Soluble PD-1 mutants

Abstract

The present invention mainly concerns a soluble PD-1 polypeptide which is a mutant of SEQ ID N:2 or 12, that binds PD-L1 and / or PD-L2, which comprises : a) at least one mutation in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1, said mutation being performed on N, A or T; b) at least one mutation in the N-glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1, said mutation being performed on N, T or S; c) at least one mutation in the N-glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1, said mutation being performed on N, Q or T; and d) at least one mutation in the N-glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1, said mutation being performed on N, D or S, wherein each of mutations a) to d) affects or prevents N-glycosylation.

Description

[0001] Soluble PD-1 mutants

[0002] The present invention mainly concerns a soluble PD-1 polypeptide which is a mutant of SEQ ID NO:2 or 12, and that binds PD-L1 and / or PD-L2, which comprises specific mutations a) to d) that affects or prevents N-glycosylation.

[0003] T cell activation is a highly regulated process, initiated by stimulation of the T cell receptor (TCR) complex through engagement of cognate peptide-major histocompatibility (MHC) complexes. Additional signals fine-tune the immune response, with costimulatory (positive) pathways that potentiate the activation and proliferation of T cells, and coinhibitory (negative) pathways that attenuate T cell activation. For example, the CD28 costimulatory receptor is an indispensable signal required for full activation of naive and effector T cells. CD28 can be ligated by CD80, CD86, and ICOS-L (B7-H2). The CTLA-4 co-inhibitor competes with CD28 for binding to CD80 and CD86.

[0004] Programmed cell death protein 1 (PD-1 , also known as PDCD1 and CD279) is a major coinhibitory receptor expressed by T cells, B cells, myeloid cells (monocytes and some dendritic cells) and natural killer (NK) cells. The ligands for PD-1 are programmed cell death 1 ligand 1 (PD-L1 ; also known as CD274 and B7-H1) and programmed cell death 2 ligand 1 (PD-L2; also known as CD273 and B7-DC), which share 34% identity and are expressed on many tumor cells and antigen-presenting cells such as monocytes, dendritic cells (DC) and macrophages. This receptor / ligand pair functions primarily to provide inhibitory signals, through the recruitment of phosphatases, such as SHP-2, to the immunoreceptor tyrosin- based switch motif (ITSM) of the cytoplasmic tail of PD-1. Notably, CD28 has been described as a preferred target over the TCR for dephosphorylation by PD-1-recruited Shp2 phosphatase (Hui, E. et al. T cell costimulatory receptor CD28 is a primary target for PD-1- mediated inhibition. Science. 355(6332):1428-1433. doi: 10.1126 / science.aaf1292 (2017)).

[0005] PD-1 has an essential role in balancing protective immunity and immunopathology, homeostasis and tolerance, and is a prominent target for cancer immunotherapy. The PD- 1 / PD-L pathway is frequently exploited as a target for immune evasion by tumor cells. Tumor cells can overexpress PD-1 ligands, which bind PD-1 on effector T cells, thereby inhibiting the anti-tumor immune response mediated by these cells.

[0006] Recently developed PD-1 checkpoint inhibitors have revolutionized cancer treatment for some patients, with FDA-approved monoclonal antibodies targeting PD-1 (Pembrolizumab, Nivolumab, Cemiplimab, Dostarlimab) or PD-L1 (Atezolizumab, Durvalumab, Avelumab). Anti-PD-L2 antibodies are currently being investigated in clinical trials.

[0007] However, the majority of patients do not show complete responses, and adverse events have been noted.

[0008] There is still a need to provide improved means having the potential to improve safety and efficacy and overcome side effects. PD-L1 or PD-L2 antagonists target ligands on tumor cells but only block one PD-1 / PD-L interaction at a time.

[0009] Moreover, antibodies may be impeded from entering tumors due to their large size and not have access to PD-1 -expressing effector T cells infiltrated within solid tissue, or to PD-L-expressing tumor cells. Antibodies may therefore fail to completely antagonize PD- 1 / PD-L pathway.

[0010] Small-molecule drugs are expected to penetrate more effectively than antibodies into the tumor microenvironment. The present invention is directed to a novel mechanism of using the soluble form of human PD-1 , to effect similar biological function and therapeutic effect. Soluble PD-1 (sPD-1) is an attractive agent for blocking the PD-1 / PD-L pathway, by targeting both PD-L1 and PD-L2 at the same time on the tumor cells, which may lead to increase in efficacy and safety of cancer treatment. However, the binding of natural sPD-1 to its ligands is weak, with a dissociation constant (KD) of 8.2 pM and 2.3 pM for the binding of hPD-1 to hPD-L1 and hPD-L2 respectively (Cheng, X. et al. Structure and interactions of the human programmed cell death 1 receptor. 288(17):11771-85. doi: 10.1074 / jbc.M112.448126. (2013)), making natural sPD-1 a poor antagonist for blocking the PD-1 / PD-L pathway.

[0011] Membrane-bound PD-1 (full-length PD-1) is a type I transmembrane glycoprotein belonging to CD28 family of receptors gene. Human PD-1 is a 55 kDa glycoprotein of 288 amino acids, the sequence of which is available in Uniprot under accession number Q15116. It is composed of the signal sequence (23 amino acids) fused to N-terminal IgV like domain (147 amino acids), a transmembrane domain (21 amino acids), and a 97 aminoacid intracytoplasmic domain that contains two tyrosine-based signaling motifs. PD-1 lacks intracellular SH2 (Src Homology 2) or SH3 binding motifs unlike CD28 and CTLA-4. PD-1 is produced as monomer as it lacks the cysteine residue that is required for homodimerization (Khan et al, Front. Immunol., 19 November 2020, Soluble PD-1 : Predictive, Prognostic, and Therapeutic Value for Cancer Immunotherapy).

[0012] Preferably, the human wild-type (WT) full-length PD-1 of the invention is SEQ ID The signal sequence fused to N-terminal IgV like domain is the extracellular fragment, or soluble form (sPD-1); it comprises 170 amino acids. The N-terminal IgV like domain is the mature soluble form and comprises 147 amino acids. However, some amino acids in N- or C-terminal may be deleted, and correspond to fragments, without any impact on the functionality of the molecule.

[0013] Thus, by « soluble PD-1 » or “sPD-1”, it is meant a protein which comprises the N- terminal IgV-like domain and optionally the signal sequence, but which does not comprise the transmembrane domain nor the intracytoplasmic domain.

[0014] Preferably, the human wild-type sPD-1 of the invention is SEQ ID NO :2.

[0015] Preferably, the human wild-type sPD-1 of the invention is SEQ ID NO :12.

[0016] Posttranslational modifications (PTMs) refer to amino acid side chain modification in some proteins after their biosynthesis: e.g. phosphorylation, acetylation, ubiquitylation, methylation, N-glycosylation, O-glycosylation, deamidation, disulfide bond formation... They have important roles in membrane protein folding, stability and other cellular functions.

[0017] However, production of recombinant proteins or peptides comprising at least one N- glycosylation site is generally a challenge.

[0018] N-linked glycosylation frequently occurs at the asparagine residues within glycosylation sequon N-X-S / T, where N is asparagine, X is any amino acid except proline, S is serine, and T is threonine. Thus, by « N-linked glycosylation site », it is meant a glycosylation sequon N-X-S / T.

[0019] Deamidation has been associated with “hot spots” such as asparagine-glycine (NG), asparagine-serine (NS), asparagine-threonine (NT) and asparagine-asparagine (NN) sites within the protein sequence.

[0020] Although the thiol side chain of cysteine (C) can be in a free form, in most cases it forms a disulfide bond either with a second cysteine (bridge) or with another thiol, as in the case of protecting groups. Efficient reduction of these disulfide bridges is a requirement for many applications of Cys-containing molecules in the fields of chemistry and biochemistry. sPD-1 has four predicted N-linked glycosylation sites within its extracellular IgV domain, at positions N49, N58, N74 and N116, wherein the amino acid numbering is the one of human WT full-length PD-1 protein (SEQ ID NO :1). Its molecular weight substantially shifted from 14 kDa, when expressed in E. coli with no glycosylation, to about 35-40 kDa, when expressed in 293T cells with glycosylation similar to that in host cells (Tan, S. et al. An unexpected N-terminal loop in PD-1 dominates binding by nivolumab. Nat Commun. 6;8:14369. doi: 10.1038 / ncomms14369. (2017)). These N-glycosylation sites are conserved in murine PD-1 protein, which shares 60% identity with human PD-1 protein. sPD-1 has one NG deamidation site at positions N102 and G103. sPD-1 contains a free cysteine residue at position 93.

[0021] Blocking N-linked glycosylation by tunicamycin, an inhibitor of N-linked glycosylation, significantly reduced the levels of cell surface PD-1. Also, glycosylation of PD-1 , in particular at the N58 site, was shown to be essential for mediating its interaction with PD-L1 (Sun, L. et al. Targeting glycosylated PD-1 induces potent anti-tumor immunity. Cancer Research. June 01 ; 80(11): 2298-2310. doi:10.1158 / 0008-5472. CAN-19-3133. (2020)).

[0022] It is an object of the present invention to provide a soluble PD-1 polypeptide, or an antigen-binding fragment or protein construct comprising said polypeptide, wherein said polypeptide has improved properties (e.g. increased binding affinity to PD-L1 and / or PD- L2, increased stability, increased manufacturability, etc.) as well as methods of making and using such sPD-1 polypeptide in treating patients with cancer and / or infections. Preferably, it is an object of the present invention to provide a soluble PD-1 polypeptide, or an antigenbinding fragment or protein construct comprising said polypeptide, wherein said polypeptide shows a simplified manufacturability while still presenting a good binding affinity to PD-L1 and / or PD-L2.

[0023] It results that the invention allows production of said polypeptides into a prokaryotic system or a yeast expression system. Moreover, the polypeptides that are obtained according to the invention show a more consistent quality profile.

[0024] Thus, the present invention targets a soluble PD-1 polypeptide (“sPD-1 polypeptide of the invention” or “sPD-1 mutant of the invention”) which is a mutant of SEQ ID NO:2 or 12, and binds PD-L1 and / or PD-L2, which comprises : a) at least one mutation in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, A or T; b) at least one mutation in the N-glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, T or S; c) at least one mutation in the N-glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, Q or T; and d) at least one mutation in the N-glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, D or S, wherein each of mutations a) to d) affects or prevents N-glycosylation.

