Anti-PD-1 antibodies and fusion proteins comprising the same

Abstract

The present invention relates to novel non-blocking anti-PD-1 antibodies, as well as fusion proteins comprising the same. The present invention also relates to the use of said anti-PD-1 antibodies or said fusion proteins as a medicament, and in particular, for treating cancer or inflammatory diseases.

Description

ANTI-PD-1 ANTIBODIES AND FUSION PROTEINS COMPRISING THE SAME FIELD OF INVENTION

[0001] The present invention relates to novel non-blocking anti-PD-1 antibodies, as well as fusion proteins comprising the same. The present invention also relates to the use of said anti-PD-1 antibodies or said fusion proteins as a medicament, and in particular, for treating cancer or inflammatory diseases.BACKGROUND OF INVENTION

[0002] PD-1, which stands for “Programmed Death-1”, is an immune checkpoint found on the surface of T cells. It plays an important role in the regulation of the immune system and in cancer. In particular, when the ligand PD-L1 binds to PD-1, it deactivates the ability of the T cells to kill cancer cells, the latter continuing to expand in the body.

[0003] PD-1 antibodies blocking PD-1 / PD-L1 interaction have been developed and have achieved promising results in cancer. For example, the blocking PD-1 antibodies Pembrolizumab and Nivolumab have been approved for a variety of cancers, including melanoma, non-small cell lung cancer, classical Hodgkin lymphoma, and head and neck cancers.

[0004] It is of particular interest to target immune cells expressing PD-1 in cancer because of the crucial role PD-1 plays in immune regulation and suppression. Blocking PD-1 allows for enhanced T cell activation, essentially removing the brake system on the T cell response to tumor invasion. However, the difficulty is that it is not possible to target cells with blocking PD-1 antibodies in patients already treated with these blocking antibodies, since the antibody binding site is already occupied.

[0005] Furthermore, the efficacy of anti -tumor activity of immune stimulating molecules such as cytokines, can be improved by the inclusion of a domain directing the molecule to the appropriate cells with anti-tumor activities in the tumor microenvironment (e.g. exhausted CD8 T cells), an effect known as ‘cis-targeting’ or ‘cis-signaling’. Examples of such cis-signaling fusion proteins with cytokines such as interleukin-2 (IL-2) have been generated to create blocking anti-PD-l / IL-2 bispecific molecules, and these molecules have demonstrated superior efficacy in mouse preclinical tumor models compared to nontargeted IL-2.

[0006] However, there are inherent pharmacological challenges associated with administering efficient amounts of blocking and agonistic therapeutic agents in a single molecule. Indeed, blocking therapies such as anti-PD-1 antibodies require precise administration (dose and frequency) to ensure full target coverage, which are different than agonistic therapies that necessitate sufficient dosing amounts and frequencies to provide immune cells the proper stimulation in a safe and effective effect.

[0007] Thus, there is a need to develop non-blocking anti-PD-1 antibodies that can target cells expressing PD-1 and that are compatible with existing therapies that block interactions between PD-1 and its ligands. Furthermore, using a non-blocking anti-PD-1 antibody for targeting cytokine delivery allows the decoupling of the two mechanisms of action and provides a route for optimized dosing of both PD-1 blockade and cytokine delivery.

[0008] The Inventors have developed new anti-PD-1 antibodies that do not block the interaction of PD-1 / PD-L1 as well as fusion proteins comprising said antibodies, which can be combined with blocking anti-PD-1 antibodies.SUMMARY

[0009] The present invention relates to an isolated anti -human PD-1 antibody or antigenbinding fragment thereof, wherein the variable region of the heavy chain (VH) comprisesat least one of, preferably two of, and more preferably the three following complementary-determining regions (CDRs):CDR1: GX₁TX₂X₃GLY (SEQ ID NO: 189), wherein X₁ is G or Y, X2is F or L, and X3 is T or S;CDR2: X₄SX₅EFGX₆X₇ (SEQ ID NO: 190), wherein X4is V or I, X5is A or V, X₆ is S or G and X7 is T or A; and / orCDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191), andwherein the variable region of the light chain (VL) comprises at least one of, preferably two of, and more preferably the three following CDRs:CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);CDR2: EVSNRPS (SEQ ID NO: 204); and / orCDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0010] In one embodiment, the VL comprises the three CDRs with SEQ ID NOs: 203-205, and the VH comprises:(a) The following CDRS:- VH-CDR1: GGTFTGLY (SEQ ID NO: 192);- VH-CDR2: VSAEFGST (SEQ ID NO: 193); and- VH-CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(b) The following CDRS- VH-CDR1: GGTLTGLY (SEQ ID NO: 194);- VH-CDR2: VSVEFGGA (SEQ ID NO: 195); and- VH-CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(c) The following CDRS- VH-CDR1: GYTLTGLY (SEQ ID NO: 196);- VH-CDR2: ISAEFGGA (SEQ ID NO: 197); and- VH-CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(d) The following CDRSCDR1: GYTLSGLY (SEQ ID NO: 198);- CDR2: VSVEFGST (SEQ ID NO: 199); and- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(e) The following CDRS- CDR1: GGTLSGLY (SEQ ID NO: 200);- CDR2: ISVEFGGA (SEQ ID NO: 201); and- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(I) The following CDRS- CDR1: GYTLSGLY (SEQ ID NO: 198);- CDR2: ISAEFGST (SEQ ID NO: 202); and- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

[0011] In one embodiment, said antibody or fragment comprises:a VH comprising or consisting of the sequence SEQ ID NO: 206, wherein X₁ is G or Y, X2 is F or L, X3 is T or S, X₄ is V or I, X₅ is A or V, X₆ is S or G, and X7 is T or A, anda VL comprising or consisting of the sequence SEQ ID NO: 207.

[0012] In one embodiment, said antibody or fragment comprises:a VH comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 208-213; anda VL comprising or consisting of SEQ ID NO: 207.

[0013] The present invention also relates to a fusion protein comprising at least one isolated anti -human PD-1 antibody or antigen-binding fragment thereof as described herein.

[0014] In one embodiment, the fusion protein as described herein comprises at least one anti -human PD-1 antibody or antigen-binding fragment thereof and at least one interleukin-2 (IL-2) or a mutant thereof.

[0015] In one embodiment, the IL-2 mutant comprises or consists of one of the sequences selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 130-143,SEQ ID NO: 145-188, SEQ ID NOs: 19-39, SEQ ID NOs: 51-97, SEQ ID NO: 99, SEQ IDNOs: 110-117, SEQ ID NOs: 119-127.

[0016] In one embodiment, the mutant IL-2 comprises or consists of one of the sequences selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 30, SEQ ID NO: 170, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 64, SEQ ID NO: 58, SEQ ID NO: 185, and SEQ ID NO: 70, preferably the mutant IL-2 comprises or consists of one of the sequences selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 64, and SEQ ID NO: 70.

[0017] In one embodiment, the fusion protein as described herein further comprises at least one masking moiety selected from the group consisting of glycans, lipids, proteins, peptides, small organic molecules and polymers.

