISA assay of a chimeric bifunctional anti-PD-1 antibody fused to cytokine protein A on both the heavy and light chains versus a humanized bifunctional anti-PD-1 antibody. Human recombinant PD-1 protein was immobilized, and anti-PD-1 Abs fused to cytokine A (IL-7) on both the heavy and light chains (chimeric anti-PD-1 Ab (▲) and humanized anti-PD-1 Ab (△)) were added at different concentrations. Color development was performed using a peroxidase-coupled anti-human Fc antibody. Colorimetric determination was performed at 450 nm using TMB substrate. In this experiment, the bifunctional molecule comprises a humanized anti-PD-1 antibody having a heavy chain variable domain as set forth in SEQ ID NO:19 and a light chain variable domain as set forth in SEQ ID NO:28. [Figure 8-1]Figure 1 shows competitive PD-1 / PD-L1 or PD-L2 ELISA assays. A: PD-1 / PD-L1 antagonist activity. PD-L1 was immobilized and antibody + biotinylated recombinant human PD-1 complexes were added. Various concentrations of anti-PD-1 antibodies were tested, and recombinant biotinylated PD1 protein was added at 0.6 μg / mL. PD1 molecules were detected using streptavidin peroxidase and colorimetrically developed at 450 nm using TMB substrate. (A) Data for anti-PD-1 antibodies fused to three type I Ig-like family proteins A or C. (B) Data for anti-PD-1 antibodies fused to type II TNF family proteins A or B. (C) Data for anti-PD-1 antibodies fused to cytokine family protein A. In this figure, the cytokine fused to the anti-PD-1 antibody is IL-7. Type I protein fusion protein A corresponds to CD86, and protein C corresponds to SIRPa. Type II protein A corresponds to OX40L, and protein B corresponds to 4-1BBL. (D): PD-1 / PD-L2 antagonist activity. PD-L2 was immobilized and complexed with biotinylated recombinant human PD-1 plus anti-PD-1 VH IL-7 (●) or anti-PD-1 VH CD80 (■). A similar protocol to the PD-L1 / PD-1 assay was used for development. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody with a heavy chain variable domain as set forth in SEQ ID NO:24 and a light chain variable domain as set forth in SEQ ID NO:28. [Figure 8-2] See Figure 8-1. [Figure 9]Figure 1 shows a bridging ELISA binding assay. (A) and (B) PD1-His recombinant protein was immobilized, and a bifunctional anti-PD-1 antibody (protein type I or type II fused to the VL domain (circle) or the VH domain (black circle)) was added at a concentration series. Protein C, A, or B soluble recombinant receptor ligands were then added at 1 μg / mL. Detection was performed using a receptor-specific mouse antibody plus a peroxidase-coupled anti-IgG mouse antibody. ELISA was developed colorimetrically at 450 nm using TMB substrate. Histograms represent recombinant protein A, B, or C immobilized on the plate, which served as a positive ELISA control. In this figure, protein A corresponds to OX40L, protein B corresponds to 4-1BBL, and protein C corresponds to SIRP-alpha. [Figure 10] Figure 1 shows T cell proliferation stimulated by bifunctional anti-PD1 molecules. CD3 CD28 preactivated T cells were restimulated on CD3 / PDL1-coated plates in the presence of anti-PD-1 bifunctional antibodies (10 μg / mL) in which type I protein (A), type II protein (B), or (C) cytokine proteins were fused to the VH or VL domain. Experiments used unfused anti-PD-1 antibodies or an isotype VH fusion antibody as a control. T cell proliferation was assessed on day 6 by H3 thymidine incorporation. Data are expressed as fold change with isotype treatment used as a control. Each point represents data from one donor. In this figure, the cytokine fused to the anti-PD-1 antibody is IL-7. Type I protein fusion protein A corresponds to CD86, protein B corresponds to CD80, and protein C corresponds to SIRPa. Type II protein A corresponds to OX40L, and protein B corresponds to 4-1BBL. [Figure 11]Figure 1 shows IFNγ secretion by T cells treated with anti-PD1 bifunctional antibodies. CD3CD28 preactivated T cells were restimulated on CD3 / PDL1-coated plates in the presence of anti-PD-1 bifunctional antibodies (10 μg / mL) in which (A) type I protein, (B) type II protein, or (C) cytokine protein was fused to the VH or VL domain. Experiments used unfused anti-PD-1 antibodies or isotype VH fusion antibodies as controls. IFNγ secretion was assessed by ELISA in supernatants collected on day 5. Data are expressed as fold change over the unfused anti-PD-1 treatment, which served as a control. Statistical analysis was performed using the Mann-Whitney test (*p<0.05). In this figure, the cytokine fused to the anti-PD-1 antibody is IL-7. Type I protein fusion protein A corresponds to CD80, protein B corresponds to CD86, and protein C corresponds to SIRPa. Type II protein A corresponds to OX40L, and protein B corresponds to 4-1BBL. [Figure 12] Figure 1 shows the pharmacokinetics of a bifunctional humanized PD-1 antibody mouse after a single injection. Balb / C mice were intravenously administered bifunctional antibody humanized variants with the IgG4 S228P isotype (●) or the IgG1 N298A isotype (◯). Plasma drug concentrations were determined by ELISA using an immobilized anti-human light chain antibody (clone NaM76-5F3) and diluted serum containing the anti-PD-1 antibody. Detection was performed using peroxidase-labeled donkey anti-human IgG. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody with a heavy chain variable domain as set forth in SEQ ID NO:24 and a light chain variable domain as set forth in SEQ ID NO:28. DETAILED DESCRIPTION OF THE INVENTION

[0030] Introduction The humanized antibodies of the present invention are bifunctional because they combine a specific anti-PD-1 effect with the effect of an immunotherapeutic agent grafted onto the humanized anti-PD-1 antibody. Indeed, the present invention relates to bifunctional molecules comprising certain humanized anti-PD-1 antibodies with an immunotherapeutic agent. More specifically, the present invention relates to bifunctional molecules comprising humanized anti-PD-1 antibodies with an immunotherapeutic agent, where the immunotherapeutic agent is covalently linked to a polypeptide chain of the humanized anti-PD-1 antibody, i.e., either the light chain or the heavy chain, or both, of the antibody or fragment thereof. More specifically, the chain of the humanized anti-PD-1 antibody or fragment thereof and the immunotherapeutic agent are prepared as a fusion protein. In this particular embodiment, the N-terminus of the immunotherapeutic agent is linked to the C-terminus of the chain of the humanized anti-PD-1 antibody or fragment thereof, optionally via a peptide linker.

[0031] The bifunctional molecules of the present invention have, in particular, one or several of the following advantages: When produced in mammalian cells (e.g., COS, CHO), the bifunctional molecules of the present invention exhibit high manufacturability and high productivity yields compared to chimeric antibodies. Furthermore, the same productivity improvements are not observed when other anti-PD1 antibodies are considered. As illustrated in Figures 1-3, bifunctional molecules containing specific humanized anti-PD-1 molecules of the present invention exhibit surprisingly better production than chimeric antibodies or two clinically approved reference anti-PD1 antibodies, i.e., pembrolizumab and nivolumab. Furthermore, this improved production was demonstrated for three different types of immunotherapeutic agents and six different immunotherapeutic agents, namely, cytokines (i.e., IL-7), type I proteins (i.e., CD80, CD86, and SIRP alpha), and type II proteins (i.e., OX40L and 4-1BBL). Furthermore, the present inventors observed that when an immunotherapeutic agent is a type I transmembrane protein, the immunotherapeutic agent retains its functionality even when grafted onto the C-terminus of a humanized anti-hPD1 antibody. This is surprising and highly intriguing. Because type I transmembrane proteins are characterized by an effective extracellular domain at the N-terminus, N-terminal grafting of type I transmembrane proteins has generally not allowed such proteins to function. In addition, improved productivity was observed when immunotherapeutic agents were fused to the C-terminus of either the heavy or light chain. Furthermore, improved productivity was observed when immunotherapeutic agents were fused to both the C-terminus of the heavy chain and the C-terminus of the light chain (Figure 1). Therefore, better production is closely associated with the humanized anti-PD1 antibodies of the present invention. This property is highly surprising and unexpected. The bifunctional molecules of the present invention are fully functional and activate innate and adaptive immune responses. Indeed, as shown in Figures 4-7, the binding of the bifunctional molecules to PD-1 is substantially unchanged. The bifunctional molecules retain substantially the same antagonistic activity as illustrated in Figures 8-9. This was demonstrated 1) with three different types of immunotherapeutic agents and six different immunotherapeutic agents, namely, cytokines (i.e., IL-7), type I proteins (i.e., CD80, CD86, and SIRPalpha), and type II proteins (i.e., OX40L and 4-1BBL), and 2) when the immunotherapeutic agents were fused to the C-terminus of the heavy or light chain or to both the light and heavy chains. Finally, the bifunctional molecules induce T cell proliferation at least as well as, or even better than, anti-PD1 antibodies alone (Figure 10). The bifunctional molecules exhibit greater efficacy in T cell activation than anti-PD1 antibodies alone (Figure 11). This ability to activate T cells better than anti-PD1 antibodies alone is a surprising property of the bifunctional molecules of the present invention, given the slight loss of binding to PD-1's ligand, namely PD-L1. - The targeting of this bifunctional molecule avoids the hematological toxicity associated with limited expression of PD-1 (it does not bind to human red blood cells (RBCs) and platelets). The bifunctional molecules of the present invention reduce tumor growth and modify the tumor microenvironment. The bifunctional molecules of the present invention potentiate a human T cell immune response and are selective antagonists of the PD-1 / PD-L1 interaction and / or the PD-1 / PD-L2 interaction. - The bifunctional molecules of the invention may exhibit additive or synergistic effects by combining anti-PD-1 and immunotherapeutic agents in a single molecule (particularly with respect to IFNγ secretion and T cell proliferation in the examples).

[0032] The humanized anti-PD-1 antibody of the present invention has a CDR sequence that is very low in similarity to other anti-PD-1 antibodies, including pembrolizumab (also known as Keytruda) and nivolumab (also known as Opdivo). Therefore, although it was unexpected and may be related to such differences in CDR sequence, the applicant has succeeded in obtaining a bifunctional molecule that exhibits advantageous effects as described in this application. The humanized anti-PD-1 antibody in which this bifunctional molecule is used has the highly unexpected ability to produce with high efficiency any immunotherapeutic agent coupled to the antibody. Therefore, it is a suitable bifunctional molecular scaffold for preparing various bifunctional molecules on an industrial scale. This is highly useful for both relevant regulatory and safety processes, as it has production volume and reproducibility compatible with drug development.

[0033] 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.

[0034] 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.

[0035] As used herein, the terms "programmed death 1," "programmed cell death 1," "PD1," "PD-1," "PDCD1," "PD-1 antigen," "human PD-1," "hPD-1," and "hPD1" are used interchangeably and refer to the programmed death-1 receptor, also known as CD279, including variants and isoforms of human PD-1 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.

[0036] 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, and even more preferably to bifunctional humanized antibodies.

[0037] 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.

[0038] 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).

[0039] 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.

[0040] "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.

[0041] 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).

[0042] 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," and are 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 (from amino terminus to carboxy terminus).

[0043] "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."

[0044] "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."

[0045] 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 -10 binds with an affinity equal to or lower than M.