[0025] Preferably, the mutation a) in the N-glycosylation site NAT, wherein N is in position 49 and the numbering is the one of SEQ I D NO: 1 , is N49T.

[0026] Preferably, the sPD-1 mutant of the invention comprises at least 70% identity with SEQ ID NO:2 or 12 and binds PD-L1 and / or PD-L2, and comprises : a) at least one mutation in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, A or T; b) at least one mutation in the N-glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, T or S; c) at least one mutation in the N-glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, Q or T; and d) at least one mutation in the N-glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, D or S, wherein each of mutations a) to d) affects or prevents N-glycosylation.

[0027] Preferably, the mutation a) in the N-glycosylation site NAT, wherein N is in position 49 and the numbering is the one of SEQ I D NO: 1 , is N49T.

[0028] The invention also relates to an antigen-binding fragment comprising a soluble PD-1 polypeptide according to the invention. It further relates to a protein construct comprising a soluble PD-1 polypeptide according to the invention that is fused, directly or via a linker, to a protein fragment.

[0029] The present invention also relates to a nucleic acid coding for a soluble PD-1 polypeptide according to the invention, or for the antigen-binding fragment according to the invention or for the protein construct according to the invention. It further relates to an expression vector comprising said nucleic acid. It further relates to a host cell comprising said nucleic acid or said expression vector. The present invention also relates to process for producing a soluble PD-1 polypeptide according to the invention, comprising:

[0030] (i) a step of expressing the nucleic acid or expression vector in the host cell, in order to express the soluble PD-1 mutant; and

[0031] (ii) optionally harvesting the soluble PD-1 mutant produced in step (i).

[0032] Finally, the invention also relates to the use of a soluble PD-1 polypeptide according to the invention, or antigen-binding fragment according to the invention or protein construct according to the invention, or nucleic acid according to the invention, or expression vector according to the invention or host cell according to the invention, as a medicament.

[0033] It further relates to a soluble PD-1 polypeptide according to the invention, or antigenbinding fragment according to the invention or protein construct according to the invention, which is conjugated with at least one compound chosen from drugs, toxins and radioactive compounds.

[0034] It further relates to the use of a soluble PD-1 polypeptide according to the invention, or antigen-binding fragment according to the invention or protein construct according to the invention, or nucleic acid according to the invention, or expression vector according to the invention or host cell according to the invention, for preventing and / or treating cancer in a subject in need thereof.

[0035] The subject is preferably a mammal. Preferably, the subject is a human, a horse, a mouse, a rat, a dog, a cat or a goat, more preferably a human.

[0036] Said use can thus be performed in the veterinary field. sPD-1 polypeptide

[0037] The sPD-1 polypeptide of the invention is a mutant of SEQ ID NO:2 or 12, and binds PD-L1 and / or PD-L2, which comprises : a) at least one mutation in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, A or T; b) at least one mutation in the N-glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, T or S; c) at least one mutation in the N-glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, Q or T; and d) at least one mutation in the N-glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, D or S, wherein each of mutations a) to d) affects or prevents N-glycosylation.

[0038] Preferably, the mutation a) in the N-glycosylation site NAT, wherein N is in position 49 and the numbering is the one of SEQ I D NO: 1 , is N49T.

[0039] Preferably the sPD-1 polypeptide of the invention is a mutant of SEQ ID NO:2 or 12, comprising at least 70% identity with SEQ ID NO:2 or 12 respectively and that binds PD-L1 and / or PD-L2, which comprises : a) at least one mutation in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, A or T; b) at least one mutation in the N-glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, T or S; c) at least one mutation in the N-glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, Q or T; and d) at least one mutation in the N-glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, D or S, wherein each of mutations a) to d) affects or prevents N-glycosylation.

[0040] Preferably, the mutation a) in the N-glycosylation site NAT, wherein N is in position 49 and the numbering is the one of SEQ I D NO: 1 , is N49T.

[0041] The sPD-1 polypeptide of the invention is a mutant of sPD-1 (SEQ ID NO :2). Preferably it comprises at least 70% identity with SEQ ID NO:2. Preferably it comprises at least 71 % identity, preferably at least 72%, preferably at least 73%, preferably at least 74% with SEQ ID NO:2; preferably the soluble PD-1 mutant is identical to SEQ ID NO:2 and comprises mutations a) to d).

[0042] Preferably the soluble PD-1 mutant is a fragment of SEQ ID NO:2 and comprises mutations a) to d). Preferably the sPD-1 polypeptide of the invention comprises at least 70% identity with SEQ ID NO:12. Preferably it comprises at least 71 % identity, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, with SEQ ID NO:12.

[0043] According to an embodiment, the soluble PD-1 mutant is identical to SEQ ID NO: 12 and comprises mutations a) to d).

[0044] The percent amino acid sequence identity is defined as the percent of amino acid residues in a Compared Sequence that are identical to the Reference Sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-). When using such software, the default parameters, e.g., for gap penalty and extension penalty, are preferably used. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10. The percent identity is determined on at least 20 consecutive amino acid residues of the reference sequence.

[0045] The sPD-1 polypeptide of the invention further binds PD-L1 and / or PD-L2. PD-L1 and PD-L2 are PD-1 ligands.

[0046] It comprises at least four mutations a) to d), i.e. at least one mutation per N- glycosylation site.

[0047] The numbering of the mutations is the one of SEQ ID NO:1 by convenience.

[0048] For sake of clarity, the N-glycosylation site NAT of mutation a) is the one for which N is in position 49 of SEQ ID NO:1 , or in position 25 of SEQ ID NO:2 or 12. The N-glycosylation site NTS of mutation b) is the one for which N is in position 58 of SEQ I D NO: 1 , or in position 34 of SEQ ID NO:2 or 12. The N-glycosylation site NQT of mutation c) is the one for which N is in position 74 of SEQ ID NO:1 , or in position 50 of SEQ ID NO:2 or 12. The N- glycosylation site NDS of mutation d) is the one for which N is in position 116 of SEQ ID NO: 1 , or in position 92 of SEQ I D NO:2 or 12. By « mutation », it is meant an amino acid insertion, an amino acid deletion or an amino acid substitution by a natural or an unnatural amino acid residue. Of course, said mutated sPD-1 retains PD-1 function. Preferably, the mutation is a substitution.

[0049] Preferably, each one of mutations a) to d) of the sPD-1 polypeptide of the invention is a substitution.

[0050] Preferably, mutations a), b) and d) are N-substitutions, and mutation c) is N- or T- substitution.

[0051] Preferably, the sPD-1 polypeptide of the invention comprises: as mutation a), at least one mutation of N or T in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 . Preferably, the mutation a) in the N-glycosylation site NAT, wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 , is N49T; as mutation b), at least one mutation of N or S in the N-glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 ; as mutation c), at least one mutation of N or T in the N-glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 ; and as mutation d), at least one mutation of N, D or S in the N-glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 , with the proviso that if D is mutated, then N is also mutated. Preferably at least two mutations of N and D are performed in the N-glycosylation site NDS.

[0052] Preferably, the sPD-1 polypeptide of the invention is such that: a) the at least one mutation in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 is chosen from N49T and T51 K, preferably N49T; b) the at least one mutation in the glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 is N58E, N58S, N58H, N58Q, S60H and S60G; c) the at least one mutation in the glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 is chosen from N74G, N74Q, T76Y and T76P; and d) the at least one mutation in the glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 is chosen from N116E, N116S, N116Q, D117A and S118R, with the proviso that if D is mutated, then N is also mutated; preferably the mutations are N116E / D117A or S118R.

[0053] Preferably, the sPD-1 polypeptide of the invention is such that: a) the at least one mutation in the glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 is N49T; b) the at least one mutation in the glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 is N58S, N58E or N58Q; c) the at least one mutation in the glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 is chosen from N74Q, T76Y and T76P; and d) the at least one mutation in the glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 is N116E, N116S or S118R.

[0054] More preferably, the sPD-1 polypeptide of the invention is such that: a) the at least one mutation in the glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 is N49T; b) the at least one mutation in the glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 is N58E or N58Q; c) the at least one mutation in the glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 is chosen from N74G or N74Q; and d) the at least one mutation in the glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 is N116E or N116Q.

[0055] Preferably, the sPD-1 polypeptide of the invention comprises at least one additional mutation in position 93, the numbering being the one of SEQ ID NO:1 ; preferably the mutation is C93S.

[0056] Preferably, the sPD-1 polypeptide of the invention comprises at least one additional mutation in position 26, 30, 32, 37, 38, 45, 57, 61 , 62, 64, 66, 67, 68, 69, 70, 77, 78, 84, 86, 87, 88, 90, 93, 96, 103, 104, 108, 109, 110, 112, 115, 122, 124, 125, 127, 129, 130, 131 , 132, 135, 139, 143, 145, 146 or 147, the numbering being the one of SEQ ID NO:1.

[0057] Preferably, the sPD-1 polypeptide of the invention comprises at least one additional mutation chosen from D26K, R30Q, W32I, W32L, F37I, S38G, T45E, S57D, E61G, S62N, V64F, N66V, N66I, W67F, Y68F, Y68N, Y68H, R69K, M70I, D77E, D77I, D77L, D77A, K78R, K78T, K78L, E84P, R86K, R86A, S87I, S87T, S87D, S87F, S87V, Q88G, Q88F, G90P, C93S, R96Q, G103K, R104K, M108F, M108P, S109T, V110I, R112N, R115I,

[0058] R115V, R115E, L122F, G124I, G124S, G124A, G124V, A125V, A125I, A125C, A125G,

[0059] A125L, S127T, S127A, S127V, A129Y, A129W, P130G, A132L, A132I, A132M, A132F,

[0060] K135L, R139F, R139V, R139Y, R139M, R143I, R143L, R143V, T145L, T145V, T145E,

[0061] E146P, R147K and R147Q, the numbering being the one of SEQ ID NO:1.