[0018] In one embodiment, the masking moiety is a water-soluble polymer, preferably a polyethylene glycol (PEG).

[0019] The present invention also relates to an isolated nucleic acid encoding the isolated anti -human PD-1 antibody or antigen-binding fragment thereof or the fusion protein as described herein.

[0020] The present invention also relates to the isolated anti -human PD-1 antibody or antigen-binding fragment thereof as described herein, or the fusion protein as described herein, for use as a medicament.

[0021] The present invention also relates to the isolated anti-human PD-1 antibody or antigen-binding fragment thereof or the fusion protein as described herein, for use in treating a cancer or an inflammatory disease in a subject in need thereof.

[0022] In one embodiment, the subject has received, is receiving or will receive an immunotherapy.

[0023] In one embodiment, the immunotherapy is a blocking anti-PD-1 antibody, preferably nivolumab or pembrolizumab.DEFINITIONS

[0024] In the present invention, the following terms have the following meanings:

[0025] “About”, preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed.

[0026] “Antigen-binding fragment”, as used herein, refers to a part or region of an antibody which comprises fewer amino acid residues than the whole antibody. An “antigen-binding fragment” binds antigen and / or competes with the whole antibody from which it derives for antigen binding (e.g., specific binding to human PD-1).

[0027] “Cancer” has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes, without limitation, diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term “cancer” further encompasses both primary and metastatic cancers.

[0028] CDR or “complementarity determining region” means the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme), or a combination thereof. More recently, a universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Nucleic Acids Res. 27: 209-212 1999). As the "location" of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues may be readily identified. This information can be used in grafting and replacement ofCDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. Correspondence between the Kabat numbering and the IMGT unique numbering system is also well known to one skilled in the art (e.g., Lefranc et al., supra).

[0029] “Consisting essentially of’, with reference to a composition, means that the at least one antibody or antigen-binding fragment thereof, fusion protein, nucleic acid or expression vector as described herein is the only one therapeutic agent or agent with a biologic activity within said composition.

[0030] “Diabodies”, as used herein, refer to small antibody fragments prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL such that inter-chain but not intra-chain pairing of the variable domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” scFv fragments in which the VH and VL of the two antibodies are present on different polypeptide chains.

[0031] “Fab” refers to a monovalent fragment containing the following regions: VH, VL, CHI and CL, linked by an intramolecular disulfide bond. As used herein, F(ab')2 refers to a fragment containing two antigen-binding regions joined by disulfides bonds. As used herein, Fab’ refers to a fragment obtained by the reduction of F(ab')2fragments.

[0032] “Fd fragment” refers to the heavy chain of the Fab fragment, comprising the VH and CHI regions.

[0033] “Fv”, as used herein, refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one VH and one VL in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the heavy and light chain) that contribute to antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0034] “Fully human”, in the context of the constant region of an antibody or antigenbinding fragment thereof, refers to an amino acid sequence identity of 100% with a human constant region.

[0035] “Host cell” generally refers to a cultured cell line. In one embodiment, whole human beings into which an expression vector encoding an antibody or antigen-binding fragment thereof or a fusion protein as described herein has been introduced are excluded from the definition of a “host cell”.

[0036] “Identity” or “identical”, when used herein in a relationship between the sequences of two or more amino acid sequences, or of two or more nucleic acid sequences, refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program / .c.. “algorithms”). Identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk A. M. (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith D. W. (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin A. M. & Griffin H. G. (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov M. R. & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo etal., 1988. SIAMJApplMath. 48(5): 1073-82. Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux etal., 1984. Nucleic Acids Res.12(1 Pt l):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol.215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm may also be used to determine identity.

[0037] “Interleukin-2” or “IL-2” refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2 (e.g., splice variants or allelic variants) as well as truncated forms of IL-2. In one embodiment, IL-2 is human IL-2, having the sequence SEQ ID NO: 1. In another embodiment, IL-2 is a truncated human IL-2, having the sequence SEQ ID NO: 50.

[0038] “Interleukin-2 receptor” or “IL-2R” refers to the heterotrimeric receptor of IL- 2. This receptor is composed by different combinations of the following chains: a (alpha) chain (also called IL-2Ra, CD25, or Tac antigen), [3 (beta) chain (also called IL-2RJ3, or CD 122), and y (gamma) chain (also called IL-2Ry, common gamma chain, or CD 132). Depending on the combinations of these chains, IL-2 receptors having low, intermediate, or high affinity for IL-2 may be generated. Low-affinity IL-2 receptors are composed of an IL-2Ra chain. Intermediate-affinity IL-2 receptors are composed of IL-2RJ3 and IL-2Ry chains. High-affinity IL-2 receptors are composed of IL-2Ra, IL-2RJ3 and IL-2Ry chains.

[0039] “IL-2 mutant” or “mutant IL-2” refers to an IL-2 that displays modifications in sequence and / or in functional properties.

[0040] “Isolated nucleic acid”, as used herein, is intended to refer to a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologoussystems. A substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.

[0041] “Mammal” refers to any mammal, including humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a human.

[0042] “Pharmaceutically acceptable excipient” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Said excipient does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a mammal, more preferably a human. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.

[0043] Peptide” refers to a linear polymer of amino acids of at least 2 amino acids and less than 50 amino acids linked together by peptide bonds.

[0044] “Polyethylene glycol” or “PEG” refers to a biocompatible, synthetic, hydrophilic poly ether compound with the formula hereinabove, wherein n > 4.nPEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The term “PEG” usually refers to oligomers and polymers with a molecular mass below 20,000 g / mol, and “PEO” for polymers with a molecular mass above 20,000 g / mol. Unless otherwise specified, the term “PEG” as used herein comprises both “PEG” and “PEO” compounds.

[0045] “Single-chain Fv”, also abbreviated as “sFv” or “scFv”, refers to antibody fragments that comprise the VH and VL antibody domains connected into a single amino acid chain. Preferably, the scFv amino acid sequence further comprises a peptide linkerbetween the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0046] “Small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0047] Subject”, as used herein, refers to a mammal, preferably a human. In one embodiment, a subject may be a “patient”, i.e., a warm-blooded animal, more preferably a human, who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a disease.

[0048] “Substantially human”, in the context of the constant region of an antibody or antigen-binding fragment thereof, refers to an amino acid sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more with a human constant region. For example, the antibodies or antigen-binding fragments thereof may be substantially human if they comprise constant domains of “human” sequence which have been altered, by one or more amino acid additions, deletions or substitutions with respect to the human sequence.

[0049] “Therapeutically effective amount” refers to the level or amount of an antibody or antigen-biding fragment thereof or a fusion protein as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively or additionally, the therapeuticallyeffective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.

[0050] “Treatment” refers to a therapeutic (or curative) treatment, to a prophylactic (or preventive) treatment, or to both a therapeutic (or curative) treatment and a prophylactic (or preventive) treatment, wherein the object is to prevent, reduce, slow down (lessen), or cure one or more of the symptom(s) or manifestation(s) of the targeted pathologic condition or disorder.