[0046] As used herein, the terms "PD-1 antibody," "anti-PD-1 antibody," "PD-1 Ab," "PD-1-specific antibody," or "anti-PD-1 Ab," or "humanized anti-PD-1 antibody" are used interchangeably and refer to an antibody, as described herein, that specifically binds to PD-1, preferably 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.

[0047] 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.

[0048] 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.

[0049] 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 form" of an 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 retaining 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 amino acids. The first amino acid in an amino acid sequence (i.e., starting from the N-terminus) shall be considered to be position 1.

[0050] 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.

[0051] [Table 1]

[0052] [Table 2]

[0053] [Table 3]

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] As used herein, the terms "subject," "host," "individual," or "patient" refer to humans, including adults and children.

[0062] 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.

[0063] "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.

[0064] As used herein, the term "medicine" refers to any substance or composition that has curative or preventative properties for a disorder or disease.

[0065] 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.

[0066] 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 of the invention (e.g., nucleic acid components or portions) can be naturally occurring, modified, engineered, isolated, and / or non-naturally occurring. Engineered nucleic acids include recombinant and synthetic nucleic acids. An "isolated nucleic acid encoding an anti-PD1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules on a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0067] As used herein, the terms "nucleic acid construct," "plasmid," and "vector" are equivalent and refer to nucleic acid molecules that serve to transfer passenger nucleic acid sequences, such as DNA or RNA, into a host cell.

[0068] 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.

[0069] "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.

[0070] 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).

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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 the humanized antibody according to the present invention can be assessed by competitive ELISA.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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%).

[0079] Anti-PD-1 antibody The bifunctional molecule according to the present invention comprises a first entity comprising a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof.

[0080] Provided herein are humanized antibodies that bind to human PD-1. In some aspects, the humanized antibodies specifically bind to human PD-1, preferably to the extracellular domain of human PD-1. In some aspects, the humanized 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.

[0081] In some aspects, the humanized anti-PD1 antibody is an isolated antibody, particularly a non-naturally occurring isolated antibody. Such an isolated humanized anti-PD1 antibody can be prepared by at least one purification step. In some embodiments, the isolated 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.

[0082] 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).

[0083] 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.

[0084] 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%.

[0085] Humanized forms of anti-PD1 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 an 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, humanized anti-hPD-1 antibodies according to the invention are derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG4.

[0086] A humanized antibody typically comprises 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. The humanized antibody 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), which describe "selection" techniques, as well as U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, 5,821,337, 7,527,791, 6,982,321, and 7,087,409; and 6,180,370.

[0087] Preferably, the humanized antibody against human PD-1 is a monoclonal antibody.

[0088] 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.

[0089] The CDR regions of the humanized antibody are derived from a murine antibody to provide a safe humanized antibody with i) a very high level of humanization (greater than 85%) and stability, and ii) a binding affinity (KD) for human PD-1 of 10 -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 and inhibition of human PD-L1 binding to human PD-1, so as to have a molecular weight less than M.

[0090] In one embodiment, the 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. For 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 domain and C H 2 domains, or at least C of the heavy chain H 1 domain, C H 2 domain, 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, or 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, and 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.

[0091] In one 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: - 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.

[0092] In another embodiment, the bifunctional molecule comprises or consists of HCDR1, HCDR2, LDCR2, and LDCR3 as specified above and the amino acid sequence of SEQ ID NO: 3, wherein either X1 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, 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, and optionally 2, and a light chain CDR1 (LCDR1) comprising or consisting 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.

[0093] In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, LDCR2, and LDCR3 as specified above, and 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 a substitution, addition, deletion, 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, and 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 a substitution, addition, deletion, 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.

[0094] In another embodiment, the bifunctional molecule comprises HCDR1, HCDR2, LDCR2, and LDCR3 as designated above, and - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain DR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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; or - a heavy chain CDR3 (HCDR3) comprising or consisting 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, and a light chain CDR1 (LCDR1) comprising or consisting 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. The present invention also includes a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising:

[0095] In a particular embodiment, the modification is a substitution, particularly a conservative substitution.

[0096] 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; or (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 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 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; or (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 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, wherein X is G or T; or (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.

[0097] 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.

[0098] 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 a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 11, 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: 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

[0099] Framework The term "antibody framework", as used herein, refers to the portion of the variable domain of either the VL and / or VH that serves as a scaffold for the antigen-binding loops (CDRs) of that variable domain.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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 have 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.

[0104] 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).

[0105] In one 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 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:

[0106] 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: 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:

[0107] 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 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:

[0108] 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:

[0109] 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:

[0110] 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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 containing one, two, or three 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, respectively; a heavy chain variable region (VH) having a modification, 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:

[0111] In a particular embodiment, the modification is a substitution, particularly a conservative substitution.

[0112] CH-CL In one embodiment, the heavy (CH) and light (CL) chains comprise the VL and VH sequences as described herein above.

[0113] 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:

[0114] 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:

[0115] Preferably, the modifications are substitutions, particularly conservative substitutions.

[0116] 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, M252Y / S254T / T256E, and 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.

[0117] Humanized 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 human immunoglobulin or humanized immunoglobulin. Preferably, the Fc region is a portion of the humanized anti-hPD-1 antibody described herein. The humanized 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 specific functions are 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 humanized 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).

[0118] 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 humanized anti-PD1 antibody comprises an IgG1 Fc region or an IgG4 Fc region.

[0119] In certain embodiments, the humanized anti-PD1 antibody optionally comprises the following sequence: 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 K322A, preferably N297A, optionally in combination with M252Y / S254T / T256E, and L234A / L235A.

[0120] More preferably, the humanized anti-hPD1 antibody comprises an IgG4 Fc region 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 humanized anti-hPD1 antibody comprised in the bifunctional molecule according to the present invention comprises an IgG4 Fc region with S228P, which stabilizes IgG4.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] [Table 4]

[0129] 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.

[0130] 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.

[0131] In one embodiment, the anti-hPD1 according to the invention has a heavy chain constant domain of SEQ ID NO: 39 and / or a light chain constant domain of SEQ ID NO: 40, in particular a heavy chain constant domain of SEQ ID NO: 39 and a light chain constant domain of SEQ ID NO: 40.

[0132] In another embodiment, the anti-hPD1 according to the invention has a heavy chain constant domain of SEQ ID NO: 57 and / or a light chain constant domain of SEQ ID NO: 40, in particular a heavy chain constant domain of SEQ ID NO: 57 and a light chain constant domain of SEQ ID NO: 40.

[0133] [Table 5]

[0134] Amino acid alterations near the junction between the Fc and non-Fc portions can dramatically increase the serum half-life of Fc molecules (WO 01 / 58957). Thus, the junction region of the proteins or polypeptides of the 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 is replaced with a non-lysine amino acid, such as alanine or leucine, to further increase serum half-life. In particular, the K444 amino acid in the IgG1 or IgG4 domain can be replaced with alanine to reduce proteolytic cleavage. Thus, in one embodiment, the anti-PD1 antibody contains at least one additional amino acid substitution consisting of K444A.

[0135] 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.

[0136] In certain embodiments, the antibody may be altered to increase, decrease, or eliminate the extent to which it is glycosylated.

[0137] Humanity "Humanity" for purposes of the present invention is measured using the T20 score analyzer for quantifying the humanity of the variable regions of monoclonal antibodies as described in Gao SH, Huang K, Tu H, Adler A S., BMC Biotechnology. 2013:13:55.

[0138] 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.

[0139] The T20 humanity score is a parameter that has been widely used in the field of antibody humanization and was 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).

[0140] 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 using the T20 score analyzer in each database. 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 the sequences in the new database.

[0141] As used herein, a "humanized antibody" is one that has a T20 humanity score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90%, and most preferably between 85% and 95%, preferably between 88% and 92%.

[0142] Thus, the humanized anti-PD1 antibody according to the present invention contained in the bifunctional molecule 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%.

[0143] Peptide Linker The present invention includes bifunctional molecules that may contain a peptide linker between the humanized anti-PD-1 antibody or fragment thereof and the immunotherapeutic agent. 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.

[0144] In an embodiment of the present disclosure, the humanized anti-hPD1 antibody is preferably linked to the immunotherapeutic agent 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 the immunotherapeutic agent via a peptide linker. As used herein, the term "linker" refers to a sequence of at least one amino acid linking the immunotherapeutic agent and the anti-PD-1 immunoglobulin sequence portion. 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.

[0145] In embodiments, the invention relates to a bifunctional molecule comprising a humanized anti-PD-1 antibody, or antigen-binding fragment thereof, as defined above, and an immunotherapeutic agent, 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 light chain, is linked to the immunotherapeutic agent, preferably the N-terminus of the immunotherapeutic agent, by a peptide linker.

[0146] In a particular aspect, the invention relates to a bifunctional molecule comprising a humanized anti-hPD-1 antibody, or antigen-binding fragment thereof, as defined above, wherein an immunotherapeutic agent 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.

[0147] In embodiments, the invention relates to a bifunctional molecule comprising a humanized anti-PD-1 antibody, or antigen-binding fragment thereof, as defined above, wherein an immunotherapeutic agent 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.

[0148] 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, especially (Gly4Ser)3.

[0149] In one embodiment, the linker contained in the bifunctional molecule is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2)3, preferably (Gly4Ser)3.

[0150] In embodiments, the invention relates to a bifunctional molecule comprising a humanized anti-PD-1 antibody or fragment thereof as defined above, wherein the antibody or fragment thereof is linked to an immunotherapeutic agent 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.

[0151] 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 the immunotherapeutic agent 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.

[0152] 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 the immunotherapeutic agent 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.

[0153] immunotherapy agents In the present invention, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or fragment thereof, as described above, linked to an immunotherapeutic agent, preferably via a peptide linker. Such humanized antibodies may also be referred to as "bifunctional antibodies," because they have two therapeutic effects: a first effect resulting from the interaction of the humanized anti-hPD-1 antibody with hPD-1, and a second effect resulting from the interaction of the immunotherapeutic agent with its ligand or receptor.

[0154] As used herein, the term "immunotherapeutic agent" refers to a substance that exerts an effect on the immune system, particularly an inhibitory or stimulatory effect, preferably a stimulatory effect. The substance may be, for example, a molecule that exerts an effect on the activation or inhibition of cells of the immune system. For example, the immunotherapeutic agent may be selected from T cell growth factors, particularly growth factors that increase the number and repertoire of naive T cells, growth factors that increase the number of dendritic cells (DCs), agonists that activate DCs and other antigen-presenting cells (APCs), adjuvants that enable and enhance cancer vaccines, agonists that activate and stimulate T cells, inhibitors of T cell checkpoint blockade, T cell growth factors that increase the growth and survival of immune T cells, and substances that inhibit, block, or neutralize cancer cell- and immune cell-derived immunosuppressive cytokines. Preferably, the immunotherapeutic agent is a peptide, polypeptide, or protein. In one embodiment, the immunotherapeutic agent is a non-antibody entity or moiety. The immunotherapeutic agent may also consist of one or more other binding molecules, antibody-binding mimetics, receptors or extracellular domains thereof, ligands of receptors, or fragments thereof that have the same functional activity. As used herein, "antibody mimic" refers to an organic compound that can specifically bind to an antigen like an antibody, but is structurally unrelated to antibodies. Antibody mimics are typically peptides or proteins with molar masses of about 3-20 kDa. Examples of antibody mimics include affilins, affimers, affitins, anticalins, and avimers.

[0155] The immunotherapeutic agent may be mutated or altered so that the biological activity of the immunotherapeutic agent is altered, for example so that the biological activity is increased, decreased, or completely inhibited.