[0062] Preferably, the sPD-1 polypeptide of the invention comprises at least one additional mutation in position 26, 30, 32, 38, 45, 57, 61 , 62, 64, 67, 69, 70, 77, 84, 86, 87, 88, 90, 93, 96, 103, 104, 108, 109, 110, 112, 115, 124, 129, 130, 132, 135, 139, 143, 145, 146 or 147, the numbering being the one of SEQ ID NO:1. Preferably, the sPD-1 polypeptide of the invention comprises at least one additional mutation chosen from D26K, R30Q, W32I, W32L, F37I, S38G, T45E, S57D, E61G, S62N, V64F, W67F, R69K, M70I, D77E, D77I, D77L, D77A, E84P, R86K, R86A, S87I, S87T, S87D, S87F, S87V, Q88G, Q88F, G90P, C93S, R96Q, G103K, R104K, M108F, M108P, S109T, V110I, R112N, R115I, R115V, R115E, L122F, G124I, G124S, G124A, G124V, A129Y, A129W, P130G, A132L, A132I, A132M, A132F, K135L, R139F, R139V, R139Y, R139M, R143I, R143L, R143V, T145L, T145V, T145E, E146P, R147K and R147Q, the numbering being the one of SEQ ID NO:1.

[0063] Alternatively, preferably, the sPD-1 polypeptide of the invention comprises at least one additional mutation in position 93, 124, 131 and / or 132, the numbering being the one of SEQ ID NO:1. Preferably, the sPD-1 polypeptide of the invention comprises at least one additional mutation chosen from C93S, G124S, G124A, G124I, G124V, K131Y, A132I, A132L, A132M and A132F, the numbering being the one of SEQ ID NO:1. Preferably, the sPD-1 polypeptide of the invention comprises at least one additional mutation chosen from C93S, G124S, K131Y and A132I, the numbering being the one of SEQ ID NO:1.

[0064] The invention further relates to a sPD-1 polypeptide which is a mutant of SEQ ID NO:2, comprising at least 70% identity with SEQ ID NO:2 and that binds PD-L1 and / or PD- L2, which comprises at least one mutation in position 26, 30, 67, 103, 117, 118, 143 or 146, the numbering being the one of SEQ ID NO:1. Preferably, said sPD-1 polypeptide comprises at least one additional mutation chosen from D26K, R30Q, W67F, G103K, D117A, S118R, R143I, R143L, R143V and E146P, the numbering being the one of SEQ ID NO:1. Glycine in position 103 corresponds to a deamidation site (NG); mutation in position 103 avoids post-translational modifications and therefore increases the consistency of the protein quality profile. The other mutations in position 26, 30, 67, 117, 118, 143 or 146 allow the increase of manufacturability and / or an increase in PD-L1 and / or PD-L2 binding (e.g. increased binding affinity to PD-L1 and / or PD-L2, increased stability, increased manufacturability). Preferably, the sPD-1 polypeptide of the invention comprises, preferably consists of, the sequence SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9.

[0065] Preferably, the sPD-1 polypeptide of the invention comprises at least 70% identity with SEQ ID NO:2, wherein mutation a) is N49T, mutation b) is N58S, N58E or N58Q, mutation c) is chosen from N74G and N74Q; and mutation d) is N116E, N116Q or N116S, the numbering being the one of SEQ ID NO:1.

[0066] The invention further relates to a sPD-1 polypeptide which is a mutant of SEQ ID NO: 12, comprising at least 70% identity (preferably at least 71 % identity, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, and preferably at least 79%) with SEQ ID NO: 12 and that binds PD-L1 and / or PD-L2, which comprises at least one mutation in position 26, 30, 32, 37, 38, 45, 57, 61 , 62, 64, 67, 69, 70, 77, 86, 87, 88, 90, 93, 96, 103, 108, 109, 110, 115, 122, 124, 129, 130, 132, 135, 139, 143, 145 or 146, the numbering being the one of SEQ ID NO:1. Preferably, said sPD-1 polypeptide comprises at least one additional mutation chosen from D26K, R30Q, W32I, W32L, F37I, S38G, T45E, S57D, E61G, S62N, V64F, W67F, R69K, M70I, D77E, D77A, R86K, R86A, S87I, S87T, S87V, Q88G, Q88F, G90P, C93S, R96Q, G103K, M108F, M108P, S109T, V110I, R115I, R115V, R115E, L122F, G124I, G124S, G124A, G124V, A129Y, A129W, P130G, A132L, A132I, A132M, A132F, K135L, R139F, R139V, R139M, R143I, R143L, T145L, T145V, T145E and E146P, the numbering being the one of SEQ ID NO:1. Glycine in position 103 corresponds to a deamidation site (NG); mutation in position 103 increases protein quality profile and therefore its manufacturability. The other mutations allow the increase of protein quality consistency and / or an increase in PD-L1 and / or PD-L2 binding (e.g. increased binding affinity to PD-L1 and / or PD-L2, increased stability, increased protein quality consistency).

[0067] Preferably, the sPD-1 polypeptide of the invention comprises, preferably consists of, a sequence chosen from SEQ ID NO: 14 to 32.

[0068] Preferably, the sPD-1 polypeptide of the invention comprises, preferably consists of, a sequence chosen from SEQ ID NO: 14 to 16.

[0069] Preferably, the sPD-1 polypeptide of the invention comprises, preferably consists of, a sequence chosen from SEQ ID NO: 17 to 23. Preferably, the sPD-1 polypeptide of the invention comprises, preferably consists of, a sequence chosen from SEQ ID NO:24 to 32.

[0070] Preferably, the sPD-1 polypeptide of the invention comprises at least 70% identity with SEQ ID NO: 12, wherein mutation a) is N49T, mutation b) is N58S, N58E or N58Q, mutation c) is N74Q; and mutation d) is N116E, N116Q or N116S, the numbering being the one of SEQ ID NO:1.

[0071] The invention also relates to an antigen-binding fragment comprising a soluble PD-1 polypeptide according to the invention. It further relates to a protein construct comprising a soluble PD-1 polypeptide according to the invention that is fused, directly or via a linker, to a protein fragment.

[0072] The term « linker » refers to any suitable peptide linker, such as a short peptide fragment. For example, a linker in accordance with the invention may comprise a short peptide, preferably from 1 to 30 amino acids, preferably from 2 to 25 amino acids. It typically comprises small amino acid residues or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.). One example of such a linker is Gly-Gly-Gly-Gly-Ser (G4S) (SEQ ID NO:33). Other examples may include permutations of these amino acids in the sequence, such as GGGSG (SEQ ID NO:34), GGSGG (SEQ ID NO:35), GSGGG (SEQ ID NO:36) or SGGGG (SEQ ID NO:37). Further examples may include peptides containing amino acid residues other than G or S, such as GGTGS (SEQ ID NO:38), GTSPGG (SEQ ID NO:39) or GNGGGS (SEQ ID NQ:40). One skilled in the art would appreciate that many commonly used peptide linkers may be used in embodiments of the invention. In accordance with some embodiments of the invention, the linkers may comprise repeat units to increase the linker length. For example, some linkers may comprise two G4S-repeated linkers, three G4S-repeated linkers, or four G4S-repeated linkers. Furthermore, some "repeat-like" linkers may comprise a mix of different peptide sequences - such as G4S - GGSGG - G4S - SGGGG (SEQ ID NO:41).

[0073] Preferably, the linker is (GGGGS)P(SEQ ID NO:33), with p being an integer from 1 to 5, preferably from 2 to 4, preferably 3.

[0074] The term « protein fragment » refers to any suitable amino acid sequence of at least 40 amino acids, preferably at least 50 amino acids. Preferably, the protein fragment is chosen from Fc fragments, Fc-VHH and single-domain antibodies (sdAb).

[0075] Fc fragments (or Fc regions) are constant regions of antibodies that mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (C1q) of the classical complement system. The term "Fc" or "Fc region" designates the constant region of an antibody with the exclusion of the first immunoglobulin constant region domain (CH1). Therefore, Fc refers to the two last domains (CH2 and CH3) of the IgG constant region, and to the N-terminal flexible hinge of these domains. The Fc may be an lgG1 Fc, an lgG2 Fc, an lgG3 Fc or an lgG4 Fc. The binding of the Fc region to a Fc receptor leads to effector functions. Said effector functions include antibody-dependent cell-mediated cytotoxicity (or ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). Preferably, the Fc region is able to bind at least one of the receptors (FcRs) of the Fc region, selected from among the C1q complement, FcgRIIIa (CD16a), FcgRIla (CD32a) and FcgRIlb (CD32b). The C1q complement is involved in CDC activity. The FcgRIIIa receptor (CD16a) is involved in ADCC. The FcgRIla receptor (CD32a) is involved in phagocytosis. Finally, the FcgRIlb receptor (CD32b) is involved in inhibition of cell activity. The Fc region can also bind a high-affinity Fc-receptor, named neonatal Fc receptor (FcRn). Indeed, in humans, the FcRn is expressed in placental cells, in intestinal, kidney and bronchial epithelial cells, in endothelial cells and in hematopoetic cells such as small intestinal macrophages, monocytes and monocyte-derived dendritic cells. Bound IgG- FcRn complexes are recycled back to the cell surface and return to circulation in the blood, whereas IgGs that do not bind to FcRn traffic into the lysosomes where they are degraded by proteases. Thus, an increase of FcRn binding leads to an increase of the half-life of the bound molecule.

[0076] The term "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains; synthetic VHH can be construed accordingly. Such VHH are also called "single domain antibody" (sdAb). Preferably, VHH can particularly be llama VHH. When the antibody comprises a VHH region instead of VH, then it is devoid of light chains.

[0077] Conjugate

[0078] The present invention also relates to a soluble PD-1 polypeptide according to the invention, or antigen-binding fragment according to the invention or protein construct according to the invention, which is conjugated with at least one compound chosen from drugs, toxins and radioactive compounds (hereafter “conjugate compound”).

[0079] The sPD-1 polypeptide or antigen-binding fragment or protein construct are as described above. The drug is preferably a chemotherapeutic drug, more preferably selected from an anthracycline, an antitumor antibiotic, an alkylating agent, an antimetabolite, an alkaloid, a topoisomerase inhibitor, an anti-mitotic agent such as a spindle poison, a DNA-intercalating agent, a taxane, a platin-based component, a specific kinase inhibitor, an androgen receptor antagonist, an hormone, a cytokine, an antiangiogenic agent, an antibody, in particular a monoclonal antibody, a modulator of the immunity system, an oncolytic virus and a TLR (Toll-Like Receptor)-3 ligand.