[0051] “Vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform a host and promote expression (e.g. transcription and translation) of the introduced sequence. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said fusion protein upon administration to a host. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukemia virus, promoter and enhancer of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the antibody, antigen-binding fragment or fusion protein can be inserted and expressed.DETAILED DESCRIPTION

[0052] The present invention relates to an isolated anti-PD-1 antibody or antigenbinding fragment thereof.

[0053] “PD-1” is also known as “Programmed cell death protein-1”, “cluster of differentiation 279”, or “CD279”. As used herein, it refers to a cell surface receptor expressed on activated T cells, which can bind to the ligands PD-L1 and PD-L2. It plays a key role in the regulation of immune response. An example of human PD-1 is Uniprot Q15116.

[0054] The isolated anti-PD- 1 antibody or antigen-binding fragment thereof as described herein may be directed against human PD-1.

[0055] The isolated anti-PD- 1 antibody or antigen-binding fragment thereof as described herein may bind human PD-1. The isolated anti-PD- 1 antibody or antigen-binding fragment thereof as described herein may bind cynomolgus monkey PD-1.

[0056] The isolated anti-PD- 1 antibody or antigen-binding fragment thereof as described herein is a non-blocking PD-1 antibody, i.e. an antibody that binds to PD-1 but does not block the PD-1 / PD-L1 interaction.

[0057] Methods for assessing whether an anti-PD- 1 antibody or antigen-binding fragment thereof is non-blocking are well known by the skilled artisan in the art, and can be found in Examples. Such non-blocking property may be assessed by PD-1 / PD-L1 blockade bioassays with effector cells expressing PD-1 (e.g. PD-1 Jurkat effector cells) and cells expressing PD-L1 (e.g. PD-L1 APC CHO-K1 cells). Other methods, include, for example, measuring IL-2 release in the presence of the antibodies to be tested, or competition assays with known blocking anti-PD-1 antibodies.

[0058] The isolated anti-PD-1 antibody or antigen-binding fragment thereof as described herein may be monoclonal. The isolated anti-PD-1 antibody or antigen-binding fragment thereof as described herein may be polyclonal.

[0059] In the following, and unless explicitly mentioned otherwise, CDR numbering and definitions are according to the IMGT numbering system.

[0060] The antibody or antigen-binding fragment thereof as described herein may comprise a heavy chain variable region (abbreviated herein as VH) which comprises at least one, preferably at least two, more preferably the three following complementary -determining regions (CDRs):- CDR1: GX₁TX₂X₃GLY (SEQ ID NO: 189), wherein X₁ is G or Y, X2is F or L, and X3 is T or S;- CDR2: X₄SX₅EFGX₆X₇ (SEQ ID NO: 190), wherein X4is V or I, X5is A or V, X₆ is S or G and X7 is T or A; and / orCDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

[0061] The antibody or antigen-binding fragment as described herein may comprise a light chain variable region (abbreviated herein as VL) which comprises at least one, preferably at least two, more preferably the three following complementary-determining regions (CDRs):- CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);- CDR2: EVSNRPS (SEQ ID NO: 204); and / or- CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0062] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: GX₁TX₂X₃GLY (SEQ ID NO: 189), wherein X₁ is G or Y, X2 is F or L, and X3 is T or S;o CDR2: X₄SX₅EFGX₆X₇ (SEQ ID NO: 190), wherein X4is V or I, X5is A or V, X₆ is S or G and X7 is T or A; and / oro CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); and / oro CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0063] In particular, the antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises the three following CDRs:o CDR1: GX₁TX₂X₃GLY (SEQ ID NO: 189), wherein X₁ is G or Y, X2is F or L, and X3 is T or S;o CDR2: X₄SX₅EFGX₆X₇ (SEQ ID NO: 190), wherein X4is V or I, X5is A or V, X₆ is S or G and X7 is T or A, and;o CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); anda VL which comprises the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); ando CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0064] The antibody or antigen-binding fragment thereof as described herein may comprise a VH comprising or consisting of the sequence SEQ ID NO: 206, wherein X1is G or Y, X2is F or L, X3is T or S, X4is V or I, X5is A or V, X6is S or G, and X7is T or A.

[0065] The antibody or antigen-binding fragment thereof as described herein may comprise a VL comprising or consisting of the sequence SEQ ID NO: 207.

[0066] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH comprising or consisting of the sequence SEQ ID NO: 206, wherein X1is G or Y, X2is F or L, X3is T or S, X4is V or I, X5is A or V, X6is S or G, and X7is T or A; anda VL comprising or consisting of the sequence SEQ ID NO: 207.

[0067] Examples of antibodies or antigen-binding fragments thereof having the CDRs of SEQ ID NOs: 189-191, 203-205 and / or the VH and VL of SEQ ID NOs: 206-207 are clones 22, 23, 24, 26, 27 and 28.

[0068] The antibody or antigen-binding fragment thereof as described herein may comprise a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:CDR1 selected from the group comprising or consisting of SEQ ID NOs: 192, 194, 196, 198 and 200;CDR2 selected from the group comprising or consisting of SEQ ID NOs: 193, 195, 197, 199, 201 and 202; and / or- CDR3 being SEQ ID NO: 191.

[0069] The antibody or antigen-binding fragment thereof as described herein may comprise a VL which comprises at least one, preferably at least two, more preferably the three CDRs as disclosed herein (SEQ ID NOs: 203-205).

[0070] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1 selected from the group comprising or consisting of SEQ ID NOs:192, 194, 196, 198 and 200;o CDR2 selected from the group comprising or consisting of SEQ ID NOs:193, 195, 197, 199, 201 and 202; and / oro CDR3 being SEQ ID NO: 191; anda VL which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1 being TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2 being EVSNRPS (SEQ ID NO: 204); and / oro CDR3 being SSYTSSSTYV (SEQ ID NO: 205).

[0071] In particular, the antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises the three following CDRs:o CDR1 selected from the group comprising or consisting of SEQ ID NOs:192, 194, 196, 198 and 200;o CDR2: selected from the group comprising or consisting of SEQ ID NOs:193, 195, 197, 199, 201 and 202; ando CDR3 being SEQ ID NO: 191; anda VL which comprises the three following CDRs:o CDR1 being TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2 being EVSNRPS (SEQ ID NO: 204); ando CDR3 being SSYTSSSTYV (SEQ ID NO: 205).

[0072] The antibody or antigen-binding fragment thereof as described herein may comprise a VH comprising or consisting of a sequence selected from the group comprising or consisting of SEQ ID NOs: 208-213.

[0073] The antibody or antigen-binding fragment thereof as described herein may comprise a VL comprising or consisting of SEQ ID NO: 207.

[0074] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH comprising or consisting of a sequence selected from the group comprising or consisting of SEQ ID NOs: 208-213; anda VL comprising or consisting of SEQ ID NO: 207.

[0075] Examples of antibodies or antigen-binding fragments thereof having the CDRs and / or the VH and VL as described herein are clones 22, 23, 24, 26, 27 and 28.