[0156] In one embodiment, the immunotherapeutic agent consists of a fragment thereof. Preferably, such a fragment retains the biological activity of the immunotherapeutic agent.

[0157] In one embodiment, the immunotherapeutic agent or fragment thereof has a size of at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa, or at least 50 kDa. Preferably, the immunotherapeutic agent or fragment thereof has a size comprised between 10 kDa and 50 kDa, 10 kDa and 40 kDa, 10 kDa and 30 kDa, 10 kDa and 20 kDa, 20 kDa and 50 kDa, 20 kDa and 40 kDa, or 20 kDa and 30 kDa.

[0158] In particular, immunotherapeutic agents useful in the context of the present invention are selected from the group consisting of immune checkpoint blockers or activators, particularly adaptive immune cells (T or B lymphocytes), therapeutic vaccines (DNA, RNA, or peptide vaccines), or immunoconjugates such as antibody-drug conjugates. In one approach, the immunotherapeutic agent is a T cell inhibitory checkpoint receptor protein on T cells (e.g., CTLA-4, BTLA, LAG-3, TIM-3, and LAIR1). In another approach, the immunotherapeutic agent is a counterreceptor or ligand on antigen-presenting cells and tumor cells (some of these counterreceptors are used for immune evasion of these cells themselves), such as B7-DC, HVEM, TIM-4, B7-H3, or B7-H4.

[0159] Other suitable immunotherapeutic agents according to the present invention can bind to, but are not limited to, hormone receptors (e.g., estrogen, progesterone), cytokine receptors (i.e., type I, such as growth hormone receptor, prolactin, erythropoietin; type II; members of the immunoglobulin superfamily, such as interleukin-1; tumor necrosis factor receptor family, such as CD27, CD30, CD40; chemokine receptors, such as interleukin-8, CCR1, CXCR4; transforming growth factor (TGF) beta receptors); cell adhesion molecules (e.g., integrins); and vascular endothelial growth factor (VEGF) receptors (e.g., neurophilin (NRP) receptors, such as NRP1, NRP2).

[0160] Preferably, the immunotherapeutic agent is human or derived from a human.

[0161] Preferably, the immunotherapeutic agent is selected from the group consisting of tumor targeting peptides, cytokines, cytokine receptor co-stimulatory molecules, inhibitory or co-inhibitory molecules, preferably type I or type II human transmembrane immune proteins, even more preferably their extracellular domains, molecular chaperone inhibitors (e.g., HSP90 inhibitors), or tubulin inhibitors (e.g., taxanes) and / or stabilizers, or DNA replication inhibitors, or any anti-cancer drug, or any derivative and / or analogue thereof, to provide additional therapeutic benefit.

[0162] In certain embodiments of the invention, the immunotherapeutic agent is a molecule that targets an Fc receptor, e.g., human FcγRI (CD64) or human Fcα receptor (CD89). Accordingly, the invention includes bispecific molecules capable of binding to both FcγR- or FcαR-expressing effector cells (e.g., monocytes, macrophages, or polymorphonuclear cells (PMNs)) and PD-1-expressing target cells. Such bifunctional molecules target PD-1-expressing cells to effector cells and induce Fc receptor-mediated effector cell activities, such as phagocytosis of PD-1-expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or superoxide anion generation.

[0163] For example, immunotherapeutic agents include ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, nectin-1, nectin-2, nectin-3, NELC-5, TLT-2, LFA-3, TIM3, TIM4, LAIR1, SIRPG, IL10R, IL6RA, IL-1R1, IL-1RAcP, IL6RB, TGFBRII, CSF1R, IL22R, VEGFR1, VEGFR2, VEGFR3, CD111, CD112, CD155, CD113, VISTA, CD244, OX40, SIRP alpha, CD80, CD24, Siglec-10, Fas, IL1 The non-exhaustive list of antibodies can be selected from Table F below, including 5RA, SIRB1, SIRB2, LTBR, ​​IL21R, GITR, CD40L, OX40L, FasL, TRAIL, TNF, LIGHT, APRIL, GITRL, CD30, CD70, CD40, CD27, CD30, CD153, RANK, CLEC3A, CLEC4A, CLEC4E, CLEC4L, CLEC51, CLEC6, CLEC7A, NKG2D, BTL-II, TGFRII, DECTIN-1, DC-SIGN, LT-alpha, LT-beta, 4-1BBL, MINCLE, TGFβ, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12A, IL12B, IL-15, IL-21, and IL-18.

[0164] In very specific embodiments, the immunotherapeutic agent is interleukin-2 (IL-2), preferably human IL-2 or a mutant or variant thereof, as disclosed, for example, in UniProt Accession No. P60568. IL-2 may be mutated in various ways to reduce its toxicity and / or increase its efficacy. Hu et al. (Blood, Vol. 101, pp. 4853-4861 (2003); U.S. Patent Application Publication No. 2003 / 0124678) substituted tryptophan for the arginine residue at position 38 of IL-2 to eliminate IL-2's vascular permeability activity. Shanafelt et al. (Nature Biotechnol, Vol. 18, No. 1, pp. 197-1202 (2000)) mutated asparagine 88 to arginine to enhance selectivity for T cells over NK cells. Heaton et al. (Cancer Res, Vol. 53, pp. 2597-602 (1993); U.S. Pat. No. 5,229,109) introduced two mutations, Arg38Ala and Phe42Lys, to reduce secretion of inflammatory cytokines from NK cells. Gillies et al. (U.S. Patent Application Publication No. 2007 / 0036752) substituted three residues in IL-2 (Asp20Thr, Asn88Arg, and Glnl26Asp) that contribute to its affinity for the medium-affinity IL-2 receptor to reduce VLS. Gillies et al. (WO 2008 / 0034473) also mutated the interface between IL-2 and CD25 with the amino acid substitutions Arg38Trp and Phe42Lys to reduce interaction with CD25 and activation of Treg cells for enhanced efficacy. In one aspect, the immunotherapeutic agent is an IL-2 variant, e.g., as described in WO 2012 / 107417 or WO 2018 / 184964. For example, variants of human IL-2 (hIL-2) with reduced affinity for CD25 can be generated by amino acid substitutions at amino acid positions 3, 35, 38, 42, 43, 45, or 72, or combinations thereof, corresponding to residue positions in human IL-2.Preferably, the mutant IL-2 is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G, and / or C125A, preferably F42A, Y45A, and L72G, more preferably T3A, F42A, Y45A, L72G, and C125A, e.g., as disclosed in WO 2018 / 184964. Even more preferably, the immunotherapeutic agent is an IL-2 mutant having the amino acid sequence set forth in SEQ ID NO: 58, with the substitutions F42A, Y45A, and L72G, preferably T3A, F42A, Y45A, L72G, and C125A.

[0165] The inventors have observed that when the immunotherapeutic agent is a type I or type II transmembrane protein, the immunotherapeutic agent retains function when grafted onto either the heavy chain or the light chain of a humanized anti-hPD-1 molecule according to the present invention.

[0166] Preferably, the bifunctional molecule comprises a humanized anti-hPD1 antibody linked to a type I or type II transmembrane protein, particularly the extracellular domain thereof. Even more preferably, the bifunctional molecule comprises a type I or type II transmembrane protein, particularly the extracellular domain thereof, linked, preferably by a peptide linker, to the carboxy terminus of a humanized anti-hPD1 antibody as described herein.

[0167] Type I transmembrane proteins are characterized by their N-terminus being on the outer side of the cell membrane and their C-terminus being on the inner side of the cell membrane. Type I transmembrane proteins typically include immunoglobulin superfamily members (e.g., CD4, CD8, CD28, CTLA4, CD86), receptor kinases (e.g., TGF-β receptor, EGFR, VEGFR, PDGFR, HGF receptor), and cytokine receptors (e.g., TNF receptor, RANK, IL-6 receptor, CSF1 receptor, c-kit). Type I transmembrane proteins can induce various biological responses after binding to their corresponding ligands. There are no particular limitations on the type I transmembrane proteins used in the present invention. Therefore, any type I transmembrane protein with biological activity can be used in the present invention. For example, type I transmembrane proteins include ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, nectin-1, nectin-2, nectin-3, NELC-5, TLT-2, LFA-3, TIM3, TIM4, LAIR1, SIRPG, IL10R, IL6RA, IL-1R1, and IL-1RA. cP, IL6RB, TGFBRII, CSF1R, IL22R, VEGFR1, VEGFR2, VEGFR3, CD111, CD112, CD155, CD113, VISTA, CD244, OX40, SIRP alpha, CD80, CD24, Siglec-10, Fas, IL15RA, SIRB1, SIRB2, LTBR, ​​IL21R, and GITR.

[0168] In a preferred embodiment, the bifunctional molecule according to the present invention comprises a type I transmembrane protein, preferably selected from the group consisting of CD86, ICOSL, PD-L1, PD-L2, ICOSL, RANK, VEGFR1, VEGFR2, CTLA4, TGF-pRII, B7-H3, B7-H4, HVEM, BTLA, LAG3, TIM3, VISTA, CD111, CD113, TIGIT, SIRP alpha, CD80, or a combination thereof.

[0169] Type II transmembrane proteins are characterized by their C-terminus being on the outer side of the cell membrane and their N-terminus being on the inner side of the cell membrane. Typically, type II transmembrane proteins are C-type lectins or C-type lectin-like receptors. The extracellular domain of the receptor contains a carbohydrate-binding domain (also called a C-type lectin domain, CTLD). Proteins with carbohydrate-binding domains are involved in various functions, such as cell adhesion, platelet activation, pathogen immunity, and apoptosis induction. There are no particular limitations on the type II transmembrane proteins used in the present invention. Therefore, any type II transmembrane protein with biological activity can be used in the present invention. For example, the type I transmembrane protein may be selected from the following non-exhaustive list: CD40L, OX40L, FasL, TRAIL, TNF, LIGHT, APRIL, GITRL, CD30, CD70, CD40, CD27, CD30, CD153, RANK, CLEC3A, CLEC4A, CLEC4E, CLEC4L, CLEC51, CLEC6, CLEC7A, NKG2D, BTL-II, TGFRII, DECTIN-1, DC-SIGN, LT-alpha, LT-beta, 4-1BBL, and MINCLE, preferably a member of the TNF family.

[0170] In a preferred embodiment, the bifunctional molecule according to the present invention comprises a type II transmembrane protein, preferably selected from the group consisting of OX40L, DECTIN-1, NKG2D, DC-SING, 4-1BBL, MINCLE, or a combination thereof.

[0171] In certain embodiments, the immunotherapeutic agent included in the bifunctional molecule is an immune checkpoint blocker or activator. It will be understood that such an immunotherapeutic agent may be distinct from h-PD1, PD-L1, PD-L2, or from molecules that target and / or bind to h-PD1, PD-L1, and / or PDL-2.

[0172] Numerous immune checkpoint blockers or activators are known in the art. In the context of the present invention, examples of immune checkpoint blockers or activators of adaptive immune cells (B or T lymphocytes) that may be useful are CTLA-4, CD86, CD28, CD40, CD40L, ICOS, ICOS-L, OX40L, GITR, HVEM, BTLA, CD160, LIGHT, TNFRSF25, 2B4, CD48, Tim1, Tim3, Tim4, Gal9, LAG-3, CD40, CD40L, CD70, CD27, VISTA, B7H3, B7H4 (B7x), TIGIT, CD112, HHLA2 (B7-H7), TMIGD2 (CD28H), butyrophilin-like 2 (BTNL2), variants and fragments thereof, in particular CD86, OX40L, ICOSL, variants and fragments thereof.