[0080] Anthracyclins include for example doxorubicin, daunorubicin, epirubicin, pirarubicin, idarubicin, zorubicin, aclarubicin, nemorubicin, sabarubicin or valrubicin.

[0081] Antitumor antibiotics include for example Bleomycin, hydroxyurea, Mitomycin C or Mitoxantrone.

[0082] Alkylating agents include for example dacarbazine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, melphalan, mechlorethamine, oxaliplatin, uramustine or temozolomide.

[0083] Examples of antimetabolites are Azathioprine, Capecitabine, Cytarabine, Floxuridine, Fludarabine, Fluorouracil, Gemcitabine, Methotrexate, Fluorouracil (5-Fll) or Pemetrexed.

[0084] Alkaloids include for example vinblastine, or vincristine (Vinorelbine).

[0085] Topoisomerase inhibitors include, for example Irinotecan, Topotecan or Etoposide.

[0086] Spindle poisons are for example selected from Vinblastine, Vincristine and Vinorelbine.

[0087] Taxanes are for example selected from docetaxel, larotaxel, cabazitaxel, paclitaxel (PG-paclitaxel and D HA- paclitaxel), ortataxel, tesetaxel, and taxoprexin.

[0088] Examples of platin-based components are CDDP and OXP.

[0089] Examples of specific kinase inhibitors are for example BRAF kinase inhibitors such as vemurafenib and dabrafenib, or MEK inhibitors such as trametinib, or Plk1 inhibitors such as volasertib.

[0090] Androgen receptor antagonists are for example bicalutamide or enzalutamide.

[0091] Tamoxifen and anti-aromatase drugs are typically used in the context of hormonotherapy.

[0092] Examples of cytokines usable in the context of an immunotherapy are IL-2 (lnterleukine-2) and IFN (Interferon) alpha (IFNa).

[0093] Antiangiogenic agents are for example VEGF inhibitors such as itraconazole, bevacizumab or ranibizumab.

[0094] Anti-CD20 (pan B-Cell antigen) and anti-Her2 / Neu (Human Epidermal Growth Factor Receptor-2 / NEU) are examples of monoclonal antibodies. Monoclonal antibodies also include anti-immune checkpoint antibodies, such as anti-PD1 , anti-PDL1 , anti-CTLA4, anti- OX40L, anti-PDL2, anti-CD73, anti-CD80, anti-CD86, anti-TIGIT, anti-Galactin-3 or anti- HVEM antibodies.

[0095] Anti-PD1 antibodies include pembrolizumab or nivolumab.

[0096] Immunity system modulators are for example IDO1 , IDO2 or TDO2 inhibitors, A2a antagonists or STING agonists.

[0097] Oncolytic viruses are for exemple Talimogene laherparepvec.

[0098] Radioactive compounds are preferably selected from radioisotopes such as111In,67Ga,68Ga,90Y,89Zr,86Y,177Lu,212Bi,213Bi,64Cu,67Cu,44Sc,44mSc and47Sc, preferably68Ga.

[0099] Toxins are preferably chosen from cyanotoxins, necrotoxins, neurotoxins, myotoxins and cytotoxins.

[0100] Preferably, the conjugate compound of the invention is conjugated with a drug, more preferably the conjugate compound of the invention is an antibody-drug conjugate (ADC).

[0101] Preferably, the conjugate compound of the invention is conjugated with a radioactive compound, and is used for visualizing a tumor, a cell or an organ in a patient.

[0102] The present invention also relates to an in vitro method for detecting cancer in a patient or in a cell, comprising detecting in a patient’s sample or in a cell the expression of PD-L1 and / or of PD-L2 thanks to the conjugate compound of the invention. By “expression of PD-L1 and / or of PD-L2”, it is meant the protein expression.

[0103] In another embodiment, the present invention relates to an in vitro method for monitoring the efficacy of a treatment comprising a PD-1 , PD-L1 and / or PD-L2 inhibitor in a patient suffering from cancer, said in vitro method comprising: a) determining PD-L1 and / or PD-L2 protein expression level(s) in a biological sample of the patient before any treatment and by using the conjugate compound of the invention. Said expression level of PD-L1 and / or PD-L2 is the reference value for PD-L1 and / or PD- L2 respectively ; b) determining PD-L1 and / or PD-L2 protein expression level(s) in a biological sample of the patient after or during the treatment, and using the conjugate compound of the invention ; c) comparing the PD-L1 and / or PD-L2 protein expression level(s) determined in b) with the PD-L1 and / or PD-L2 protein expression level(s) determined in a), wherein a PD-L1 and / or PD-L2 protein expression level(s) determined in b) higher than the PD-L1 and / or PD-L2 protein expression level(s) determined in a) is indicative that the treatment is efficient, and wherein a PD-L1 and / or PD-L2 protein expression level(s) determined in b) lower than the PD-L1 and / or PD-L2 protein expression level(s) determined in a) is indicative that the treatment is not efficient.

[0104] Preferably, step b) is repeated over time. Preferably, each step b) is performed within a time period of at least one month, preferably at least 3 months, preferably at least 6 months.

[0105] The present invention also relates to an in vitro method for detecting the presence and / or the expression of PD-L1 and / or of PD-L2 in a biological sample, thanks to the conjugate compound of the invention.

[0106] In another embodiment, the present invention relates to an in vitro method for determining whether a patient suffering from cancer is susceptible to be a good-reponder to anti-PD-1 , anti-PD-L1 or anti-PD-L2 immunotherapy, said in vitro method comprising: a) determining PD-L1 and / or PD-L2 protein expression level(s) in a biological sample of the patient before any immunotherapy and by using the conjugate compound of the invention; b) comparing the PD-L1 and / or PD-L2 protein expression level(s) determined in a) with reference value(s), wherein a PD-L1 and / or PD-L2 protein expression level(s) determined in a) higher than the reference value(s) is indicative that the patient is susceptible to be a goodresponder to anti-PD-1 , anti-PD-L1 or anti-PD-L2 immunotherapy, and wherein a PD-L1 and / or PD-L2 protein expression level(s) determined in a) lower than the reference value(s) is indicative that the patient is not susceptible to be a good-responder, or is a bad- responder, to anti-PD-1 , anti-PD-L1 or anti-PD-L2 immunotherapy .

[0107] The reference value of step b) may correspond to PD-L1 and / or PD-L2 concentration(s) in a tumor that is sensitive to PD1 .

[0108] In the above methods, the biological sample is preferably a blood sample, a plasma sample, a saliva sample, a urine sample or a biopsy. The biological sample may also be a cell sample from an animal or cell model used in laboratories.

[0109] All the above methods preferably comprise mixing the sample with the conjugate compound of the invention, and then detecting complex formation between the conjugate compound of the invention and PD-L1 and / or PD-L2 present in the sample.

[0110] Nucleic acids, expression vectors and host cells The present invention also relates to a nucleic acid coding for a soluble PD-1 polypeptide according to the invention, or for the antigen-binding fragment according to the invention or for the protein construct according to the invention. It further relates to an expression vector comprising said nucleic acid. It further relates to a host cell comprising said nucleic acid or said expression vector.

[0111] Expression vectors typically include a sPD-1 polypeptide according to the invention operably linked, that is, placed in a functional relationship, with control or regulatory sequences, selectable markers, any fusion partners, and / or additional elements. The sPD- 1 polypeptide of the present invention may be produced by culturing a host cell transformed with nucleic acid, preferably an expression vector, containing nucleic acid encoding the sPD-1 polypeptide, under the appropriate conditions to induce or cause its expression. A wide variety of appropriate host cell lines may be used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, a variety of mammalian cell lines that may find use are described in the ATCC cell line catalog, available from the American Type Culture Collection. Host cells may be, but not limited to, YB2 / 0 (YB2 / 3HL.P2.GII.IGAg.2O cell, deposit to the American Type Culture Collection, ATCC n°CRL-1662), SP2 / 0, YE2 / 0, Namalwa, CHO cell lines, particularly CHO-K-1 , CHO-LeclO, CHO-Lecl, CHO-Lecl3, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293- HEK, BHK, KGH6, NSO, SP2 / 0-Ag 14, C127, JC, LA7, ZR-45-30, hTERT, NM2C5, UACC- 812 and the like. The methods of introducing exogenous nucleic acid into host cells are well known in the art, and will vary with the host cell used.

[0112] Therapeutic uses

[0113] Finally, the invention also relates to the use of a soluble PD-1 polypeptide according to the invention, or antigen-binding fragment according to the invention or protein construct according to the invention, or nucleic acid according to the invention, or expression vector according to the invention or host cell according to the invention, as a medicament.

[0114] It further relates to the use of a soluble PD-1 polypeptide according to the invention, or antigen-binding fragment according to the invention or protein construct according to the invention, or nucleic acid according to the invention, or expression vector according to the invention or host cell according to the invention, for preventing and / or treating cancer in a subject in need thereof.

[0115] The subject is preferably a mammal. Preferably, the subject is a human, a horse, a mouse, a rat, a dog, a cat or a goat, more preferably a human. Said use can thus be performed in the veterinary field.

[0116] Preferably the cancer is a solid or a liquid tumor.

[0117] It is also described a method for treating a cancer in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a soluble PD-1 polypeptide, or an antigen-binding fragment or protein construct comprising said polypeptide according the invention.

[0118] Cancer refers to tumors. The tumors to be treated include primary tumors and metastatic tumors, as well as refractory tumors. Refractory tumors include tumors that fail to respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone or combinations thereof. Refractory tumors also encompass tumors that appear to be inhibited by treatment with such agents, but recur up to five years, sometimes up to ten years or longer after treatment is discontinued.

[0119] Examples of cancers that may be treated by the soluble PD-1 polypeptide, or an antigen-binding fragment or protein construct comprising said polypeptide of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangio sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non- Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0120] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0121] By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen.

[0122] The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.