[0076] The antibody or antigen-binding fragment thereof as described herein may comprise a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:- CDR1: GGTFTGLY (SEQ ID NO: 192);- CDR2: VSAEFGST (SEQ ID NO: 193); and / or- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

[0077] The antibody or antigen-binding fragment as described herein may comprise a VL which comprises at least one, preferably at least two, more preferably the three CDRs as disclosed herein (SEQ ID NOs: 203-205).

[0078] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: GGTFTGLY (SEQ ID NO: 192);o CDR2: VSAEFGST (SEQ ID NO: 193); and / oro CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); anda VL which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); and / oro CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0079] In particular, the antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises the three following CDRs:o CDR1: GGTFTGLY (SEQ ID NO: 192);o CDR2: VSAEFGST (SEQ ID NO: 193); ando CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); ando CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0080] The antibody or antigen-binding fragment thereof as described herein may comprise a VH comprising or consisting of SEQ ID NO: 208.

[0081] The antibody or antigen-binding fragment thereof as described herein may comprise a VL comprising or consisting of SEQ ID NO: 207.

[0082] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH comprising or consisting of SEQ ID NO: 208; anda VL comprising or consisting of SEQ ID NO: 207.

[0083] An example of antibodies or antigen-binding fragments thereof having the CDRs of SEQ ID NOs: 191-193, 203-205 and / or the VH and VL of SEQ ID NOs: 207-208 is clone 22.

[0084] The antibody or antigen-binding fragment thereof as described herein may comprise a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:- CDR1: GGTLTGLY (SEQ ID NO: 194);- CDR2: VSVEFGGA (SEQ ID NO: 195); and / or- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

[0085] The antibody or antigen-binding fragment as described herein may comprise a VL which comprises at least one, preferably at least two, more preferably the three CDRs as disclosed herein (SEQ ID NOs: 203-205).

[0086] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: GGTLTGLY (SEQ ID NO: 194);o CDR2: VSVEFGGA (SEQ ID NO: 195); and / oro CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191), and a VL which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); and / oro CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0087] In particular, the antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises the three following CDRs:o GGTLTGLY (SEQ ID NO: 194);o CDR2: VSVEFGGA (SEQ ID NO: 195); ando CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191), and a VL which comprises the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); ando CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0088] The antibody or antigen-binding fragment thereof as described herein may comprise a VH comprising or consisting of SEQ ID NO: 209.

[0089] The antibody or antigen-binding fragment thereof as described herein may comprise a VL comprising or consisting of SEQ ID NO: 207.

[0090] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH comprising or consisting of SEQ ID NO: 209; anda VL comprising or consisting of SEQ ID NO: 207.

[0091] An example of antibodies or antigen-binding fragments thereof having the CDRs of SEQ IDNOs:191, 194-195, 203-205 and / or the VH andVL of SEQ IDNOs: 207, 209 is clone 23.

[0092] The antibody or antigen-binding fragment thereof as described herein may comprise a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:- CDR1: GYTLTGLY (SEQ ID NO: 196);- CDR2: ISAEFGGA (SEQ ID NO: 197); and / or- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

[0093] The antibody or antigen-binding fragment as described herein may comprise a VL which comprises at least one, preferably at least two, more preferably the three CDRs as disclosed herein (SEQ ID NOs: 203-205).

[0094] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: GYTLTGLY (SEQ ID NO: 196);o CDR2: ISAEFGGA (SEQ ID NO: 197); and / oro CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); and / oro CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0095] In particular, the antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises the three following CDRs:o CDR1: GYTLTGLY (SEQ ID NO: 196);o CDR2: ISAEFGGA (SEQ ID NO: 197); ando CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); ando CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0096] The antibody or antigen-binding fragment thereof as described herein may comprise a VH comprising or consisting of SEQ ID NO: 210.

[0097] The antibody or antigen-binding fragment thereof as described herein may comprise a VL comprising or consisting of SEQ ID NO: 207.

[0098] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH comprising or consisting of SEQ ID NO: 210; anda VL comprising or consisting of SEQ ID NO: 207.

[0099] An example of antibodies or antigen-binding fragments thereof having the CDRs ofSEQ IDNOs:191, 196-197, 203-205 and / or the VH andVL ofSEQ IDNOs: 207, 210 is clone 24.

[0100] The antibody or antigen-binding fragment thereof as described herein may comprise a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:- CDR1: GYTLSGLY (SEQ ID NO: 198);- CDR2: VSVEFGST (SEQ ID NO: 199); and / or- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

[0101] The antibody or antigen-binding fragment as described herein may comprise a VL which comprises at least one, preferably at least two, more preferably the three CDRs as disclosed herein (SEQ ID NOs: 203-205).

[0102] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: GYTLSGLY (SEQ ID NO: 198);o CDR2: VSVEFGST (SEQ ID NO: 199); and / oro CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); and / oro CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0103] In particular, the antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises the three following CDRs:o CDR1: GYTLSGLY (SEQ ID NO: 198);o CDR2: VSVEFGST (SEQ ID NO: 199); ando CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); ando CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0104] The antibody or antigen-binding fragment thereof as described herein may comprise a VH comprising or consisting of SEQ ID NO: 211.

[0105] The antibody or antigen-binding fragment thereof as described herein may comprise a VL comprising or consisting of SEQ ID NO: 207.

[0106] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH comprising or consisting of SEQ ID NO: 211; anda VL comprising or consisting of SEQ ID NO: 207.

[0107] An example of antibodies or antigen-binding fragments thereof having the CDRs of SEQ IDNOs:191, 198-199, 203-205 and / or the VH andVL of SEQ IDNOs: 207, 211 is clone 26.

[0108] The antibody or antigen-binding fragment thereof as described herein may comprise a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:- CDR1: GGTLSGLY (SEQ ID NO: 200);- CDR2: ISVEFGGA (SEQ ID NO: 201); and / or- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

[0109] The antibody or antigen-binding fragment as described herein may comprise a VL which comprises at least one, preferably at least two, more preferably the three CDRs as disclosed herein (SEQ ID NOs: 203-205).

[0110] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: GGTLSGLY (SEQ ID NO: 200);o CDR2: ISVEFGGA (SEQ ID NO: 201); and / oro CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); and / oro CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0111] In particular, the antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises the three following CDRs:o CDR1: GGTLSGLY (SEQ ID NO: 200);o CDR2: ISVEFGGA (SEQ ID NO: 201); ando CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); ando CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0112] The antibody or antigen-binding fragment thereof as described herein may comprise a VH comprising or consisting of SEQ ID NO: 212.

[0113] The antibody or antigen-binding fragment thereof as described herein may comprise a VL comprising or consisting of SEQ ID NO: 207.

[0114] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH comprising or consisting of SEQ ID NO: 212; anda VL comprising or consisting of SEQ ID NO: 207.

[0115] An example of antibodies or antigen-binding fragments thereof having the CDRs of SEQ ID NOs:191, 200-201, 203-205 and / or the VH and VL of SEQ ID NOs: 207, 212 is clone 27.