[0173] Preferably, a bifunctional molecule according to the invention comprises a humanized anti-PD-1 antibody, or an antigen-binding fragment thereof, as defined above, linked, preferably by a peptide linker, to an immunotherapeutic agent selected from the group consisting of CD86, ICOSL, PD-L1, PD-L2, ICOSL, RANK, VEGFR1, VEGFR2, CTLA4, TGF-pRII, B7-H3, B7-H4, HVEM, BTLA, LAG3, TIM3, VISTA, CD111, CD113, TIGIT, OX40L, DECTIN-1, NKG2D, DC-SIGN, and MINCLE.

[0174] In a preferred embodiment, the bifunctional molecule according to the present invention comprises a cytokine as an immunotherapeutic agent, preferably selected from the group consisting of TGFβ, IL-1, IL-2, IL-6, IL-10, IL-12A, IL12B, IL-15, IL-21, and IL-18.

[0175] In one embodiment, the type I transmembrane protein, type II protein, or cytokine, or fragment thereof, has a size of at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa, or at least 50 kDa. Preferably, the immunotherapeutic agent has a size comprised between 10 kDa and 50 kDa, 10 kDa and 40 kDa, 10 kDa and 30 kDa, 10 kDa and 20 kDa, 20 kDa and 50 kDa, 20 kDa and 40 kDa, or 20 kDa and 30 kDa.

[0176] In one embodiment, the immunotherapeutic agent comprised in the bifunctional molecule according to the invention is selected in Table F below.

[0177] [Table 6A]

[0178] [Table 6B]

[0179] [Table 6C]

[0180] [Table 6D]

[0181] [Table 6E]

[0182] [Table 6F]

[0183] [Table 6G]

[0184] In particular, the bifunctional molecules of the invention can be obtained by linking a functional variant or functional fragment of an immunotherapeutic agent, particularly an immune checkpoint blocker or activator, to a humanized anti-hPD-1 antibody described herein. Preferably, the immunotherapeutic agent corresponds to the extracellular domain (ECD) of the immune checkpoint blocker or activator.

[0185] In embodiments, the invention relates to a humanized anti-PD-1 antibody or antigen-binding fragment thereof as defined above, wherein the immunotherapeutic agent is selected from the group consisting of CD86, PD-L1, PD-L2, ICOSL, RANK (tumor necrosis factor receptor superfamily member 11A), VEGF-R1, VEGF-R2, CTLA4, B7-H3, B7-H4, HVEM, CD40, CD111, CD112, CD155, CD113, IL1-R1, IL1-RAcP, LFA-3, CD28, ICOS, BTLA, LAG3, TIGIT, VISTA, CD244, OX40, The fragments of the immunotherapeutic agents of the present invention comprise or consist of the extracellular domain (ECD) of a protein selected from the group consisting of SIRP alpha, CD80, CD24, Siglec-10, Fas, IL15RA, SIRB1, SIRB2, LTBR, ​​IL21R, CD27, TIM3, TIM4, GITR, LAIR1, CD30, OX40L, TGFRII, DECTIN-1, NKG2D, DC-SIGN, LT-alpha, LT-beta, 4-1BBL, MINCLE, CD70, CD40L, CD153, GITRL, BTL-II, variants and fragments thereof. In particular, the fragments of the immunotherapeutic agents of the present invention have a size less than or equal to 500, 400, 300, 200, 100, or 50 amino acids.

[0186] The reference sequence of the extracellular domain of human OX40L used in the examples of the present application corresponds to the sequence associated with SEQ ID NO:51.

[0187] The reference sequence of the extracellular domain of human ICOSL used in the examples of the present application corresponds to the sequence associated with SEQ ID NO:52.

[0188] The reference sequence of the extracellular domain of human CD86 used in the examples of this application corresponds to the sequence associated with SEQ ID NO:53.

[0189] [Table 7]

[0190] Bifunctional molecules according to the present invention include antibodies and their fragments, but may also include macromolecules such as artificial proteins, peptides, and any compounds capable of mimicking antibody binding and binding to antigens, herein referred to as "antigen-binding antibody mimics." Such proteins include affitins and anticalins. Affitins are artificial proteins capable of selectively binding to antigens. Affitins are structurally derived from the DNA-binding protein Sac7d found in Sulfolobus acidocaldarius, a microorganism belonging to the archaea domain. Affitin libraries can be generated by randomizing the amino acids on the binding surface of Sac7d, for example, by generating variants corresponding to random substitutions of 11 residues in the binding interface of Sac7d. The resulting protein library can then be subjected to rounds of ribosome display, and affinity may be tested for various targets, such as peptides, proteins, viruses, and bacteria. Affitins are antibody mimics and are being developed as biotechnology tools. Affitins have also been used as specific inhibitors of various enzymes (Krehenbrink et al., J. Mol. Biol., 383:5, 2008). Those skilled in the art can easily develop anticalins with the required binding properties using methods known in the art, in particular the generation of phage display libraries and / or ribosome display libraries, as disclosed in WO 2008068637 and the publications cited above, and screening them with antigens as disclosed herein. Anticalins are artificial proteins capable of binding to antigens, either proteins or small molecules. Anticalins are antibody mimics derived from human lipocalins, a family of natural binding proteins. Anticalins are approximately eight times smaller, with a size of approximately 180 amino acids and a mass of approximately 20 kDa (Skerra, Febs J., 275:11, 2008). In particular, anticalin phage display libraries have been generated that allow screening and selection of anticalins with specific binding properties.Those skilled in the art can readily develop affitins with the required binding properties using methods known in the art, particularly the generation of phage and / or ribosome display libraries and screening them with antigens as disclosed herein, as disclosed in, inter alia, EP 1270725 B1, U.S. Pat. No. 8,536,307 B2 (Schlehuber and Skerra, Biophys. Chem., 96:2-3, 2002), and the publications cited above. Both anticalins and affitins can be produced in several expression systems, including bacterial expression systems. Thus, the present invention provides affitins, anticalins, and other similar antibody mimetics that have the characteristics of the humanized antibodies described herein, particularly with respect to binding to PD-1, inhibiting the interaction of PD-1 with PD-L1 and / or PD-L2, not inhibiting T cell proliferation, and increasing T cell proliferation, all of which are contemplated as macromolecules of the present invention. All embodiments disclosed herein relating to antibodies or fragments thereof may be substituted, mutatis mutandis, with the macromolecules of the present invention, particularly antigen-binding antibody mimetics. Previously known protein drugs include growth factors, hormone proteins, enzyme proteins (zymoproteins), cytokines, interferons, erythropoietin, and molecules. Except for molecules, all other protein pharmaceuticals are homogeneous proteins containing only one type of protein component. Existing molecular pharmaceuticals (e.g., etanercept and ilonacept) produced by fusing the extracellular domain of a receptor protein with the Fc fragment of human IgG are composed of two protein components, but perform only one function: blocking the binding of endogenous receptors to their corresponding ligands.

[0191] Bifunctional molecules The present invention particularly provides bifunctional molecules comprising or consisting of a humanized anti-hPD1 antibody or antibody fragment thereof and an immunotherapeutic agent, where the humanized anti-hPD1 antibody or antibody fragment thereof is covalently linked to the immunotherapeutic agent as a fusion protein. In particular, bifunctional molecules according to the present invention comprise two entities: a first entity comprising or consisting essentially of a humanized anti-hPD1 antibody or fragment thereof; and a second entity comprising or consisting essentially of an immunotherapeutic agent, optionally linked by a peptide linker.

[0192] In particular, bifunctional molecules according to the present invention contain one, two, three, or four immunotherapeutic agent molecules. In particular, a bifunctional molecule may contain only one immunotherapeutic agent molecule linked to only one of the light or heavy chains of an anti-PD-1 antibody. Alternatively, a bifunctional molecule may contain two immunotherapeutic agent molecules linked to either the light or heavy chain of an anti-PD-1 antibody. Alternatively, a bifunctional molecule may contain two immunotherapeutic agent molecules, the first of which is linked to the light chain of an anti-PD-1 antibody and the second of which is linked to the heavy chain of an anti-PD-1 antibody. Alternatively, a bifunctional molecule may contain three immunotherapeutic agent molecules, two of which are linked to either the light or heavy chain of an anti-PD-1 antibody and the last of which is linked to the other chain of an anti-PD-1 antibody. Finally, the bifunctional molecule may also contain four immunotherapeutic 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. Thus, the bifunctional molecule contains from one to four immunotherapeutic molecules as disclosed herein.

[0193] In one embodiment, only one of the light chains comprises one immunotherapeutic agent molecule (e.g., the bifunctional molecule comprises one immunotherapeutic agent molecule), only one of the heavy chains comprises one immunotherapeutic agent molecule (e.g., the bifunctional molecule comprises one immunotherapeutic agent molecule), each light chain comprises one immunotherapeutic agent molecule (e.g., the bifunctional molecule comprises two immunotherapeutic agent molecules), each heavy chain comprises one immunotherapeutic agent molecule (e.g., the bifunctional molecule comprises two immunotherapeutic agent molecules), or only one of the light chains and only one of the heavy chains comprises one immunotherapeutic agent molecule. Each light chain contains one immunotherapeutic agent molecule and only one of the heavy chains contains one immunotherapeutic agent molecule (e.g., a bifunctional molecule contains two immunotherapeutic agent molecules), each light chain contains one immunotherapeutic agent molecule and only one of the heavy chains contains one immunotherapeutic agent molecule (e.g., a bifunctional molecule contains three immunotherapeutic agent molecules), each heavy chain contains one immunotherapeutic agent molecule and only one light chain contains one immunotherapeutic agent molecule (e.g., a bifunctional molecule contains three immunotherapeutic agent molecules), or both the light and heavy chains contain one immunotherapeutic agent molecule (e.g., a bifunctional molecule contains four immunotherapeutic agent molecules).

[0194] In one 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, 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; A humanized anti-human PD-1 antibody or an antigen-binding fragment thereof; and (b) an immunotherapeutic agent, preferably 1 to 4 molecules of the immunotherapeutic agent, or a fragment thereof; comprising or consisting of The antibody heavy or light chain or fragment thereof is covalently linked to the immunotherapeutic agent as a fusion protein, preferably by a peptide linker.

[0195] More specifically, the immunotherapeutic agent can be any immunotherapeutic agent or extracellular portion thereof as previously disclosed in the section titled "Immunotherapeutic Agents," and the humanized anti-human PD-1 antibody or antigen-binding fragment thereof can be any humanized anti-human PD-1 antibody or antigen-binding fragment thereof as previously disclosed in the section titled "Anti-PD-1 Antibodies."

[0196] Preferably, the N-terminus of the immunotherapeutic agent is connected to the C-terminus of the heavy or light chain of the humanized anti-human PD-1 antibody, optionally via a peptide linker. Optionally, such bifunctional molecules include at least one peptide linker connecting the N-terminus of the immunotherapeutic agent to the C-terminus of the heavy or light chain of the humanized 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.

[0197] In another embodiment, the present invention provides (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 bifunctional molecule comprising a humanized anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising or consisting of: The heavy and / or light chains are linked at their C-terminus, preferably via a peptide linker, to an immunotherapeutic agent, preferably the extracellular domain (or ECD) of an immunotherapeutic agent, preferably selected from the group consisting of tumor targeting peptides, cytokines, cytokine receptors, stimulatory or costimulatory molecules, inhibitory or co-inhibitory molecules, preferably type I or type II, in particular type I, human transmembrane immune protein immunotherapeutics.