[0123] The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0124] By a "therapeutically effective amount" is meant a sufficient amount of the soluble PD- 1 polypeptide, or an antigen-binding fragment or protein construct comprising said polypeptide of the invention to treat the disease (e.g. cancer) at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the soluble PD-1 polypeptide, or an antigen-binding fragment or protein construct comprising said polypeptide of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the soluble PD-1 polypeptide, or an antigen-binding fragment or protein construct comprising said polypeptide; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1 ,000 mg per adult per day. Typically, the compositions contain 0.01 , 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day. The soluble PD-1 polypeptide, or an antigen-binding fragment or protein construct comprising said polypeptide of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0125] "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The soluble PD-1 polypeptide, or an antigen-binding fragment or protein construct comprising said polypeptide can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0126] Preparation methods

[0127] The present invention also relates to process for producing a soluble PD-1 polypeptide according to the invention, comprising:

[0128] (i) a step of expressing the nucleic acid or expression vector in the host cell, in order to express the soluble PD-1 mutant; and

[0129] (ii) optionally harvesting the soluble PD-1 mutant produced in step (i).

[0130] For example, a method for producing a sPD-1 polypeptide of the invention may comprise the following steps : preparing a cDNA expression vector encoding the sPD-1 polypeptide ; transfecting the cDNA obtained in the previous step in host cells, such as CHO cells; incubating said transfected host cells ; and then collecting the obtained soluble PD-1 polypeptide, for example by using affinity chromatography and / or size-exclusion chromatography and / or hydrophobic interaction chromatography and / or ion exchange chromatography. The sPD-1 polypeptide of the invention may be prepared as described in the examples.

[0131] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0132] Sequences of the application

[0133] Brief description of figures

[0134] The figures of the present application are as follows : Figure 1. Production in CHO cells: SEC HPLC chromatogram of bispecific constructs (anti-CD28 / sPD-1), including high affinity sPD-1 protein mutated on one or more N- glycosylation sites and free cysteine removed, compared to high affinity sPD-1 protein with 4 N-glycosylation sites conserved

[0135] Figure 1 depicts the purification by size exclusion chromatography (SEC-HPLC). Anti- CD28 / sPD-1 bispecific constructs containing sPD-1 variants (as described in Table 1) or sPD-1 WT were produced in Chinese hamster ovary (CHO) cells, and purified through protein A affinity chromatography and preparative SEC. Capture eluates from previous chromatography steps were loaded on a Waters BioSuite 250 4um UHR SEC 4.6x300mm column and eluted using 0.2 M Potassium phosphate, 0.25 M KCI for quality control analysis. The percentage of monomer content is indicated on each graph.

[0136] Figure 2. Binding of anti-CD28 / sPD-1 constructs, including high affinity sPD-1 protein with 4 N-glycosylation sites conserved, or high affinity sPD-1 protein mutated on one or more N-glycosylation sites and free cysteine, to human PD-L1 and PD-L2 by ELISA. Figure 2 shows the binding by ELISA of anti-CD28 / sPD-1 bispecific constructs containing sPD-1 variants (as described in Table 1) or sPD-1 WT to immobilized human PD-L1 protein, His Tag (Acrobiosystems, Cat. No. PD1-H5229, coating concentration 1 pg / ml) (Figure 2A) and human PD-L2 protein, His Tag (Acrobiosystems, Cat. No. PD2-H5220, coating concentration 2 pg / ml) (Figure 2B). Binding was detected though a secondary anti-human IgG-HRP antibody. Binding values are reported as absorbance at 450 nm with blank substracted. Experiment was conducted twice independently.

[0137] Figure 3:

[0138] 3A: Expression of sPD-1 WT and high affinity sPD-1 in yeast model and binding to human PD-L1 or PD-L2 :

[0139] Figure 3A shows dot plots of flow cytometry data. Yeast cells expressing sPD-1 WT (SEQ ID NO :2) or high affinity (HA) sPD-1 (SEQ ID NO :4) were incubated with 100 nM or 20 nM PD-L1 or PD-L2 human recombinant proteins (human PD-L1 protein, Fc Tag, Peprotech, Cat. No. 310-35; human PD-L2 protein, Fc tag, Peprotech, Cat. No. 310-38). PD-1 expression is plotted on the y-axis. PD-L1 (top) or PD-L2 (bottom) binding is plotted on the x-axis. Bottom left cells correspond to non-induced cells. Top left cells correspond to cells expressing PD-1 but not binding to PD-L1 or PD-L2. Top right cells correspond to cells expressing PD-1 and binding to PD-L1 or PD-L2.

[0140] 3B: Expression of sPD-1 variants mutated on one or more N-glycosylation site (N49, N58, N74, N116) in yeast model and binding to human PD-L1 or PD-L2 :

[0141] Figure 3B depicts dot plots of flow cytometry data showing the impact of abrogating each of the 4 N-glycosylation sites in the human sPD-1 sequence. Yeast cells expressing high affinity sPD-1 variants mutated on one or more N-glycosylation sites (positions 49, 58, 74, 116) (clones 7 to 21) were incubated with 100 nM PD-L1 or PD-L2 human recombinant proteins (human PD-L1 protein, Fc Tag, Peprotech, Cat. No. 310-35; human PD-L2 protein, Fc tag, Peprotech, Cat. No. 310-38). PD-1 expression is plotted on the y-axis. PD-L1 (top) or PD-L2 (bottom) binding is plotted on the x-axis. Results are depicted in dark grey and were compared with high affinity sPD-1 (G124S, K131Y, A132I; SEQ ID NO :4) protein with the 4 N-glycosylation sites conserved (light grey).

[0142] Figure 4 : PD-1 amino acid substitutions at position 49 (N-glycosylation site) and effect on binding to hPD-L1 and hPD-L2

[0143] Figure 4 shows the impact of each mono-substitution of residue N49 by each of the other 19 amino acids on binding to PD-L1 and PD-L2. DMS (Deep Mutationnal Scanning) is a mutagenesis method which aims to perform all possible mono-substitutions on all selected residues within a given protein sequence. The DMS library is obtained in the form of DNA coding for the variable parts of the protein under study. In this library, each DNA strand has a codon mutated from the parental sequence. This DMS library is then integrated into an expression plasmid specifically designed to express proteins on the yeast surface. Yeast are then transformed and induced to allow the expression of the single-mutated proteins on their surface. This new library (called display library) is screened by flow cytometry using fluorescent reporters to reveal the expression of the protein as well as the binding of the protein with its target (fluorescent target). Here, yeast cells expressing the display library were washed twice with PBSF buffer (PBS. BSA 0.1 %), incubated 2 hours with PD-L1 or PD-L2 human recombinant proteins, then with fluorescent reporters for 15 minutes on ice (anti-HA DyLight 488 for PD-1 expression, and anti-human Fc PE for PD-L1 or PD-L2 detection). Cells were washed once and resuspended in PBSF buffer, then sorted by flow cytometry with 2 consecutive cell sortings (first, 100nM hPD-L1-Fc or hPD-L2-Fc ; second, 10nM hPD-L1-Fc or hPD-L2-Fc). The recombinant proteins used were : human PD-L1 protein, Fc Tag, Peprotech, Cat. No. 310-35; human PD-L2 protein, Fc tag, Peprotech, Cat. No. 310-38.

[0144] The plasmids contained in these yeast populations were extracted and sequenced by high- throughput sequencing. The analysis of the sequencing data allowed to identify the impact of each mutation on the functionality of the protein and to produce positive and negative functionality maps of the studied protein. The values in the table indicate Iog2 enrichment of the single mutant after sorting compared to before sorting divided by enrichment of the reference sequence. A positive value indicates mutations that may improve affinity and / or folding. A negative value indicates mutations that may be deleterious to affinity and / or folding. « D » indicates that the mutant is present in the unsorted library but absent in the sorted library, thus a very negative impact of the mutation.

[0145] Figure 5 : Expression of sPD-1 variants mutated at each N-glycosylation site (N49, N58, N74, N116), with N49Q or N49T, in yeast model and binding to human PD-L1 or PD-L2

[0146] Figure 5 depicts dot plots of flow cytometry data showing the impact of abrogating each of the 4 N-glycosylation sites in the human sPD-1 sequence. Yeast cells expressing high affinity sPD-1 variants mutated on one or more N-glycosylation sites (positions 49, 58, 74, 116) (clones 21 to 24) were incubated with 1 pM PD-L1 or PD-L2 human recombinant proteins (human PD-L1 protein, Fc Tag, Peprotech, Cat. No. 310-35; human PD-L2 protein, Fc tag, Peprotech, Cat. No. 310-38). PD-1 expression is plotted on the y-axis. PD-L1 (top) or PD-L2 (bottom) binding is plotted on the x-axis. Results are depicted in dark grey and were compared with high affinity sPD-1 (G124S, K131Y, A132I ; SEQ ID NO :4) protein with the 4 N-glycosylation sites conserved (light grey) (Figure 5A). The raw data (MFI) are indicated in Figure 5B.

[0147] Figure 6:

[0148] Figure 6A: Combinatorial library screening

[0149] Figure 6A depicts the strategy for the combinatorial library screening. The mutations identified as being interesting for the optimization objectives (affinity optimization, manufacturability... ) were combined in one combinatorial library. This library was generated and transformed into YSD. Here, yeast cells were washed twice with PBSF buffer (PBS. BSA 0.1 %), incubated 2 hours with PD-L1 or PD-L2 human recombinant proteins, then with fluorescent reporters for 15 minutes on ice (anti-V5 antibody FITC for PD-1 expression, and anti-human Fc PE or Streptavidin-PE for PD-L1 or PD-L2 detection). Cells were washed once and resuspended in PBSF buffer, then sorted by flow cytometry. Selections based on binding were performed through six consecutive cell sortings : binding on 200 nM human PD-L1-Fc, 200 nM human PD-L1 monovalent, 20 nM human PD-L2 monovalent, 2 nM human PD-L2 monovalent, 2 nM human PD-L1 monovalent and on this last gate, 2 nM human PD-L1 monovalent and 2 nM human PD-L2 monovalent. The recombinant proteins used were : human PD-L1 protein, Fc Tag, Peprotech, Cat. No. 310-35; human PD-L2 protein, Fc tag, Peprotech, Cat. No. 310-38; human biotinylated monovalent PD-L1 protein, Acrobiosystems, Cat. No. BV2088-78GR1-ZH ; human biotinylated monovalent PD-L2 protein, Acrobiosystems, Cat. No. BV1055-96HF1-17G.