[0116] The antibody or antigen-binding fragment thereof as described herein may comprise a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:- CDR1: GYTLSGLY (SEQ ID NO: 198);- CDR2: ISAEFGST (SEQ ID NO: 202); and / or- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

[0117] The antibody or antigen-binding fragment as described herein may comprise a VL which comprises at least one, preferably at least two, more preferably the three CDRs as disclosed herein (SEQ ID NOs: 203-205).

[0118] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: GYTLSGLY (SEQ ID NO: 198);o CDR2: ISAEFGST (SEQ ID NO: 202); and / oro CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises at least one, preferably at least two, more preferably the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); and / oro CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0119] In particular, the antibody or antigen-binding fragment thereof as described herein may comprise:a VH which comprises the three following CDRs:o CDR1: GYTLSGLY (SEQ ID NO: 198);o CDR2: ISAEFGST (SEQ ID NO: 202); ando CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191); and a VL which comprises the three following CDRs:o CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);o CDR2: EVSNRPS (SEQ ID NO: 204); ando CDR3: SSYTSSSTYV (SEQ ID NO: 205).

[0120] The antibody or antigen-binding fragment thereof as described herein may comprise a VH comprising or consisting of SEQ ID NO: 213.

[0121] The antibody or antigen-binding fragment thereof as described herein may comprise a VL comprising or consisting of SEQ ID NO: 207.

[0122] The antibody or antigen-binding fragment thereof as described herein may comprise:a VH comprising or consisting of SEQ ID NO: 213; anda VL comprising or consisting of SEQ ID NO: 207.

[0123] An example of antibodies or antigen-binding fragments thereof having the CDRs of SEQ ID NOs:191, 198, 202-205 and / or the VH and VL of SEQ ID NOs: 207, 213 is clone 28.

[0124] It will be understood to the skilled artisan in the art that any one of CDR1, CDR2 and / or CDR3 of the VH and / or the VL as described herein can be characterized as having 1, 2, 3 or more amino acids being substituted by a different amino acid.

[0125] In particular, any one of CDR1, CDR2 and / or CDR3 of the VH and / or of the VL as described herein can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with the particular CDR or sets of CDRs listed above.

[0126] It will be understood to the skilled artisan in the art that any of the VH and / or VL as described herein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids substituted by a different amino acid.

[0127] In particular, any of the VH and / or VL as described herein can have an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with the VH and / or VL listed above.

[0128] In particular, any of the VH and / or VL as described herein can have an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with the VH and / or VL listed above outside the CDRs.

[0129] In one embodiment, the antibody or antigen-binding fragment thereof has a set of CDRs as defined hereinabove, and a framework region of VH and / or VL having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with the VH and / or VL listed above.

[0130] The antibody or antigen-binding fragment thereof as described herein may be selected from the group comprising or consisting of a whole antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a Fab', a Fab'-SH, a F(ab)’2, a Fd, a defucosylated antibody, a bispecific antibody, a diabody, a triabody, a tetrabody and a unibody.

[0131] In one embodiment, the antibody or antigen-binding fragment thereof as described herein is a whole antibody (i.e. a full-length antibody). In one embodiment, the antibody or antigen-binding fragment thereof as described herein is a Fab.

[0132] Antigen-binding fragments of antibodies can be obtained using standard methods. For instance, Fab or F(ab')2fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques.

[0133] The antibody or antigen-binding fragment thereof as described herein may be a fully or substantially human antibody or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof as described herein is fully human. In one embodiment, the antibody or antigen-binding fragment thereof as described herein is substantially human.

[0134] It will be appreciated that antibodies or antigen-binding fragments thereof as described herein can be modified using known methods to modify the properties of the antibodies or fragments thereof.

[0135] For example, to slow clearance in vivo and obtain a more desirable pharmacokinetic profile, the antibody or antigen-binding fragment thereof as describedherein may be modified with polyethylene glycol (PEG). Methods for coupling and site-specifically conjugating PEG to an antibody or an antigen-binding fragment thereof are well known in the art.

[0136] Modifications can also be made to framework residues within the VH and / or the VL to improve the properties of the antibody or fragment. Typically, such framework modifications are made to decrease the immunogenicity of the antibody or fragment. For example, one approach is to “back-mutate” one or more framework residues to the corresponding germline sequence.

[0137] As another example, antibodies or antigen-binding fragments thereof can be aglycosyled (i.e., the antibody or fragment lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody or fragment for the antigen or alter the antibody-dependent cellular cytotoxicity activity of the antibody or fragment thereof. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.

[0138] As compared to the anti-PD-1 antibodies of the prior art, the anti-PD-1 antibodies or antigen-binding fragments thereof as described herein may present one or several of the following advantages:The anti-PD-1 antibodies or fragments as described herein can be combined with blocking anti-PD-1 antibodies.The antibodies or fragments as described herein are of lower affinity for PD-1, so it will reduce the binding to low expressing cells and only allow tight binding when binding in cis to other targets.The antibodies or fragments as described herein present a cross-reactivity for the cynomolgus monkey PD-1, allowing to perform pre-clinical studies on monkeys.

[0139] The antibody or the antigen-binding fragments thereof as described herein can be fused to another molecule, such as a protein. Thus, the present invention also relates to a fusion protein comprising at least one antibody or an antigen-binding fragments thereof as described herein.

[0140] The fusion protein as described herein may comprise at least one antibody or antigen-binding fragment thereof as described herein, and at least one cytokine.

[0141] Said cytokine may be an interleukin. In particular, said cytokine may be an interleukin-2 (IL-2) or a mutant thereof. Thus, in one embodiment, the fusion protein comprises at least one antibody or antigen-binding fragment thereof as described herein, and at least one IL-2 or a mutant thereof.

[0142] In one embodiment, the interleukin is human IL-2. An example of human IL-2 is SEQ ID NO: 1.

[0143] In one embodiment, the interleukin is a mutant IL-2, preferably an IL-2 mutant derived from human IL-2.

[0144] In one embodiment, the IL-2 mutant as described herein is derived from human IL-2 with SEQ ID NO: 1 or a variant thereof, preferably wherein said variant comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 1.

[0145] Said mutant may comprise the same number of amino acids as SEQ ID NO: 1, and thus the mutations and positions described herein are the same for the mutant. Alternatively, said mutant may comprise a different number of amino acids as SEQ ID NO: 1. In this case, the skilled artisan in the art will know how to place the mutations and positions described herein in the mutant.

[0146] The IL-2 mutant may comprise at least one non-endogenous transglutaminase motif. As used herein, a transglutaminase motif is a motif comprising at least one Q residue, in particular a surface exposed Q residue, whose is recognized by a transglutaminase (i.e. the side chain acts as a substrate for the transglutaminase enzyme), which catalyzes the formation of an isopeptide bond between y-carboxamide groups (-(C=O)NH2) of glutamine residue side chains and various primary amines (acyl-acceptors), notably the e-amino groups (-NH2) of lysine residue side chains, with subsequent release of ammonia (NH3). Transglutaminase motifs may be found endogenously in proteins, and in particular, in IL-2. As an example, human IL-2 withSEQ ID NO: 1 comprises an endogenous transglutaminase motif with the Q74 residue, and with the motif consisting of AQS.