[0198] In another embodiment, the present invention provides (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. (iii) optionally, a peptide linker selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3; A bifunctional molecule comprising a humanized anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising or consisting of: The heavy chain and / or light chain may be linked at its C-terminus, optionally via a peptide linker, to one of the following: ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, nectin-1, nectin-2, nectin-3, NELC-5, TLT-2, LFA-3, TIM3, TIM4, LAIR1, SIR PG, IL10R, IL6RA, IL-1R1, IL-1RAcP, IL6RB, TGFBRII, CSF1R, IL22R, VEGFR1, VEGFR2, VEGFR3, CD111, CD11 2, CD155, CD113, VISTA, CD244, OX40, SIRP Alpha, CD80, CD24, Siglec-10, Fas, IL15RA, SIRB1, SIRB2, LTBR, ​​I and IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12A, IL-12B, IL-15, IL-18, and IL-21.

[0199] In a very particular embodiment, the present invention provides (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein 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, 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. (iii) optionally, a peptide linker selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3; A bifunctional molecule comprising a humanized anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising or consisting of: The heavy and / or light chains relate to bifunctional molecules, wherein the C-terminus is linked, optionally via a peptide linker, to IL-2 or a variant thereof, preferably as disclosed above, more preferably having the amino acid sequence shown in SEQ ID NO: 58.

[0200] 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 using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, humanized anti-hPD-1 antibody / immunotherapeutic agent conjugates can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the immunotherapeutic agent itself or a ligand of the immunotherapeutic agent, depending on the nature of the immunotherapeutic agent.

[0201] 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.

[0202] 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%.

[0203] In some examples, the bifunctional molecules described herein inhibit or reduce 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.

[0204] In some instances, the bifunctional molecules described herein stimulate IFN-gamma secretion.

[0205] In another example, the bifunctional molecules described herein enhance T cell activation, stimulate the secretion of IFNg by T cells, and / or stimulate the proliferation of immune cells such as T cells.

[0206] Preparation of bifunctional molecules - Nucleic acid molecules encoding bifunctional molecules, recombinant expression vectors and host cells containing them To generate the bifunctional molecules of the invention, a humanized anti-hPD1 antibody of the invention is operably linked to an immunotherapeutic agent. Both entities of the bifunctional molecule are encoded by the same vector and produced as a fusion protein. Accordingly, nucleic acids encoding any of the bifunctional molecules described herein, vectors such as expression vectors or recombinant viruses containing such nucleic acids, and host cells containing the nucleic acids and / or vectors are also disclosed herein.

[0207] 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.

[0208] In general, such methods include: (1) transfecting or transforming a suitable host cell with a polynucleotide encoding a recombinant bifunctional molecule of the present 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:

[0209] 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.

[0210] Nucleic acids, vectors, and host cells are described in more detail herein below.

[0211] 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.

[0212] Antibody DNA sequences may be, for example, amplified from RNA of immunoglobulin-synthesizing cells, synthesized using PCR with cloned immunoglobulins, or synthesized with oligonucleotides encoding known signal peptide amino acid sequences. Preferably, the peptide signal comprises or consists of the amino acid sequence of SEQ ID NO: 49 for VH and / or CH and / or 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.

[0213] 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.

[0214] In particular, the nucleic acid molecule encoding the 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 nucleic acid encoding an immunotherapeutic agent operably linked to either the first nucleic acid or the second nucleic acid, or both, optionally via a nucleic acid encoding a linker; Includes:

[0215] In one embodiment, 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 nucleic acid encoding an immunotherapeutic agent, 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:

[0216] In another embodiment, the nucleic acid molecule encoding the bifunctional molecule as defined above comprises: - a first nucleic acid molecule encoding a variable heavy chain domain of SEQ ID NO: 17, in which 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 in which 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 optionally having a peptide signal of 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 nucleic acid encoding an immunotherapeutic agent, 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:

[0217] 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 nucleic acid encoding an immunotherapeutic agent, 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:

[0218] In very particular embodiments, the nucleic acid molecule encoding the variable heavy chain domain has the sequence set forth in SEQ ID NO: 61 and / or the nucleic acid molecule encoding the variable light chain domain has the sequence set forth in SEQ ID NO: 62.

[0219] By "operably linked" is intended a nucleic acid encoding a protein fusion comprising a variable heavy or light chain domain, optionally a linker peptide, and an immunotherapeutic agent. 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.

[0220] In one embodiment, the nucleic acid molecule is an isolated, particularly a non-naturally occurring, nucleic acid molecule.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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 and light chains, which can then be purified and assembled to form an intact antibody in vitro.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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).

[0229] 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.

[0230] 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.

[0231] 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.

[0232] In another embodiment, the host cell contains (e.g., has been transformed with) a vector that contains both entities of the bifunctional molecule. Preferably, the host cell contains (e.g., has been transformed with) a vector that includes 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 an immunotherapeutic agent disclosed herein.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] Methods for selecting suitable bifunctional molecules In one aspect, the present invention provides a method for selecting a bifunctional molecule of the present invention, comprising: a. testing the ability of the bifunctional molecule to bind to PD-1 (e.g., by the methods described in Example X); b. testing the ability of the bifunctional molecule to inhibit the binding of human PD-L1 and / or PD-L2 to human PD-1 (e.g., by the methods described in Example X); c. testing the ability of the bifunctional molecule not to inhibit T cell proliferation, preferably to increase proliferation of T cells, particularly regulatory T cells (e.g., by the methods described in Example X); d. testing the ability of the bifunctional molecule not to inhibit T cell activation, but preferably to increase T cell activation (e.g., by the methods described in Example X); e. testing the ability of the bifunctional molecule to increase IFNγ secretion by human PBMCs (e.g., by the method described in Example X); f. Testing the ability of the bifunctional molecule to bind to a ligand or receptor for the immunotherapeutic agent (e.g., by the methods described in Example X). comprising or consisting of at least one of and optionally g. Selecting a bifunctional molecule that specifically binds to PD-1 and / or significantly inhibits the binding of PD-L1 and / or PD-L2 to PD-1, and / or does not significantly inhibit, but preferably increases, the proliferation and / or activation of human T cells, and / or increases the secretion of IFNγ by human PBMCs, and / or is capable of binding to a ligand or receptor of an immunotherapeutic agent. The present invention relates to a method comprising:

[0239] The method for selecting modified antibodies of the present invention can advantageously be carried out in addition to the method for producing bifunctional molecules according to the present invention, as described herein above.

[0240] 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.

[0241] 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)).

[0242] 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.

[0243] 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).

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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 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 humanized anti-PD1 antibody or antibody fragment 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 humanized anti-PD1 antibody or antibody fragment thereof. Exemplary treatments are described in more detail herein below in the "Methods and Uses" section.

[0257] 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.

[0258] In particular, the subject suffers from a disease in which the PD-1 / PDL-1 pathway may be involved, particularly a disease in which at least one of the ligands of PD-1 (e.g., PDL-1 and / or PDL-2) 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.

[0259] In certain embodiments, the subject has already undergone at least one treatment, preferably several courses of treatment, prior to administration of the bifunctional molecule according to the invention or the pharmaceutical composition according to the invention.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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. In particular, any of the bifunctional molecules, nucleic acids, groups of nucleic acids, vectors, host cells, or pharmaceutical compositions provided herein may be used in methods of treatment 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.

[0267] Preferably, the present invention relates to methods for treating 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.

[0268] Even more preferably, the present invention relates to a method for the treatment of a disease and / or disorder selected from the group consisting of cancer and infectious diseases, preferably chronic infectious diseases, in a subject in need thereof, comprising administering to said subject an effective amount of a bifunctional molecule or pharmaceutical composition as defined above. Examples of such diseases are described more specifically herein below.

[0269] 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 another signaling pathway.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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 an anti-PD-1 antibody 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).

[0275] In some embodiments, the amount of humanized anti-hPD-1 antibody described herein is effective to inhibit the binding of human PD-L1 and / or PD-L2 to human PD-1 (e.g., inhibit binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control).

[0276] In some embodiments, the amount of a humanized anti-hPD-1 antibody described herein is sufficient to have antagonist activity of human PD-L1 and / or PD-L2 binding to human PD-1 (e.g., inhibit binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control).

[0277] The present invention also relates to the bifunctional molecules described herein; nucleic acids or vectors encoding same, or pharmaceutical compositions comprising same, for use in treating disorders and / or diseases in a subject and / or for use as a medicament or vaccine. The present invention relates to the use of the bifunctional molecules described herein; nucleic acids or vectors encoding same, or pharmaceutical compositions comprising same, in the manufacture of a medicament for treating a disease and / or disorder in a subject. Finally, the present invention relates to a method of treating a disease or disorder in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition or bifunctional molecule to the subject.

[0278] Disclosed herein are methods of treating a patient having a disease and / or disorder, the methods comprising: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of any of the bifunctional molecules, nucleic acids, vectors, or pharmaceutical compositions described herein.

[0279] A subject in need of treatment may be a human who has, is at risk for, or is suspected of having a disease associated with a PD-1-mediated signaling pathway. Such patients can be identified by routine medical examination. For example, subjects suitable for treatment can be identified by determining whether such subjects have PD-1-, PD-L1-, and / or PD-L2-positive cells. In one embodiment, the subject in need of treatment is a patient who has, is suspected of having, or is at risk for a disease, preferably a PD-1-, PDL1-, and / or PDL2-positive disease, even more preferably a disease in which PD-1 and / or at least one ligand of PD-1 is overexpressed. Disruption of the PD-1 / PD-L1 and / or PD-1 / PD-L2 interaction in such a subject by administration of a bifunctional molecule or pharmaceutical composition according to the present invention may enhance the subject's immune response. In some embodiments, any of the humanized anti-PD-1 antibodies or pharmaceutical compositions described herein can be used to treat PD-1-positive cells.

[0280] cancer It is known in the art that antibody blockade of PD-1 can enhance the immune response against cancerous cells in a patient. Accordingly, in one aspect, the invention provides a bifunctional molecule or pharmaceutical composition for use in treating a subject with cancer, comprising administering to the individual an effective amount of a bifunctional molecule or pharmaceutical composition, preferably a bifunctional molecule or pharmaceutical composition for disrupting or inhibiting PD1 / PD-L1 and / or PD1 / PD-L2 interactions.

[0281] In one embodiment, the subject in need of treatment is a patient having, suspected of having, or at risk of having a disease, preferably a PD-1 or PD-L1 positive cancer, even more preferably a cancer in which PD-1 or PD-L1 is expressed or overexpressed. For example, patients suitable for treatment can be identified by testing whether such patients have PD-L1 positive tumor cells. Additionally or alternatively, subjects suitable for treatment are those with tumor-infiltrating T cells that express or overexpress PD-1.

[0282] In another embodiment, the subject is a patient who has, is suspected of having, or is at risk for developing cancer, preferably a PD-L1- and / or PD-L2-positive cancer. In some embodiments, any of the bifunctional molecules or pharmaceutical compositions described herein can be used to treat PD-L1- and / or PD-L2-positive tumors. For example, human patients suitable for treatment can be identified by determining whether such patients have PD-L1- and / or PD-L2-positive cancer cells.