[0150] Figure 6B: Sequences of sPD-1 variants and affinity to hPD-L1 and hPD-L2 by yeast surface display

[0151] After the consecutive cell sortings, the plasmids contained in the yeast populations were extracted and sequenced by high-throughput sequencing. The table reflects the sequences of the engineered high affinity sPD-1 variants that were produced. Each numbered column represents the amino acid position for each shown residue relative to the native sPD-1 fragment (fragment of sPD-1 of SEQ ID NO : 12), using the numbering of SEQ ID NO:1. For each PD-1 variant recovered, divergence from the wild-type amino acid residue is indicated in bold with the single-letter code for the resulting mutation for each variant. The N- glycosylation site positions are indicated in bold and underlined. The measured apparent KD affinity for human PD-L1 and PD-L2 is indicated (when measured) on the last column. Figure 7: Apparent KD evaluation of sPD-1 variants to hPD-L1 and hPD-L2 by yeast surface display

[0152] Figure 7 depicts graphs of binding analyzed by flow cytometry for each engineered high affinity sPD-1 variant expressed on yeast cells to human PD-L1 and PD-L2. Yeast displaying each sPD-1 variant were incubated with increasing doses of human PD-L1 or PD-L2 monovalent proteins, and cells were analyzed by flow cytometry (human biotinylated monovalent PD-L1 protein, Acrobiosystems, Cat. No. BV2088-78GR1-ZH ; human biotinylated monovalent PD-L2 protein, Acrobiosystems, Cat. No. BV1055-96HF1-17G). The apparent KD values and confidence intervals are indicated in the table.

[0153] Figure 8: HPLC-SEC chromatograms - production of Fc-sPD-1 variants in CHO cells Figure 8 is a graph depicting the purification by size exclusion chromatography (SEC- HPLC). Fc-sPD-1 variants, with sPD-1 variants as described in Figure 6B, were produced in Chinese hamster ovary (CHO) cells, and purified through protein A affinity chromatography and preparative SEC. Capture eluates from previous chromatography steps were loaded on a Waters BioSuite 2504um UHR SEC 4.6x300mm column and eluted using 0.2 M Potassium phosphate, 0.25 M KCI for quality control analysis. The percentage of monomer content is indicated on each graph.

[0154] The human wild-type (WT) full-length PD-1 is SEQ ID NO :1.

[0155] The human WT soluble PD-1 (sPD-1) is SEQ ID NO :2. This sequence contains four N- glycosylation sites, defined as N-X-S / T, with N at positions 49, 58, 74 and 116, wherein the amino acid numbering is the one of human WT full-length PD-1 protein (SEQ ID NO :1). Due to the low affinity of natural human sPD-1 for its ligands, G124S, K131Y and A132I mutations were used, known to increase the affinity of PD-1 to its ligands as described before (Li, Y. et al. High-affinity PD-1 molecules deliver improved interaction with PD-L1 and PD-L2. Cancer Science. 109(8):2435-2445. doi: 10.1111 / cas.13666 (2018)), to generate high affinity (HA) sPD-1 protein (HA sPD-1), sPDI-001 , of SEQ ID NO :4.

[0156] EXAMPLE 1 : PRODUCTION OF GLYCAN-CONTROLLED sPD-1 VARIANTS IN MAMMALIAN (CHO) CELLS AND BINDING TO HUMAN PD-L1 AND PD-L2 In order to evaluate the effect of abrogating the N-glycosylation sites of sPD-1 on its production, manufacturability and activity, anti-CD28 / sPD-1 bispecific constructs containing glycan-controlled or non-glycosylated sPD-1 variants, or sPD-1 WT, were designed. They comprise SEQ ID NO:10 as VL, and SEQ ID NO:11 as VH.

[0157] For each sPD-1 variant tested, the amino acid residues at positions 124-131-132 (for which mutations have been previously described to enhance the affinity of PD-1 to its ligands, (Li, Y. et al. High-affinity PD-1 molecules deliver improved interaction with PD-L1 and PD-L2. Cancer Science. 109(8):2435-2445. doi: 10.1111 / cas.13666 (2018)), positions 49-58-74- 116 (N-glycosylation sites) and position 93 (free cysteine), were summarized in Table 1 below. The amino acid residues in sPD-1 WT protein are also indicated.

[0158] Table 1 : Amino acid residues at positions 124, 131, 132 (for which mutations have been described as enhancing the affinity of PD- 1 to its ligands in Li Y et al. Cancer Science. 2018), positions 49, 58, 74, 116 (N-glycosylation sites) and position 93 (free cysteine) in each of sPD-1 variant sPD-1-001 variant corresponds to high affinity (HA) sPD-1 protein, with mutations known to increase PD-1 affinity to its ligands (G124S, K131Y and A132I), with N-glycosylation sites conserved and free cysteine conserved. sPD-1 -003 variant includes the affinity mutations, along with 3 N-glycosylation sites abrogated by replacing asparagine (N) with glutamine (Q), which is structurally the closest amino acid, (N49-Q58-Q74-Q116) and free cysteine mutated to serine. sPD-1-004 variant includes the affinity mutations, along with 4 N-glycosylation sites abrogated by replacing asparagine (N) with threonine (T) or glutamine (Q) (T49-Q58-Q74- Q116) and free cysteine mutated to serine. It is a mutant of the invention. sPD-1-005 variant includes the affinity mutations, along with 4 N-glycosylation sites abrogated by replacing asparagine (N) with threonine (T), glutamic acid (E) or glycine (G) (T49-E58-G74-E116) and free cysteine mutated to serine. It is a mutant of the invention.

[0159] Anti-CD28 / sPD-1 bispecific constructs were produced in mammalian cells (Chinese hamster ovary (CHO) cells), and purified through protein A affinity chromatography and preparative SEC. Capture eluates from previous chromatography steps were loaded on a Waters BioSuite 2504um UHR SEC 4.6x300mm column and eluted using 0.2 M Potassium phosphate, 0.25 M KCI for quality control analysis. Figure 1 shows the SEC HPLC chromatogram for each construct. The percentage of monomer content is indicated on each graph. Results show that the N-glycosylation sites could be abrogated (sPD-1-003: N49- Q58-Q74-Q116 ; sPD-1-004 : T49-Q58-Q74-Q116 ; sPD-1-005: T49-E58-G74-E116) and the free cysteine mutated to serine (C93S), while maintaining an excellent high postproduction purity profile (> 90% monomer content).

[0160] The binding of anti-CD28 / sPD-1 bispecific constructs containing glycan-controlled or nonglycosylated sPD-1 variants or sPD-1 WT was then assessed by enzyme-linked immunosorbent assay (ELISA). As seen in Figure 2, anti-CD28 / sPD-1 bispecific constructs containing sPD-1 variants with affinity mutations (G124S, K131Y and A132I) , i.e. sPD-1- 001 , sPD-1-003, sPD-1-004 and sPD-1-005, showed increased binding to human PD-L1 and PD-L2 compared to bispecific constructs containing sPD-1 WT protein. Furthermore, anti-CD28 / sPD-1 bispecific constructs with the N-glycosylations sites abrogated (sPD-1- 003: N49-Q58-Q74-Q116 ; sPD-1-004 : T49-Q58-Q74-Q116 ; sPD-1-005: T49-E58-G74- E116) and the free cysteine mutated to serine (C93S), showed similar affinity to PD-L1 compared to HA sPD-1 protein (sPD-1-001), in which the N-glycosylations sites and the free cysteine are conserved (Figure 2A), and a slightly increased affinity to PD-L2 (Figure 2B). The affinity was similar whether sPD-1 variant contains one conserved and three abrogated N-glycosylation sites (sPD-1-003), or four abrogated N-glycosylation sites (sPD- 1-004 and sPD-1-005).

[0161] Altogether, these results confirmed that the 4 N-glycosylation sites of sPD-1 can be abrogated while maintaining a good post-production purity profile in a mammalian system. These results were unexpected as previous findings showed that glycosylation was be essential for protein folding, structure, function, and N58 in particular was essential for mediating PD-1 interaction with PD-L1.

[0162] EXAMPLE 2 : PRODUCTION OF GLYCAN-CONTROLLED sPD-1 VARIANTS IN YEAST SURFACE DISPLAY MODEL AND BINDING TO HUMAN PD-L1 AND PD-L2

[0163] In order to evaluate in more detail the effect of abrogating the N-glycosylation sites, sPD-1 constructs were expressed by yeast surface display (YSD), which offers the advantages of eukaryotic systems such as post-translational modifications, correct folding and glycosylation of proteins.

[0164] Cells were washed twice with PBSF buffer (PBS BSA 0.1 %), and incubated 2h with 100 nM of PD-L1 or PD-L2 (or 20 nM for clones supposed to be weak binders). Yeast cells were then incubated with fluorescent reporters for 15 min on ice : anti-HA APC (for PD1 expression) and anti-human Fc PE (for PD-L1 or PD-L2 detection). Cells were washed once and resuspended in PBSF buffer. Yeasts cells were then analyzed on a BD FACSAria III cytometer. Results are shown in Figure 3. PD-1 expression is plotted on the y-axis. PD-L1 (top) or PD-L2 (bottom) binding is plotted on the x-axis.

[0165] As expected, no binding was observed with sPD-1 WT, given the low affinity for its ligands. As described before, HA sPD-1 protein showed increased binding to PD-L1 and PD-L2 (Figure 3A). This latter was then used for the next experiments as the reference clone.

[0166] Then, glycan-controlled sPD-1 variants, with one or more N-glycosylation sites (N49, N58, N74, N166) abrogated by replacing asparagine (N) with glutamine (Q) were compared to HA sPD-1 protein with the 4 N-glycosylation sites conserved. These clones all contain G124S, K131Y, A132I mutations so that the binding could be detectable. These clones (7 to 21) show the impact of each of the glycosylation sites (Figure 3B).

[0167] Clone 7 (N49-Q58-Q74-Q116; SEQ ID NO: 13), clone 8 (Q49-N58-Q74-Q116), clone 9 (Q49-Q58-N74-Q116) and clone 10 (Q49-Q58-Q74-N116) have 3 N-glycosylation sites abrogated and 1 site conserved.

[0168] Clone 11 (N49-N58-Q74-Q116), clone 12 (N49-Q58-N74-Q116), clone 13 (N49-Q58-Q74- N116), clone 14 (Q49-N58-N74-Q116), clone 15 (Q49-N58-Q74-N116) and clone 16 (Q49- Q58-N74-N116) have 2 N-glycosylation sites abrogated and 2 sites conserved.