[0147] The IL-2 mutant may comprise one non-endogenous transglutaminase motif. The mutant IL-2 may comprise two or more non-endogenous transglutaminase motifs.

[0148] The at least one non-endogenous transglutaminase motif as described herein may be inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, H79, E95, E100, E110 and S130.

[0149] The at least one non-endogenous transglutaminase motif as described herein may be characterized by the presence of a glutamate (Q) residue at position Q57 by reference to SEQ ID NO: 1. In this embodiment, it will be understood that the Q57 residue is already present in SEQ ID NO: 1 and that the at least one non-endogenous transglutaminase motif is inserted by substitution of the amino acids surrounding Q57 to form a transglutaminase site.

[0150] The at least one non-endogenous transglutaminase motif as described herein may be one of the motifs of Table 3. In particular, the at least one non-endogenous transglutaminase motif as described herein may be selected from the group comprising or consisting of SEQ ID NOs: 3-18, VIQ, SEQ ID NOs: 40-49 and SEQ ID NOs: 102-109.

[0151] The at least one non-endogenous transglutaminase motif as described herein may be LTQG (SEQ ID NO: 118).

[0152] The IL-2 mutant may comprise or consist of one of the sequences selected from the group comprising or consisting of SEQ ID NOs: 19-39, SEQ ID NOs: 51-97, SEQ ID NO: 99, SEQ ID NOs: 110-117, SEQ ID NOs: 119-127, and SEQ ID NOs: 130-188.

[0153] In particular, the IL-2 mutant may comprise or consist of one of the sequences selected from the group comprising or consisting of SEQ ID NO: 30, SEQ ID NO: 170, SEQ ID NO: 144, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 64, SEQ ID NO: 58 SEQ ID NO: 185, and SEQ ID NO: 70.

[0154] In one embodiment, the mutant IL-2 comprises or consists of one of the sequences selected from the group comprising or consisting of SEQ ID NO: 30, SEQ ID NO: 144, SEQ ID NO: 32, SEQ ID NO: 64, SEQ ID NO: 185 and SEQ ID NO: 70.

[0155] In one embodiment, the mutant IL-2 comprises or consists of one of the sequences selected from the group comprising or consisting of SEQ ID NO: 144, SEQ ID NO: 64, and SEQ ID NO: 70.

[0156] The mutant IL-2 may comprise at least one non-endogenous transglutaminase motif inserted by insertion of a glutamate (Q) residue at position H79. The mutant IL-2 may comprise at least one non-endogenous transglutaminase motif of sequence SEQ ID NO:14, preferably with an insertion of a glutamate (Q) residue at position H79.

[0157] In one embodiment, the fusion protein as described herein comprises at least one antibody or an antigen-binding fragment thereof as described herein, and at least one mutant IL-2 comprising at least one non-endogenous transglutaminase motif of sequence SEQ ID NO:14, preferably with an insertion of a glutamate (Q) residue at position H79.

[0158] In one embodiment, the mutant IL-2 comprises or consists of SEQ ID NO: 144. Thus, in one embodiment, the fusion protein as described herein comprises at least one antibody or the antigen-binding fragments thereof as described herein, and the mutant comprising or consisting of SEQ ID NO: 144.

[0159] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 189-191, 203-205 and / or VH and VL with SEQ ID NOs: 206-207, and the mutant comprising or consisting of SEQ ID NO: 144.

[0160] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191-193, 203-205 and / or VH and VL with SEQ ID NOs: 207-208, and the mutant comprising or consisting of SEQ ID NO: 144. An example of such fusion protein is the fusion protein

[0161] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 194-195, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 209, and the mutant comprising or consisting of SEQ ID NO: 144. An example of such fusion protein is the fusion protein 2-233.

[0162] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 196-197, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 210, and the mutant comprising or consisting of SEQ ID NO: 144. An example of such fusion protein is the fusion protein 2-234 (also named fusion-34 herein).

[0163] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 198-199, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 211, and the mutant comprising or consisting of SEQ ID NO: 144. An example of such fusion protein is the fusion protein 2-230.

[0164] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 200-201, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 212, and the mutant comprising or consisting of SEQ ID NO: 144. An example of such fusion protein is the fusion protein 2-231.

[0165] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 198, 202-205 and / or VH and VL with SEQ ID NOs: 207 and 213, and the mutant comprising or consisting of SEQ ID NO: 144. An example of such fusion protein is the fusion protein 2-232.

[0166] The mutant IL-2 may comprise at least one non-endogenous transglutaminase motif inserted by insertion of a glutamate (Q) residue at position H79. The mutant IL-2 may comprise at least one non-endogenous transglutaminase motif of sequence SEQ ID NO:15, preferably with an insertion of a glutamate (Q) residue at position H79.

[0167] In one embodiment, the fusion protein as described herein comprises at least one antibody or an antigen-binding fragment thereof as described herein, and at least one mutant IL-2 comprising at least one non-endogenous transglutaminase motif of sequence SEQ ID NO:15, preferably with an insertion of a glutamate (Q) residue at position H79.

[0168] In one embodiment, the mutant IL-2 comprises or consists of SEQ ID NO: 64. Thus, in one embodiment, the fusion protein as described herein comprises at least one antibody or the antigen-binding fragments thereof as described herein, and the mutant comprising or consisting of SEQ ID NO: 64.

[0169] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 189-191, 203-205 and / or VH and VL with SEQ ID NOs: 206-207, and the mutant comprising or consisting of SEQ ID NO: 64.

[0170] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191-193, 203-205 and / or VH and VL with SEQ ID NOs: 207-208, and the mutant comprising or consisting of SEQ ID NO: 64.

[0171] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 194-195, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 209, and the mutant comprising or consisting of SEQ ID NO: 64.

[0172] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 196-197 and / or VH and VL with SEQ ID NOs: 207 and 210, and the mutant comprising or consisting of SEQ ID NO: 64.

[0173] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 198-199, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 211, and the mutant comprising or consisting of SEQ ID NO: 64.

[0174] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 200-201, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 212, and the mutant comprising or consisting of SEQ ID NO: 64.

[0175] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 198, 202-205 and / or VH and VL with SEQ ID NOs: 207 and 213, and the mutant comprising or consisting of SEQ ID NO: 64.

[0176] The mutant IL-2 may comprise at least one non-endogenous transglutaminase motif inserted by insertion of at least one non-endogenous transglutaminase motif of sequence SEQ ID NO: 47. Said motif may be inserted at the C-terminus of the sequence with SEQ ID NO: 1 or the C-terminus of the sequence with SEQ ID NO: 50, preferably at the C-terminus of the sequence with SEQ ID NO: 50.

[0177] In one embodiment, the fusion protein as described herein comprises at least one antibody or an antigen-binding fragment thereof as described herein, and at least one mutant IL-2 comprising at least one non-endogenous transglutaminase motif of sequence SEQ ID NO: 47, preferably inserted at the C-terminus of the sequence with SEQ ID NO: 1 or the C-terminus of the sequence with SEQ ID NO: 50, more preferably at the C-terminus of the sequence with SEQ ID NO: 50.