[0283] In a further aspect, there is provided a bifunctional molecule or pharmaceutical composition for use in treating cancer, preferably a PD-1, PD-L1 and / or PD-L2 positive cancer, even more preferably a cancer in which PD-1, PD-L1 and / or PD-L2 is overexpressed.

[0284] In a particular embodiment, the bifunctional molecule or pharmaceutical composition according to the invention is for use in the treatment of cancer by activating exhausted T cells.

[0285] In another embodiment, the present invention provides the use of a bifunctional molecule or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating cancer, e.g., for inhibiting the growth of tumor cells, preferably PD-L1 or PD-L2 positive tumor cells, in a subject.

[0286] In an embodiment of the present disclosure, the cancer to be treated is associated with exhausted T cells.

[0287] Preferably, "PD-L1-positive tumor cells" or "PD-L2-positive tumor cells" are intended to refer to a population of tumor cells in which PD-L1 or PD-L2, respectively, is expressed by at least 10% of the tumor cells, preferably by at least 20, 30, 40, or 50% of the tumor cells.

[0288] Thus, in one embodiment, the present invention provides a method of treating cancer in a subject, e.g., inhibiting the growth of tumor cells, comprising administering to the subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition according to the present invention. In particular, the present invention relates to treating a subject with a bifunctional molecule such that the growth of cancerous cells is inhibited.

[0289] Any suitable cancer that can be treated with the bifunctional molecules provided herein may be a hematopoietic cancer or a solid cancer. Such cancers include carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, renal cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, stomach cancer, urethral cancer, environmentally induced cancer, and any combination of such cancers. The present invention is also useful for treating metastatic cancers, particularly metastatic cancers that express PD-L1 (Iwai et al., (2005), Int. Immunol. 17:133-144). In addition, the present invention induces refractory or recurrent malignancies.

[0290] In certain embodiments, the cancer is a hematological or solid tumor with high expression of PD-1 and / or PD-L1. Such cancer may be selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndromes, and acute myeloid leukemia.

[0291] In certain embodiments, the cancer is a virus-induced cancer or a cancer associated with immunodeficiency. Such cancers can be selected from the group consisting of Kaposi's sarcoma (e.g., associated with Kaposi's sarcoma herpesvirus); squamous cell carcinoma of the cervix, anus, penis, and vulva, and oropharyngeal carcinoma (e.g., associated with human papillomavirus); 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 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 cancer associated with human immunodeficiency virus (HIV) infection.

[0292] Preferably, the cancer to be treated or prevented is 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.

[0293] Preferred cancers for treatment typically include cancers that are responsive to immunotherapy. Alternatively, preferred cancers for treatment are cancers that are not responsive to immunotherapy.

[0294] By way of example, and without wishing to be bound by theory, treatment with anti-cancer antibodies or anti-cancer immunoconjugates or other current anti-cancer therapies that induce cancer cell death enhances the immune response mediated by PD-1. Thus, treatment of hyperproliferative diseases (e.g., cancer tumors) may involve combining bifunctional molecules as disclosed herein, which can enhance the host's anti-tumor immune response, with anti-cancer therapy, either simultaneously, sequentially, or in any combination thereof. Preferably, the bifunctional molecules can be used in combination with other immunogenic agents, standard cancer therapies, or other antibodies as described herein below.

[0295] infectious disease The bifunctional molecules, nucleic acids, groups of nucleic acids, vectors, host cells, or pharmaceutical compositions of the invention can be used to treat patients exposed to a particular toxin or pathogen. Accordingly, one aspect of the invention is a method for treating an infectious disease in a subject, preferably comprising administering to the subject a humanized anti-PD-1 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising such, such that the infectious disease in the subject is treated.

[0296] Any suitable infection may be treated with the bifunctional molecules, nucleic acids, groups of nucleic acids, vectors, host cells or pharmaceutical compositions provided herein.

[0297] Some examples of pathogenic viruses that cause infections treatable by the methods of the present invention include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0298] In particular, the bifunctional molecules or pharmaceutical compositions of the present invention are used to treat patients with chronic viral infections, such as retroviruses, anelloviruses, circoviruses, herpesviruses, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), polyomavirus BK, polyomaviruses, adeno-associated viruses (AAV), herpes simplex type 1 (HSV-1), adenoviruses, herpes simplex type 2 (HSV), and the like. -2), Kaposi's sarcoma herpesvirus (KSHV), hepatitis B virus (HBV), GB virus C, papillomavirus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), hepatitis D virus (HDV), human T-cell leukemia virus type 1 (HTLV1), xenotropic murine leukemia virus-related virus (XMLV), rubella virus, rubella, parvovirus B19, measles virus, and coxsackievirus.

[0299] Some examples of pathogenic bacteria that cause infections that can be treated by the methods of the present invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospirosis, and lyme disease bacteria.

[0300] Some examples of pathogenic fungi that cause infections treatable by the methods of the present invention include Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger), Mucor (e.g., mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and the like. immitis and Histoplasma capsulatum.

[0301] Some examples of pathogenic parasites that cause infections treatable by the methods of the present invention are Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba spp., Giardia lambia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and the like. gondi, and Brazilian hookworm (Nippostrongylus brasiliensis).

[0302] In all of the above methods, the bifunctional molecules can be combined with other forms of immunotherapy, such as cytokine treatment (e.g., interferon, GM-CSF, G-CSF, IL-2), or any therapy that results in enhanced presentation of tumor antigens.

[0303] Combination therapy In particular, the bifunctional molecules of the present invention can be combined with several other potential strategies to overcome immune evasion mechanisms using agents in clinical development or already on the market (Antonia et al., Immuno-oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258-6268, 2014). Such combinations with the bifunctional molecules of the present invention can: 1-Reversing the inhibition of adaptive immunity (blocking T-cell checkpoint pathways); 2- Switching adaptive immunity (using agonist molecules, especially antibodies, to enhance T cell costimulatory receptor signaling); 3-improving the function of innate immune cells; 4- Activating the immune system (enhancing immune-cell effector functions), for example through vaccine-based strategies; This can be clearly useful.

[0304] Accordingly, also provided herein is a combination therapy for any of the diseases associated with PD-1 signaling described herein, using any of the bifunctional molecules described herein or pharmaceutical compositions comprising same and a suitable second agent. In embodiments, the bifunctional molecule and second agent can be present in the pharmaceutical compositions described above. Alternatively, as used herein, the term "combination therapy" or "combination therapy" encompasses the administration of these two agents (e.g., a bifunctional molecule described herein and an additional or second suitable therapeutic agent) in a sequential manner, i.e., each therapeutic agent is administered at a different time, and the administration of at least two of these therapeutic agents or agents is in a substantially simultaneous manner. The sequential or substantially simultaneous administration of each agent can be effected by any suitable route. The agents can be administered by the same route or by different routes. For example, a first agent (e.g., a bifunctional molecule) can be administered orally, and an additional therapeutic agent (e.g., an anti-cancer agent, an anti-infective agent; or an immunomodulator) can be administered intravenously. Alternatively, selected agents of the combination may be administered intravenously, while other agents of the combination may be administered orally.

[0305] In another aspect, the present invention relates to a therapeutic means, in particular a combination product means, which comprises as active ingredients a bifunctional molecule as defined above and an additional therapeutic agent, said active ingredients being formulated for separate, sequential or combined therapy, in particular combined or sequential use.

[0306] As used herein, the term "sequential" refers to a sequence or order in which the two substances are administered, unless otherwise specified. For example, if a dosing regimen includes the administration of a bifunctional molecule and a second agent, the sequential dosing regimen may include the administration of the bifunctional molecule of the present invention before, simultaneously, substantially simultaneously, or after the administration of the second agent, but both agents are administered in the sequence or order. The term "separate" refers to a sequence in which one substance is maintained apart from the other, unless otherwise specified. The term "concurrently" refers to a sequence occurring or occurring at the same time, i.e., the agents of the present invention are administered at the same time, unless otherwise specified. The term "substantially simultaneously" refers to the administration of agents within minutes of each other (e.g., within 15 minutes of each other) and is intended to encompass both conjoint administration and sequential administration, although when administration is sequential, it is separated in time by a short period of time (e.g., the time it takes a physician to administer the two compounds separately).

[0307] It should be understood that any combination described herein can be used in any sequence to treat the disorders or diseases described herein. The combinations described herein may be selected based on a number of factors, including, but not limited to, efficacy in inhibiting or preventing progression of the target disease, efficacy in mitigating side effects of other substances in the combination, or efficacy in mitigating symptoms associated with the target disease. For example, the combination therapies described herein may reduce any of the side effects associated with each individual member of the combination.

[0308] The present invention also relates to a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent.

[0309] When the bifunctional molecules or pharmaceutical compositions described herein are used contemporaneously with an additional therapeutic agent, a sub-therapeutic dosage of either the bifunctional molecule or pharmaceutical composition of the additional or second agent, or a sub-therapeutic dosage of both, can be used in treating a subject, preferably a subject having, or at risk of developing, a disease or disorder associated with PD-1-mediated cell signaling.

[0310] In aspects, the additional or second therapeutic agent is an alkylating agent, an angiogenesis inhibitor, an antibody, an antimetabolite, an antimitotic, an antiproliferative agent, an antiviral, an Aurora kinase inhibitor, a pro-apoptotic agent (e.g., a Bcl-2 family inhibitor), an activator of the death receptor pathway, a Bcr-Abl kinase inhibitor, a BiTE (bispecific T cell engager) antibody, an antibody drug conjugate, a biological response modifier, a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase inhibitor, a cell cycle inhibitor, a cyclooxygenase-2 inhibitor, a DVD, a leukemia viral oncogene homolog (ErbB2) receptor inhibitor, a growth factor inhibitor, a heat shock protein (HSP)-90 inhibitor, a histone deacetylase (HDAC) inhibitor, a hormone therapy, an immunological agent, an inhibitor of inhibitor of apoptosis proteins (IAPs), an intercalating antibiotic, a kinase inhibitor, a kinesin inhibitor, a steroid hormone receptor ... 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, peptide vaccines, etc., epitopes or neoepitopes derived from tumor antigens, as well as combinations of one or more of these substances.

[0311] For example, the additional therapeutic agent can be selected from the group consisting of chemotherapy, radiation therapy, targeted therapy, anti-angiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes derived from tumor antigens, myeloid checkpoint inhibitors, other immunotherapies, and HDAC inhibitors.

[0312] In a preferred embodiment, the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, an immunotherapeutic agent, a cell therapy agent (e.g., CAR-T cells), an antibiotic, and a probiotic. The immunotherapeutic agent may be an antibody targeting a tumor antigen, particularly an antibody targeting a tumor antigen selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti-CD52.

[0313] In an embodiment, the invention relates to a combination therapy as defined above, wherein the second therapeutic agent is in particular selected from the group consisting of a therapeutic vaccine, an immune checkpoint blocker or activator, in particular a therapeutic vaccine, an immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) and an antibody drug conjugate. Preferably, suitable substances for co-use with any of the humanized anti-hPD-1 antibodies or fragments thereof or pharmaceutical compositions according to the invention include antibodies that bind to costimulatory receptors (e.g., OX40, CD40, ICOS, CD27, HVEM or GITR), substances that induce immunogenic cell death (e.g., chemotherapeutic agents, radiotherapeutic agents, anti-angiogenic agents, or agents for targeted therapy), substances that inhibit checkpoint molecules (e.g., CTLA4, LAG3, TIM3, B7H3, B7H4, BTLA, or TIGIT), cancer vaccines, substances that modulate immunosuppressive enzymes (e.g., IDO1 or iNOS), substances that target Treg cells, agents for adoptive cell therapy, or substances that modulate myeloid cells.