[0169] Clone 17 (Q49-N58-N74-N116), clone 18 (N49-Q58-N74-N116), clone 19 (N49-N58-Q74- N116) and clone 20 (N49-N58-N74-Q116) have 1 N-glycosylation site abrogated and 3 sites conserved.

[0170] Clone 21 (Q49-Q58-Q74-Q116) has 4 N-glycosylation sites abrogated. As described for Figure 3A, each sPD-1 clone was expressed by YSD and PD-L1 / 2 binding was assessed by flow cytometry. Results are shown in Figure 3B (in dark grey for the clones) and were compared with those of HA sPD-1 protein with the 4 N-glycosylation sites conserved (in light grey).

[0171] As seen in Figure 3B, the N-glycosylation site in the position 49 appeared to be the most important for sPD-1 folding and binding to PD-L1 and PD-L2. If this first N-glycosylation site N49 was mutated to Q49, as in clones 8, 9 and 10, 14, 15, 16, 17, then sPD-1 folding and binding to PD-L1 and PD-L2 were reduced compared to clones 7, 11 , 12, 13, 18, 19, 20 in which the N49 site was conserved.

[0172] Clones 18, 19, and 20 show that substitution of N58, N74 and N116 to glutamine, respectively, did not affect or only slightly affect binding to PD-L1 and PD-L2.

[0173] Finally, results show that clone 21 , in which the 4 N-glycosylation sites were abrogated, displays strongly reduced, though still detectable, binding to PD-L1 and PD-L2.

[0174] These results show that abrogation of the N-glycosylation sites, by replacing asparagine with glutamine, in PD-1 sequence, with N at position 58, 74 et 116, is possible with minimal effect on binding to its ligands. While abrogation of N49 is critical and strongly reduces PD- 1 binding to PD-L1 and PD-L2.

[0175] EXAMPLE 3: EFFECT OF EACH MONO-SUBSTITUTION AT POSITION N49 OF sPD-1 ON FOLDING AND BINDING TO HUMAN PD-L1 AND PD-L2

[0176] The impact of each mono-substitution at position N49 of PD-1 sequence was assessed through deep mutation scanning (DMS). This DMS library was then integrated into an expression plasmid specifically designed to express proteins on the yeast surface. Yeast are then transformed and induced to allow the expression of the single-mutated proteins on their surface. The display library was incubated 2 hours with PD-L1 or PD-L2 to reveal the binding of the protein to its targets, then with fluorescent reporters, then sorted by flow cytometry with 2 consecutive cell sortings (first, 100nM hPD-L1-Fc or hPD-L2-Fc ; second, 10nM hPD-L1-Fc or hPD-L2-Fc). Figure 4 indicates mutations that may improve affinity and / or folding (positive value), mutations that may be deleterious to affinity and / or folding (negative value). « D » indicates that the mutant is present in the unsorted library but absent in the sorted library, thus a very negative impact of the mutation. The results show that only Asparagine (N) substitution to a Threonine (T) at position 49 was able to improve PD-1 affinity to PD-L1 (Iog2 enrichment: 1.9) and PD-L2 (0.7). All the other mono-substitutions had a negative impact on PD-L1 and PD-L2 binding. Thus, sPD-1 variants, with asparagine (N) at residue 49 replaced with glutamine (Q) were compared to variants with asparagine (N) at residue 49 replaced with threonine (T). Each sPD-1 clone was expressed by YSD and PD-L1 / 2 binding was assessed by flow cytometry. Results are shown in Figure 5A (in dark grey for the clones) and were compared with those of HA sPD-1 protein with the 4 N-glycosylation sites conserved (in light grey). The raw data (MFI) are indicated in Figure 5B.

[0177] Clone 21 (Q49-Q58-Q74-Q116) has 4 N-glycosylation sites abrogated.

[0178] Clone 22 (N49-N58-N74-N116) has 4 N-glycosylation sites conserved.

[0179] Clone 23 (T49-Q58-Q74-Q116) has 4 N-glycosylation sites abrogated, with threonine at position 49.

[0180] Clone 24 (T49-N58-N74-N116) has 1 N-glycosylation site abrogated, with threonine at position 49, and 3 sites conserved.

[0181] As seen in Figure 5A and 5B, clone 21 , in which the 4 N-glycosylation sites are abrogated (N to Q) shows strongly reduced binding to human PD-L1 and PD-L2. However, the simple substitution of N49 by T, as in clone 23, allows to recover the binding of clone 22, with the 4 N-glycosylation sites conserved, or even increases it slightly.

[0182] This is confirmed by clone 24, in which only the first N-glycosylation site was abrogated (N to Q), that shows increased binding to hPD-L1 compared to clone 22, in which the 4 N- glycosylation sites are conserved.

[0183] Altogether, these results show that N49 substitution to T in sPD-1 sequence allows to remove all the 4 N-glycosylation sites, while maintaining binding to hPD-L1 and hPD-L2.

[0184] EXAMPLE 4: GENERATION OF HIGH AFFINITY NON-GLYCOSYLATED sPD-1 VARIANTS IN YEAST SURFACE DISPLAY MODEL

[0185] In order to generate sPD-1 variants with increased properties, the mutations identified as being interesting for the optimization objectives (affinity optimization for PD-L1 and PD-L2, manufacturability...) were combined in one combinatorial library, expressed by yeast surface display.

[0186] To screen for PD-1 polypeptides having even greater affinity for PD-L1 and PD-L2, six consecutive cell sortings were performed, as shown in Figure 6A, first on 200 nM divalent recombinant PD-L1 protein, then on monovalent proteins with decreasing concentrations to select the best binders (200 nM monovalent PD-L1 , 20 nM monovalent PD-L2, 2 nM monovalent PD-L2, 2 nM monovalent PD-L1). After the consecutive cell sortings, the plasmids contained in the yeast populations were extracted and sequenced by high- throughput sequencing. Figure 6B reflects the sequences of the engineered high affinity sPD-1 variants that were produced : clones 147_1 , 147_2, 147_3, 147_4, 147_5, 147_6, 147_7, clone A and clone A1 (respectively of SEQ ID NO:17 to 23, 14 and 15). Each numbered column represents the amino acid position for each shown residue relative to the native sPD-1 fragment (fragment of sPD-1 of SEQ ID NO : 12), using the numbering of SEQ I D NO: 1. First, all these clones contain the substitution N49T at the first N-glycosylation site, and the 3 other N-glycosylation sites (N58, N74, N116) were also abrogated. In addition, selected after the consecutive cell sortings, these non-glycosylated clones contain additional mutations identified to increase PD-1 binding to PD-L1 and PD-L2. For each sPD- 1 variant recovered, divergence from the wild-type amino acid residue is indicated in bold with the single-letter code for the resulting mutation for each variant. E.g. clone 147_1 (SEQ ID NO: 17) contains a R30Q substitution: Arginine (R) at position 30 was mutated to Glutamine (Q). The measured apparent KD by yeast surface display for human PD-L1 and PD-L2 is indicated (when measured) on the last column.

[0187] EXAMPLE 5: BINDING OF HIGH AFFINITY NON-GLYCOSYLATED sPD-1 VARIANTS TO HUMAN PD-L1 AND PD-L2 IN YEAST SURFACE DISPLAY MODEL

[0188] The high-affinity non-glycosylated sPD-1 variants were then expressed on the surface of yeast cells and the binding of human monovalent PD-L1 and PD-L2 was evaluated by flow cytometry to determine the apparent KD of each clone (Figure 7). The KD varies from 0.39 nM to 121 nM (binding to PD-L1) and 0.28 nM to 5.7 nM (binding to PD-L2).

[0189] EXAMPLE 6: PRODUCTION OF HIGH AFFINITY NON-GLYCOSYLATED Fc-sPD-1 VARIANTS IN MAMMALIAN (CHO) CELLS

[0190] By displaying an optimized library on the surface of yeast cells and analyzing ligand binding propensity of the variants using deep mutational scanning and high-throughput sequencing, several mutations that enhance binding to PD-L1 and PD-L2 were identified.

[0191] These high-affinity non-glycosylated sPD-1 variants were then produced in mammalian cells (Chinese Hamster Ovary (CHO) cells). In addition to clones identified in Figure 6B, additional clones were produced and evaluated, with some variations at residues 124 and 132.

[0192] Clone 147_3_G124 (SEQ ID NO:24) corresponds to clone 147_3 with wild-type residue Glycine (G) at position 124. Clone 147_4_G124 (SEQ ID NO:25) corresponds to clone 147_4 with wild-type residue Glycine (G) at position 124. Clone 147_5_G124 (SEQ ID NO:26) corresponds to clone 147_5 with wild-type residue Glycine (G) at position 124.

[0193] Clone 147_6_L132 (SEQ ID NO:27) corresponds to clone 147_6 with Leucine (L) at position 132. Clone 147_6_M132 (SEQ ID NO:29) corresponds to clone 147_6 with Methionine (M) at position 132. Clone 147_6_F132 (SEQ ID NO:31) corresponds to clone 147_6 with Phenylalanine (F) at position 132.

[0194] Clone 147_7_L132 (SEQ ID NO:28) corresponds to clone 147_7 with Leucine (L) at position 132. Clone 147_7_M132 (SEQ ID NQ:30) corresponds to clone 147_7 with Methionine (M) at position 132. Clone 147_7_F132 (SEQ ID NO:32) corresponds to clone 147_7 with Phenylalanine (F) at position 132.

[0195] Fc-sPD-1 constructs containing each of sPD-1 variants were designed using a human IgG 1 Fc region.

[0196] High affinity non-glycosylated Fc-sPD-1 constructs were produced in CHO cells, and purified through protein A affinity chromatography and preparative SEC. Capture eluates from previous chromatography steps were loaded on a Waters BioSuite 2504um UHR SEC 4.6x300mm column and eluted using 0.2 M Potassium phosphate, 0.25 M KCI for quality control analysis. Figure 8 shows the SEC HPLC chromatogram for each construct. The percentage of monomer content is indicated on each graph. Results show that the four N- glycosylation sites could be abrogated, while maintaining an excellent high post-production purity profile in a mammalian system (> 95% monomer content, except for Fc-sPD-1-147_1 (94.09%) and Fc-sPD-1-Clone A1 (86.44%)).