[0178] In one embodiment, the mutant IL-2 comprises or consists of SEQ ID NO: 70. Thus, in one embodiment, the fusion protein as described herein comprises at least one antibody or the antigen-binding fragments thereof as described herein, and the mutant comprising or consisting of SEQ ID NO: 70.

[0179] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 189-191, 203-205 and / or VH and VL with SEQ ID NOs: 206-207, and the mutant comprising or consisting of SEQ ID NO: 70.

[0180] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191-193, 203-205 and / or VH and VL with SEQ ID NOs: 207-208, and the mutant comprising or consisting of SEQ ID NO: 70.

[0181] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 194-195, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 209, and the mutant comprising or consisting of SEQ ID NO: 70.

[0182] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 196-197 and / or VH and VL with SEQ ID NOs: 207 and 210, and the mutant comprising or consisting of SEQ ID NO: 70.

[0183] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 198-199, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 211, and the mutant comprising or consisting of SEQ ID NO: 70.

[0184] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 200-201, 203-205 and / or VH and VL with SEQ ID NOs: 207 and 212, and the mutant comprising or consisting of SEQ ID NO: 70.

[0185] In one embodiment, the fusion protein as described herein comprises an antibody or the antigen-binding fragments thereof having CDRs with SEQ ID NOs: 191, 198, 202-205 and / or VH and VL with SEQ ID NOs: 207 and 213, and the mutant comprising or consisting of SEQ ID NO: 70.

[0186] The fusion protein as described herein may further comprise at least one masking moiety, wherein the at least one masking moiety is linked to a Q residue of the at least one non-endogenous transglutaminase motif of the mutant IL-2 as defined hereinabove.

[0187] When the mutant IL-2 comprises two or more non-endogenous transglutaminase motifs, the fusion protein may comprise at least two masking moieties, wherein the at least two masking moieties are linked to a Q residue of each non-endogenous transglutaminase motif of the mutant IL-2 as defined hereinabove.

[0188] When the fusion protein comprises at least two masking moieties, the at least two masking moieties may be different. When the fusion protein comprises at least two masking moieties, the at least two masking moieties may be similar.

[0189] As used herein, the masking moiety refers to a moiety that, when linked to a Q residue of the non-endogenous transglutaminase motif of the mutant IL-2, prevents or reduces the signaling of the mutant IL-2, or the binding or affinity of the mutant IL-2 to its receptor.

[0190] Methods for measuring the IL-2 signaling are well known in the art and comprise, for example, the measurement of the induction of IL-2 receptor signaling (e.g., by detection of phosphorylated STAT5a), the measurement of the induction of T cell proliferation (e.g., by detection of Ki-67 using in particular CellTrace™ Cell Proliferation Kits, by direct assessment of T cell proliferation in the presence of IL-2, in T cell activation assay experiments (comprising, for example, the activation of cells with CD3 and CD28 in the presence of IL-2), or using cell lines that depend on IL-2 to proliferate, such as, for example CTLL2 cell line) and / or the measurement of an up-regulation of expression of activation markers (such as e.g., CD25, CD69, cytotoxic molecules, such as, for example, granzyme B, and the like).

[0191] Affinity of a protein is commonly expressed as an equilibrium dissociation constant (KD). Thus, the binding kinetics, such as the KD, of the mutant IL-2 to its receptor may be determined by surface plasmon resonance (SPR, e.g., using BIAcore®).

[0192] Other methods for measuring binding or affinity of proteins are well known in the art, and include, for example, cell-binding assays, such as competitive binding assays.

[0193] The mutant IL-2 comprised in the fusion protein as defined hereinabove may have a reduced binding or affinity for its receptor as compared to the mutant IL-2 that isfree (i. e., not associated with a masking moiety). In particular, the mutant IL-2 comprised in the fusion protein as defined hereinabove may have a reduced binding or affinity for its receptor by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more as compared to the mutant IL-2 that is free (i.e., not associated with a masking moiety).

[0194] The mutant IL-2 comprised in the fusion protein as defined hereinabove may have a reduced binding or affinity for the IL-2R alpha, beta and / or gamma chain as compared to the mutant IL-2 that is free (i.e., not associated with a masking moiety). In particular, the mutant IL-2 comprised in the fusion protein as defined hereinabove may have a reduced binding or affinity for the IL-2R alpha, beta and / or gamma chain by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more as compared to the mutant IL-2 that is free (i.e., not associated with a masking moiety).

[0195] The mutant IL-2 comprised in the fusion protein as defined hereinabove may have a reduced binding or affinity for the low-affinity, intermediate-affinity, and / or high-affinity IL-2 receptor as compared to the mutant IL-2 that is free (i.e., not associated with a masking moiety). In particular, the mutant IL-2 comprised in the fusion protein as defined hereinabove may have a reduced binding or affinity for the low-affinity, intermediate-affinity, and / or high-affinity IL-2 receptor by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more as compared to the mutant IL-2 that is free (i.e., not associated with a masking moiety).

[0196] The mutant IL-2 as described herein may exhibit reduced or eliminated affinity for CD25, coupled with modulated affinity for CD122, in which the modulated affinity for CD122 is achieved via transglutaminase-mediated conjugation at one or more single site(s) newly introduced to the IL-2 structure via mutagenesis. The mutant IL-2 may be conjugated to a moiety, such as PEG via a linker which is, or is not, selectively cleaved in diseased tissue. Without willing to be bound to any theory, it is hypothesized that this form of mutant IL-2 may exhibit reduced ability to stimulate T regulatory (Treg) cells (which are CD25-high) while effectively stimulate T effector (Teff) cells (CD25-low). This is a potentially beneficial phenotype in a cancer setting where one would seek to stimulate Teff cells over Treg, thereby stimulating anti-cancer therapeutic activity.

[0197] Alternatively, the mutant IL-2 as described herein may exhibit native (high) affinity for CD25, coupled with modulated affinity for CD122, in which the modulated affinity for CD122 is achieved via transglutaminase-mediated conjugation at one mor more single site(s) newly introduced to the IL-2 structure via mutagenesis. The mutant IL-2 may be conjugated to a moiety, such as PEG via a linker which is, or is not, selectively cleaved in diseased tissue. Without willing to be bound to any theory, it is hypothesized that the mutant IL-2 may exhibit enhanced ability to stimulate Treg cells (which are CD25-high) and minimized stimulation of Teff cells (CD25-low). This is a potentially beneficial phenotype in an inflammatory disease setting where one would seek to stimulate Treg cells over Teff, thereby stimulating anti-inflammatory therapeutic activity.

[0198] As disclosed herein, the at least one masking moiety may be any type of molecule. In particular, the at least one masking moiety may be selected from the group comprising or consisting of glycans, lipids, proteins, peptides, small organic molecules and polymers.

[0199] Thus, in one embodiment, the fusion protein further comprises at least one masking moiety selected from the group consisting of glycans, lipids, proteins, peptides, small organic molecules and polymers.