[0314] In an embodiment, the invention relates to a combination therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) selected from the group consisting of anti-CTLA4, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonists, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists.

[0315] In one embodiment, the additional or second therapeutic agent is an antibody that specifically targets a tumor antigen selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti-CD52.

[0316] Specific examples of second therapeutic agents are provided on pages 36-43 of WO 2018 / 053106, the disclosure of which is incorporated herein by reference.

[0317] Combination therapy may also rely on a combination of surgery, chemotherapy (such as docetaxel or dacarbazine), radiation therapy, immunotherapy (such as antibodies targeting CD40, CTLA-4), gene targeting and modulation, and / or administration of the bifunctional molecule with other agents such as immune-modulators, angiogenesis inhibitors, and any combination thereof.

[0318] kit Any of the bifunctional molecules or compositions described herein may be included in kits provided by the present disclosure. The present disclosure provides kits for use in enhancing immune responses and / or treating diseases associated with PD-1 signaling (e.g., cancer and / or infectious diseases), among other things.

[0319] In the context of the present invention, the term "kit" refers to two or more components (one of which corresponds to a bifunctional molecule, nucleic acid molecule, vector, or cell of the present invention) packaged in a container, recipient, or vice versa. A kit may be described herein as a set of products and / or implements sufficient to achieve a particular goal, which can be marketed as a single unit.

[0320] Specifically, the kit according to the present invention comprises: - a bifunctional molecule as defined above, - a humanized anti-hPD1 antibody or antigen-binding fragment thereof conjugated to an immunotherapeutic agent; - a nucleic acid molecule or group of nucleic acid molecules encoding said bifunctional molecule, - a vector containing said nucleic acid molecule or group of nucleic acid molecules, and / or - a cell containing said vector or nucleic acid molecule or group of nucleic acid molecules may include:

[0321] Thus, the kit may comprise, in suitable container means, the pharmaceutical composition of the invention, and / or the bifunctional molecule, and / or the host cell, and / or the vector encoding the nucleic acid molecule of the invention and / or the nucleic acid molecule of the invention or related reagents. In some embodiments, means for obtaining a sample from an individual and / or means for assaying the sample may be provided. In certain embodiments, the kit includes cells, buffers, cell culture media, vectors, primers, restriction enzymes, salts, etc. The kit may also include sterile, pharmaceutically acceptable buffers and / or other diluents.

[0322] The containers may be unit doses, bulk packages (e.g., multi-dose packages), or semi-unit doses. In embodiments, the present invention relates to kits as defined above for single-administration dose units. The kits of the present invention may also include a first recipient comprising the dried / lyophilized bifunctional molecule and a second recipient comprising the aqueous formulation. In certain embodiments of the present invention, kits containing single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are provided.

[0323] The kits of the present invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packaging for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., sprayers), or infusion devices, such as minipumps, is also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a bifunctional molecule described herein, comprising a humanized anti-hPD1 antibody conjugated to an immunotherapeutic agent.

[0324] Compositions included in kits according to the present invention may also be formulated into syringe-compatible compositions. In this case, the container means itself may be a syringe, pipette, and / or other such device, from which the formulation may be applied to an infected area of ​​the body and / or applied to and / or mixed with other components of the kit. Alternatively, kit components may be provided as a dried powder. When reagents and / or components are provided as a dry powder, a soluble composition can be reconstituted by the addition of a suitable solvent. It is anticipated that the solvent may also be provided in a separate container means and be suitable for administration.

[0325] In some embodiments, the kit further includes an additional substance for cancer or infectious disease, which may be combined with the bifunctional molecule or other components of the kit of the present invention, or may be provided separately in the kit. In particular, the kits described herein may include one or more additional therapeutic agents, such as those described in the "combination therapy" section described hereinabove. The kit may be tailored to a particular cancer for an individual and may include a respective second cancer therapy for the individual, as described hereinabove.

[0326] Instructions for use of the bifunctional molecules or pharmaceutical compositions described herein generally include information regarding dosage, administration schedule, route of administration for the intended treatment, means for reconstituting the bifunctional molecule, and / or means for diluting the bifunctional molecules of the invention. Instructions provided in kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual). In some embodiments, the kit can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administering a pharmaceutical composition comprising the bifunctional molecule to enhance an immune response and / or treat a disease described herein. The kit can further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has a disease associated with PD-1 signaling, such as those described herein. [Example]

[0327] The following figures and examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. While the present invention has been described in terms of specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, method step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0328] result Example 1 Production and binding characteristics of a bifunctional anti-PD1 molecule and its interaction with PDL1 and towards ligands Figure 1 shows that bifunctional molecules of the present invention having a humanized anti-PD1 antibody fused to both the heavy and light chains exhibit improved production yields compared to bifunctional molecules having a chimeric anti-PD1 antibody. As shown in Figure 1, bifunctional molecules having a type 1 protein (A (i.e., CD86), B (i.e., CD80), and C (i.e., SIRPa)) or a type 2 protein (A (i.e., OX40L) and B (i.e., 4-1BBL)) and a cytokine (i.e., IL-7) fused to either the heavy chain (A) or light chain (B) are produced better than bifunctional molecules having a chimeric anti-PD1 antibody. Figure 1C shows that fusion of a cytokine (i.e., IL-7) to the heavy and light chains similarly improves production yields. This molecule contains four cytokines fused to a dimeric antibody.

[0329] The production yield of bifunctional molecules with a humanized anti-PD-1 scaffold was compared with two other anti-PD-1 scaffolds, Keytruda (pembrolizumab) and Opdivo (nivolumab). Figures 2A and 2B show that the humanized anti-PD-1 antibodies of the present invention exhibit better production yields of bifunctional molecules fused to cytokines (i.e., IL-7) or type I protein B (i.e., CD80) compared with other anti-PD-1 scaffolds. This improvement was observed using two different producer cell lines (CHO and HEK freestyle) and two different production methods (shake flasks and 12-well plates). To confirm this increase with other fusion type I proteins, the present inventors generated bifunctional molecules with a pembrolizumab scaffold fused to SIRPa. Furthermore, as shown in Figure 2C, higher cellular production yields were obtained with the humanized anti-PD-1 scaffold of the present invention compared with the Keytruda scaffold. In a fed-batch manufacturing process (non-optimized fed-batch CHO cell production in a bioreactor), the productivity of a bifunctional molecule containing humanized anti-PD-1 fused to CD80 type I protein was good (1 g / L).

[0330] In parallel, the present inventors constructed bifunctional molecules with various non-anti-PD-1 scaffold antibodies in which cytokines or type I or type II proteins were fused to the heavy or light chain, and compared their productivity with that of bifunctional molecules with humanized anti-PD-1 of the present invention. Figure 3 shows that all bifunctional molecules with the anti-PD-1 humanized scaffold of the present invention had significantly higher production yields, in contrast to all other scaffolds, confirming the high manufacturability of the humanized anti-PD-1 described in the present invention.

[0331] In parallel, we compared the PD1 binding of the bifunctional molecule with that of a chimeric or humanized anti-PD1 antibody. Figure 4 and Table 1 below show that good binding to the PD1 antigen is maintained regardless of the type of immunotherapeutic agent fused to the antibody heavy or light chain. The bifunctional molecule slightly loses binding efficacy compared to the control anti-PD1 antibody alone, but remains a potent binder to PD-1 (EC50<10ng / ml).

[0332] However, using surface plasmon resonance experiments (Biacore assays), the bifunctional molecules exhibited a similar range of affinity for PD-1 compared to the control anti-PD-1 alone. Biacore assays are a standard method used to measure protein affinity and are more sensitive and accurate than ELISA assays. In these assays, anti-PD-1 alone or an anti-human Fc antibody was placed on a sensor chip to capture the bifunctional molecules. Various concentrations of PD-1 recombinant protein (6.25–100 nM) were then added to measure affinity. Anti-PD-1 alone yielded a KD of 3.46 nM, anti-PD-1 IL-7 yielded a KD of 2.61 nM, and anti-PD-1 SIRPa yielded a KD of 3.83 nM.

[0333] Figures 5 and 6 compare the binding of bifunctional molecules containing chimeric and humanized anti-PD1 antibodies fused to different immunotherapeutic agents (type I proteins, type II proteins, or cytokine proteins) in the heavy chain (Figure 5) or light chain (Figure 6). The results and Tables 2 and 3 demonstrate that binding is comparable for both chimeric and humanized forms of the bifunctional molecules, and for immunotherapeutic agents fused to either the heavy or light chain of the antibody. We also tested bifunctional molecules containing anti-PD1 antibodies fused to the heavy and light chains, resulting in four proteins of the same type on a single antibody. Comparing the data in Tables 1, 2, 3, and 4, the EC50 values ​​are comparable. However, as shown in Figure 1C, production yields are still better for bifunctional molecules containing humanized anti-PD1 antibodies than for bifunctional molecules containing chimeric anti-PD1 antibodies.

[0334] [Table 8]

[0335] [Table 9]

[0336] [Table 10]

[0337] [Table 11]

[0338] The antagonistic properties of each bifunctional molecule with an anti-PD1 antibody fused to different immunotherapeutic agents against the PD1-PDL1 interaction were then analyzed compared to the anti-PD1 antibody alone. Figure 8A and Table 5 (Table 12) present the results of an ELISA competition assay. As previously suggested, the antagonistic properties of bifunctional molecules with an anti-PD1 antibody are not altered by fusing the antibody to an immunotherapeutic agent, although binding to PD1 is slightly reduced, as shown in Figure 4. The antagonistic properties of the bifunctional molecules anti-PD-1 VH IL7 or anti-PD-1 VH CD80 against PD1-PDL2 were evaluated by ELISA (Figure 8B). Both molecules efficiently block PD-L2 binding to PD-1.

[0339] [Table 12]

[0340] To test the binding of immunotherapeutics to their ligands, we performed a bridging ELISA assay. Figure 9 shows the results for type I and type II proteins. The histograms are positive controls for binding of immunotherapeutics alone to the ligand. Ligand binding of immunotherapeutics is preserved when the immunotherapeutics are fused to the heavy or light chain of an anti-PD1 antibody.

[0341] Example 2 Ex vivo characterization of bifunctional molecules fused to anti-PD1 antibodies and immunotherapeutic agents on T cell proliferation and activation To measure the efficacy of the anti-PD1 bifunctional molecule on T cell activation and proliferation, we examined T cell proliferation after treatment with an anti-PD1 antibody alone (pembrolizumab was used as an anti-PD1 antibody control), an isotype control, or a bifunctional molecule with an anti-PD1 antibody fused to the VH or VL chain. The results, shown in Figure 10, indicate that any bifunctional molecule with an anti-PD1 antibody induces T cell proliferation at least as well as, or even better than, anti-PD1 alone. Assays measuring T cell activation, as indicated by IFNg secretion in Figure 11, show very good efficacy for T cell activation. Indeed, the bifunctional molecule with an anti-PD1 antibody induces T cell activation at least as well as, or even better than, the control anti-PD1 antibody. This indicates that the bifunctional molecule format can enhance the efficacy of anti-PD1 antibodies on T cell proliferation and activation in vivo and may be a better format for targeting T cells to tumors.