[0197] EXAMPLE 7: BINDING OF HIGH AFFINITY NON-GLYCOSYLATED Fc-sPD-1 VARIANTS TO HUMAN AND MOUSE PD-L1 AND PD-L2

[0198] The binding of high-affinity non-glycosylated Fc-sPD-1 variants was then assessed by SPR on human and mouse, monovalent and divalent PD-L1 / L2 proteins.

[0199] As seen in Table 2, the mean KD for Fc-sPD-1 variants range from 10'5to 10'9M on human monovalent PD-L1 protein, and 10'6to 10'1° M on human monovalent PD-L2 protein. On human divalent PD-L1 and PD-L2 proteins, the binding was even increased due to avidity.

[0200] Table 2: SPR binding of Fc-sPD-1 variants to monovalent human PD-L1 and human PD-L2

[0201] Table 2 depicts the binding kinetics measured by surface plasmon resonance (SPR) for each Fc-sPD-1 variant to human PD-L1 and PD-L2 monovalent proteins. SPR allows for analysis of association and dissociation rate constants, and modeling of biomolecular interaction kinetics. The table presents statistically averaged kineticparameters : association (Ka) and dissociation (Kd) rate constants, and the equilibrium constant (KD), calculated through the defining relation KD= kd / ka. Fc-sPD- 1 variants were immobilized to a HC30M chip and the binding of recombinant human PD-L1 and PD-L2 was evaluated by Carterra LSA (human PD-L1 protein, His tag, Acrobiosystems, Cat. No. PD1-H5229; human PD-L2 protein, His tag, Acrobiosystems, Cat. No. PD2-H5220).

[0202] N / D not detectable

[0203] As seen in Table 3, the mean KD for Fc-sPD-1 variants range from 10'9to 10'10M on human divalent PD-L1 protein, and 10'9to 10'1° M on human divalent PD-L2 protein.

[0204] Table 3: SPR binding of Fc-sPD-1 variants to divalent human PD-L1 and human PD- L2

[0205] Table 3 depicts the binding kinetics measured by surface plasmon resonance (SPR) for each Fc-sPD-1 variant to human PD-L1 and PD-L2 divalent proteins. Fc-sPD-1 variants were immobilized to a HC30M chip and the binding of recombinant human PD-L 1 and PD- L2 was evaluated by Carterra LSA (human PD-L1 protein, Fc Tag, Peprotech, Cat. No. 310- 35; human PD-L2 protein, Fc tag, Peprotech, Cat. No. 310-38). hPD-L2 divalent

[0206] As seen in Table 4, the mean KD for Fc-sPD-1 variants range from 10'5to 10'9M on mouse monovalent PD-L1 protein, and 10'7to 10'11M on mouse monovalent PD-L2 protein. On mouse divalent PD-L1 and PD-L2 proteins, the binding was even increased due to avidity.

[0207] Table 4: SPR binding of Fc-sPD-1 variants to monovalent mouse PD-L1 and mouse PD-L2

[0208] Table 4 depicts the binding kinetics measured by surface plasmon resonance (SPR) for each Fc-sPD- 1 variant to mouse PD-L 1 and PD-L2 monovalent proteins. Fc-sPD- 1 variants were immobilized to a HC30M chip and the binding of recombinant mouse PD-L1 and PD-

[0209] L2 was evaluated by Carterra LSA (mouse PD-L1 protein, His tag, Acrobiosystems, Cat.

[0210] No. PD1-M5220; mouse PD-L2 protein, His tag, Acrobiosystems, Cat. No. PD2-M52E3).

[0211] N / D not detectable

[0212] As seen in Table 5, the mean KD for Fc-sPD-1 variants range from 10'9to 10'1° M on mouse divalent PD-L1 protein, and 10'10M on mouse divalent PD-L2 protein.

[0213] Table 5: SPR binding of Fc-sPD-1 variants to divalent mouse PD-L1 and mouse PD- L2

[0214] Table 5 depicts the binding kinetics measured by surface plasmon resonance (SPR) for each Fc-sPD-1 variant to mouse PD-L1 and PD-L2 divalent proteins. Fc-sPD-1 variants were immobilized to a HC30M chip and the binding of recombinant mouse PD-L1 and PD- L2 was evaluated by Carterra LSA (mouse PD-L1 protein, Fc tag, Acrobiosystems, Cat. No. PD1-M5251; mouse PD-L2 protein, Fc tag, Acrobiosystems, Cat. No. PD2-M5254). N / D not detectable Altogether, these results show that the high-affinity non-glycosylated sPD-1 variants according to the invention bound with much higher affinity to PD-L1 and PD-L2 than the native PD-1 mimic polypeptide. These high-affinity non-glycosylated sPD-1 variants according to the invention are cross-reactive on human and mouse (except for clone A1) PD-L1 and PD-L2 proteins.

Claims

CLAIMS1. Soluble PD-1 polypeptide which is a mutant of SEQ ID NO:2 or 12, that binds PD- L1 and / or PD-L2, which comprises : a) at least one mutation in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ I D NO: 1 , said mutation being N49T ; b) at least one mutation in the N-glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, T or S; c) at least one mutation in the N-glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, Q or T; and d) at least one mutation in the N-glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 , said mutation being performed on N, D or S, wherein each of mutations a) to d) affects or prevents N-glycosylation.

2. Soluble PD-1 polypeptide according to claim 1 , which comprises at least 70% identity, preferably at least 71% identity, preferably at least 72%, preferably at least 73%, preferably at least 74% with SEQ ID NO:2 or which comprises at least 70% identity, preferably at least 71% identity, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79% with SEQ ID NO: 12; preferably the soluble PD-1 mutant is identical to SEQ ID NO:2 or 12 and comprises mutations a) to d), and / or the mutations a) to d) increase the affinity of the mutant for PD-L1 and / or PD-L2.

3. Soluble PD-1 polypeptide according to claim 1 or 2, wherein mutations b) and d) are N-substitutions, and mutation c) is N- or T-substitution.

4. Soluble PD-1 polypeptide according to any one of claims 1 to 3, which comprises: a) at least the mutation of N in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 , said mutation being N49T; b) at least one mutation of N or S in the N-glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 ;c) at least one mutation of N or T in the N-glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 ; and d) at least one mutation of N or D or S in the N-glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 , with the proviso that if D is mutated, then N is also mutated, preferably at least two mutations of N and D in the N-glycosylation site NDS.

5. Soluble PD-1 polypeptide according to any one of claims 1 to 4, which comprises: a) the mutation in the N-glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ ID NO:1 is N49T; b) the at least one mutation in the glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 is N58E, N58Q, N58S, N58H, S60H and S60G; c) the at least one mutation in the glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 is chosen from N74G, N74Q, T76Y and T76P; and d) the at least one mutation in the glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 is chosen from N116E, N116Q, N116S, D117A and S118R, with the proviso that if D is mutated, then N is also mutated; preferably the mutations are N116E / D117A or S118R.

6. Soluble PD-1 polypeptide according to any one of claims 1 to 5, wherein: a) the mutation in the glycosylation site NAT wherein N is in position 49 and the numbering is the one of SEQ I D NO: 1 is N49T ; b) the at least one mutation in the glycosylation site NTS wherein N is in position 58 and the numbering is the one of SEQ ID NO:1 is N58S, N58E or N58Q; c) the at least one mutation in the glycosylation site NQT wherein N is in position 74 and the numbering is the one of SEQ ID NO:1 is chosen from N74G, N74Q, T76Y and T76P; and d) the at least one mutation in the glycosylation site NDS wherein N is in position 116 and the numbering is the one of SEQ ID NO:1 is N116E, N116Q, N116S or S118R.

7. Soluble PD-1 polypeptide according to any one of claims 1 to 6, which comprises at least one additional mutation chosen from D26K, R30Q, W32I, W32L, F37I, S38G, T45E, S57D, E61G, S62N, V64F, W67F, R69K, M70I, D77E, D77I, D77L, D77A, E84P, R86K, R86A, S87I, S87T, S87D, S87F, S87V, Q88G, Q88F, G90P, C93S,R96Q, G103K, R104K, M108F, M108P, S109T, V110I, R112N, R115I, R115V, R115E, L122F, G124I, G124S, G124A, G124V, A129Y, A129W, P130G, A132L, A132I, A132M, A132F, K135L, R139F, R139V, R139Y, R139M, R143I, R143L, R143V, T145L, T145V, T145E, E146P, R147K and R147Q, the numbering being the one of SEQ I D NO: 1.

8. Soluble PD-1 polypeptide according to any one of claims 1 to 5, which comprises, preferably consists of, the sequence SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9, or of a sequence chosen from SEQ ID NO:14 to 32.

9. Antigen-binding fragment comprising a soluble PD-1 polypeptide according to any one of claims 1 to 8; or protein construct comprising a soluble PD-1 polypeptide according to any one of claims 1 to 8 that is fused, directly or via a linker, to a protein fragment.

10. Protein construct according to claim 9, wherein the protein fragment is chosen from Fc fragments, Fc-VHH and single-domain antibodies (sdAb).11 . Nucleic acid coding for a soluble PD-1 polypeptide according to any one of claims 1 to 8, or for an antigen-binding fragment according to claim 9 or for a protein construct according to claim 9 or 10.

12. Expression vector comprising the nucleic acid of claim 11 .

13. Host cell comprising a nucleic acid of claim 9 or an expression vector of claim 12.

14. Soluble PD-1 polypeptide according to any one of claims 1 to 8, or antigen-binding fragment according to claim 9 or protein construct according to claim 9 or 10, or nucleic acid according to claim 11 , or expression vector according to claim 12 or host cell according to claim 13, for use as a medicament.

15. Soluble PD-1 polypeptide according to any one of claims 1 to 8, or antigen-binding fragment according to claim 9 or protein construct according to claim 9 or 10, which is conjugated with at least one compound chosen from drugs, toxins and radioactive compounds.

16. Soluble PD-1 polypeptide according to any one of claims 1 to 8, or antigen-binding fragment according to claim 9 or protein construct according to claim 9 or 10, or nucleic acid according to claim 11, or expression vector according to claim 12 or host cell according to claim 13, for use for preventing and / or treating cancer in a subject in need thereof.

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