[0342] Bifunctional molecules of the present invention comprising anti-PD1 antibodies fused with one, two, three, or four immunotherapeutic agents exhibit good binding to PD1 and good inhibitory properties against PD1-PDL1 interaction. These bifunctional molecules are produced better than bifunctional molecules comprising chimeric antibodies. Ex vivo, these bifunctional molecules induce better proliferation and activation of human T cells. This indicates that these bifunctional molecules may also be more effective in vivo, particularly against intratumoral T cells, compared with anti-PD1 antibodies alone.

[0343] Example 3 In vivo pharmacokinetics of Bicki anti-PD1 antibody To analyze the pharmacokinetics of bifunctional molecules with humanized anti-PD-1, mice were intravenously injected with 5 mg / kg of bifunctional molecules with humanized anti-PD-1 fused to SIRPa in the heavy chain. Two different isotypes were compared: the IgG1 N298A isotype and the IgG4 S228P isotype. Plasma concentrations were assessed by ELISA at multiple time points after injection. As shown in Figure 12, the bifunctional molecules with humanized anti-PD-1 constructed with IgG1 N298A exhibited a better pharmacokinetic profile than the bifunctional molecules with humanized anti-PD-1 constructed with the IgG4 S228P isotype.

[0344] Materials and Methods PD1 binding ELISA and bridging ELISA assays For the PD-1 binding ELISA assay, recombinant hPD1 (Sino Biologicals, Beijing, China; reference number 10377-H08H) was immobilized to plastic at 0.5 μg / ml in carbonate buffer (pH 9.2), and purified antibody was added to measure binding. After incubation and washing, peroxidase-conjugated donkey anti-human IgG (Jackson Immunoresearch, USA; reference number 709-035-149) was added, and color development was performed colorimetrically at 450 / 650 nm using TMB substrate (3,3',5,5'-tetramethylenzidine, BD Bioscience, San Jose, CA, USA).

[0345] A similar method was used for the bridging ELISA assay. Recombinant hPD1 was immobilized and purified bifunctional antibodies were added in serial dilutions. After incubation and washing, recombinant receptors for proteins C, A, or B were then added at 1 μg / mL. Detection was performed using anti-receptor specific mouse antibodies and peroxidase-labeled donkey anti-mouse IgG antibodies (reference number 715-036-151). Color development was performed using conventional methods.

[0346] ELISA antagonist: Competition between PDL1 or PD-L2 and humanized anti-PD1 A competitive ELISA assay was performed using a PD-1:PD-L1 inhibitor screening ELISA assay pair (AcroBiosystems, USA; Reference No. EP-101). In this assay, recombinant hPDL1 was immobilized to plastic at 2 μg / ml in PBS pH 7.4 buffer. Purified antibodies (at various concentrations) were mixed with biotinylated human PD1 (AcroBiosystems, USA; Reference No. EP-101) at a final (fixed) concentration of 0.66 μg / ml, and competitive binding was measured at 37°C for 2 hours. After incubation and washing, peroxidase-labeled streptavidin (Vector Laboratories, USA; Reference No. SA-5004) was added to detect biotin-CD47Fc binding, followed by conventional color development. The same ELISA protocol was performed to evaluate the PDL2 / PD1 antagonist activity by coating PD-L2 Fc recombinant protein (Sinobiological, #10292-H02H).

[0347] IFN-gamma secretion and T cell proliferation assays Human T cells were purified from peripheral blood mononuclear cells using an untouched pan T cell isolation kit (reference number 130-096-535, MACS Miltenyi Biotech, USA) and an Automacs pro separator (Miltenyi). T cells were stimulated and PD-1 expression was induced by incubation on CD3 / CD28-coated plates (anti-CD3 clone OKT3 and anti-CD28 clone CD28.2, each at a concentration of 3 μg / mL). Twenty-four hours after stimulation, T cells were harvested, counted, and restimulated with anti-CD3 (clone OKT3, 2 μg / mL) plus recombinant human PD-L1 (Sinobiological, reference number 10084-H02H, 5 μg / mL) in the presence of isotype control, unfused anti-PD-1, or an anti-PD-1 bifunctional antibody. Supernatants were collected on day 6 to quantify IFNg secretion (Human IFNg ELISA Set, BD Bioscience, USA, Ref. 555142) and T cell proliferation was assessed by H3 thymidine incorporation.

[0348] Pharmacokinetics and pharmacodynamics of a humanized anti-PD1 antibody in mice BalbcRJ (female, 6-9 weeks old) received a single intraorbital dose (34.4 nM / kg) of a bifunctional humanized anti-PD-1 antibody. Plasma drug concentrations were determined by ELISA using immobilized anti-human light chain antibody (clone NaM76-5F3) and diluted serum containing anti-PD-1 antibody. Detection was performed by adding peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch, USA; reference number 709-035-149) and developing by conventional methods.

[0349] Antibodies and bifunctional molecules The following antibodies and bifunctional molecules were used in the different experiments disclosed herein: pembrolizumab (Keytrudra, Merck), nivolumab (Opdivo, Bristol-Myers Squibb), and bifunctional molecules disclosed herein comprising an anti-PD1 humanized antibody comprising a heavy chain defined in SEQ ID NO: 19, 22 or 24 and a light chain defined in SEQ ID NO: 28 or an anti-PD1 chimeric antibody comprising a heavy chain defined in SEQ ID NO: 59 and a light chain defined in SEQ ID NO: 60.

Claims

1. (a) a humanized anti-human PD-1 antibody or an antigen-binding fragment thereof, - a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 19, 22 or 24; and a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28 Including, an antagonist of the binding of human PD-L1 and / or PD-L2 to human PD-1; a humanized anti-human PD-1 antibody or an antigen-binding fragment thereof; and (b) an immunotherapeutic agent or a fragment thereof, wherein the fragment retains the biological activity of the immunotherapeutic agent; A bifunctional molecule consisting of A bifunctional molecule, wherein 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 the immunotherapeutic agent as a fusion protein.

2. 2. The bifunctional molecule of claim 1, wherein 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 the immunotherapeutic agent as a fusion protein by a peptide linker.

3. 3. The bifunctional molecule of claim 1 or 2, wherein the immunotherapeutic agent or fragment thereof is selected from the group consisting of a tumor-targeting peptide, a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a costimulatory molecule, an inhibitory or co-inhibitory molecule, a molecular chaperone inhibitor, and a type I or type II human transmembrane immune protein.

4. The bifunctional molecule of any one of claims 1 to 3, wherein the immunotherapeutic agent or fragment thereof has a size comprised between 10 kDa and 50 kDa.

5. The immunotherapeutic agent may be ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, nectin-1, nectin-2, nectin-3, NELC-5, TLT-2, LFA-3, TIM3, TIM4, LAIR1, SIRPG, IL10R, IL6RA, IL-1R1, IL-1RAcP, IL6RB, or TGFBRI.

5. The bifunctional molecule of claim 3 or 4, which is a type I human transmembrane immunity protein or a fragment thereof selected from the group consisting of I, CSF1R, IL22R, VEGFR1, VEGFR2, VEGFR3, CD111, CD112, CD155, CD113, VISTA, CD244, OX40, CD80, CD24, Siglec-10, Fas, IL15RA, SIRB1, SIRB2, LTBR, ​​IL21R, and GITR.

6. 5. The bifunctional molecule of claim 3 or 4, wherein the immunotherapeutic agent is a type II human transmembrane immune protein or a fragment thereof selected from the group consisting of CD40L, OX40L, FasL, TRAIL, TNF, LIGHT, APRIL, GITRL, CD30, CD70, CD40, CD27, CD30, CD153, RANK, CD96, CLEC1, CLEC2 / CLE1B, CLEC3A, CLEC4A, CLEC4E, CLEC4L, CLEC51, CLEC6, CLEC7A, NKG2D, BTL-II, TGFRII, DECTIN-1, DC-SIGN, LT-alpha, LT-beta, 4-1BBL, and MINCLE.

7. 5. The bifunctional molecule of claim 3 or 4, wherein the immunotherapeutic agent is a cytokine or a fragment thereof selected from the group consisting of TGFβ, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12A, IL12B, IL-15, IL-21, and IL-18.

8. The bifunctional molecule of claim 3 or 4, wherein the immunotherapeutic agent is human IL-2 or a variant thereof.

9. 5. The bifunctional molecule of claim 3 or 4, wherein the immunotherapeutic agent is human IL-2 as set forth in SEQ ID NO: 58, with substitutions F42A, Y45A, and L72G.

10. 10. The bifunctional molecule of claim 1, 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.

11. 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 10. The bifunctional molecule of any one of claims 1 to 9, 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.

12. 10. The bifunctional molecule of claim 1, 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.

13. 13. An isolated nucleic acid molecule or group of isolated nucleic acid molecules encoding the bifunctional molecule of any one of claims 1 to 12.

14. A vector comprising the nucleic acid molecule or group of nucleic acid molecules according to claim 13.

15. 15. A host cell comprising a vector according to claim 14, or a nucleic acid molecule or group of nucleic acid molecules according to claim 13.

16. 16. A method for producing a bifunctional molecule according to any one of claims 1 to 12, comprising culturing a host cell according to claim 15 and isolating the bifunctional molecule.

17. 16. A pharmaceutical composition comprising a bifunctional molecule according to any one of claims 1 to 12, a nucleic acid molecule or a group of nucleic acid molecules according to claim 13, a vector according to claim 14, or a host cell according to claim 15, and a pharmaceutically acceptable carrier.

18. 18. The pharmaceutical composition of claim 17, further comprising an additional therapeutic agent.

19. 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 19. The pharmaceutical composition of claim 18, wherein the therapeutic agent is selected from the group consisting of an antitumor agent, a mammalian target of rapamycin inhibitor, 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.

20. A pharmaceutical composition according to any one of claims 17 to 19, or a bifunctional molecule according to any one of claims 1 to 12, or a nucleic acid molecule or group of nucleic acid molecules according to claim 13, or a vector according to claim 14, or a host cell according to claim 15, for use as a medicament.

21. 21. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 20 for use in the treatment of cancer.

22. The cancer is selected from the group consisting of hematological malignancies or solid tumors with PD-1 and / or PD-L1 expression, hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndrome, 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.

22. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 21, wherein the cancer is selected from the group consisting of: Hodgkin's lymphoma, and lymphoproliferative disorders; hepatocellular carcinoma; Merkel cell carcinoma; and cancer associated with human immunodeficiency virus (HIV) infection, and a cancer 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.

23. 23. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell according to any one of claims 20 to 22 for use in combination with radiation therapy or an additional therapeutic agent.

24. 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 24. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 23, selected from the group consisting of a target, microRNA, mitogen-activated extracellular signal-regulated kinase inhibitor, multivalent binding protein, nonsteroidal anti-inflammatory drug (NSAID), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitor, platinum chemotherapeutic agent, polo-like kinase (Plk) inhibitor, phosphoinositide-3 kinase (PI3K) inhibitor, proteasome inhibitor, purine analog, pyrimidine analog, receptor tyrosine kinase inhibitor, retinoid / deltoid plant alkaloid, small inhibitory ribonucleic acid (siRNA), topoisomerase inhibitor, ubiquitin ligase inhibitor, hypomethylating agent, checkpoint inhibitor, peptide vaccine, epitope or neoepitope derived from a tumor antigen, and combinations of one or more of these substances.

25. 21. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 20 for use in the treatment of an infectious disease.

26. 26. The pharmaceutical composition, bifunctional molecule, nucleic acid molecule or group of nucleic acid molecules, vector, or host cell of claim 25, 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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