[0200] The at least one masking moiety may be a glycan.

[0201] The at least one masking moiety may be a polymer, preferably a water-soluble polymer.

[0202] In particular, the water-soluble polymer may be selected from the group comprising or consisting of polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), and combinations thereof.

[0203] Preferably, the water-soluble polymer may be a polyethylene glycol (PEG).

[0204] The at least one masking moiety may be a linear PEG. As used herein, a linear PEG refers to a PEG composed of one linear chain.

[0205] The at least one masking moiety may be a linear PEG up to about 20kDa, preferably of about 10 kDa.

[0206] The at least one masking moiety may be a branched PEG. As used herein, a branched PEG refers to a PEG composed of at least two linear PEGs that are linked together. An example of a branched PEG is a Y-shaped PEGs, such as, for example, a Y-shaped PEG with two PEGs of about 20 kDa.

[0207] The at least one masking moiety may be a PEG having a weight comprised from about 5 to about 50 kDa, preferably from about 10 to about 40 kDa.

[0208] It will be understood that, when the fusion protein comprises at least two masking moieties, the at least two masking moieties may be PEG that are similar or different.

[0209] The fusion protein as described herein may comprise at least one mutant IL-2, at least one masking moiety and at least one antibody or antigen-binding fragment thereof as described herein, wherein the at least one masking moiety is linked to the at least one mutant IL-2 with a linker and wherein the at least one mutant IL-2 is linked to the at least one antibody or antigen-binding fragment thereof with another linker.

[0210] The linkers linking (i) the at least one masking moiety to the at least one mutant IL-2 and (ii) the at least one mutant IL-2 to the at least one antibody or antigen-binding fragment thereof may be similar. Alternatively, the linkers linking (i) the at least one masking moiety to the at least one mutant IL-2 and (ii) the at least one mutant IL-2 to the at least one antibody or antigen-binding fragment thereof may be different.

[0211] The fusion protein may comprise at least one mutant IL-2, at least two masking moieties and at least one antibody or antigen-binding fragment thereof as described herein, wherein the at least two masking moieties are linked to the at least one mutant IL-2 with a linker.

[0212] The linkers linking the at least two masking moieties to the at least one mutant IL-2 may be similar. The linkers linking the at least two masking moieties to the at least one mutant IL-2 may be different.

[0213] The linker linking the at least one mutant IL-2 to the at least one antibody or antigen-binding fragment thereof as described herein may be a cleavable linker. Alternatively, the linker linking the at least one mutant IL-2 to the at least one antibody or antigen-binding fragment thereof as described herein may be an uncleavable linker.

[0214] It will be understood that the nature of the linker (i.e., cleavable or uncleavable) will depend on the therapeutic applications, since, depending on the conditions, it may be desirable to have either a releasable IL-2 or an IL-2 irreversibly linked to the antibody or antigen-binding fragment thereof as described herein.

[0215] The linker linking the at least one masking moiety to the at least one mutant IL-2 may be a cleavable linker. Alternatively, the linker linking the at least one masking moiety to the at least one mutant IL-2 may be an uncleavable linker.

[0216] It will be understood that the nature of the linker (i.e., cleavable or uncleavable) will depend on the therapeutic applications, since, depending on the conditions, it may be desirable to have either a releasable masking moiety or an IL-2 irreversibly linked to the masking moiety.

[0217] The fusion protein may comprise at least one or at least two masking moiety (ies), at least one mutant IL-2 and at least one antibody or antigen-binding fragment thereof as described herein, wherein said elements are linked by cleavable linkers.

[0218] The fusion protein may comprise at least one or at least two masking moiety (ies), at least one mutant IL-2 and at least one antibody or antigen-binding fragment thereof as described herein, wherein said elements are linked by uncleavable linkers.

[0219] The fusion protein may comprise at least one or at least two masking moiety (ies), at least one mutant IL-2 and at least one an antibody or antigen-binding fragment thereof as described herein, wherein the at least one or at least two masking moiety(ies) are linked to the at least one mutant IL-2 with an uncleavable linker, and wherein the at least oneantibody or antigen-binding fragment thereof as described herein is linked to the at least one mutant IL-2 with a cleavable linker.

[0220] The fusion protein may comprise at least one or at least two masking moiety(ies), at least one mutant IL-2 and at least one antibody or antigen-binding fragment thereof as described herein, wherein the at least one or at least two masking moiety(ies) are linked to the at least one mutant IL-2 with a cleavable linker, and wherein the at least one antibody or antigen-binding fragment thereof as described herein is linked to the at least one mutant IL-2 with an uncleavable linker.

[0221] The uncleavable linker may be a peptide having a length ranging from 2 to 10 amino acids.

[0222] For example, a glycine-serine doublet provides a particularly suitable hinge domain (GS linker). The hinge domain may be a Gly / Ser linker. Examples of Gly / Ser linkers include, but are not limited to, GS linkers, G2S linkers, G3S linkers, G4S linkers.

[0223] Examples of G2S linkers include, but are not limited to, GGS.

[0224] G3S linkers comprise the amino acid sequence (Gly-Gly-Gly-Ser)nalso referred to as (GGGS)n (SEQ ID NO: 100), where n is a positive integer equal to or greater than 1 (such as, example, n=l, n=2, n=3. n=4, n=5, n=6, n=7, n=8, n=9 or n=10).

[0225] G4S linkers comprise the amino acid sequence (Gly-Gly-Gly-Gly-Ser)nalso referred to as (GGGGS)n(SEQ ID NO: 101), where n is a positive integer equal to or greater than 1 (such as, example, n=l, n=2, n=3. n=4, n=5, n=6, n=7, n=8, n=9 or n=10).

[0226] As another example, linkers A4T (SEQ ID NO: 129) may be also used in the context of the present invention.

[0227] The cleavable linker may be a cleavable peptide having a length ranging from 2 to 10 amino acids.

[0228] The cleavable linker may be cleavable by enzymes, photo-irradiation, pH, or chemical agents.

[0229] The cleavable linker may be cleavable by enzymes, such as, for example, linkers cleavable by proteases, linkers cleavable by beta glucuronidase or peptide linkers.

[0230] The cleavable linker may be cleavable by pH. Examples of linker cleavable by pH include, without limitation, hydrazine linker, maleic acid amide linker or cis-aconityl linker.

[0231] Another object of the invention is an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof or a fusion protein as described herein.

[0232] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as for example plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0233] Thus, another object of the present invention is an expression vector comprising a nucleic acid encoding an antibody or an antigen-binding fragment thereof or a fusion protein as described herein.

[0234] Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSGl beta d2-4 and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, Gpenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found in the art.

[0235] Another object of the invention is an isolated host cell comprising said vector. Said host cell may be used for the recombinant production of an antibody or an antigenbinding fragment thereof or a fusion protein as described herein.

[0236] Host cells may be prokaryote, yeast, or eukaryote cells, preferably mammalian cells, such as, for example: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growthin suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581; ATCC CRL 8287) or NSO (HPA culture collections no. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM’s PERC-6 cell line. Expression vectors suitable for use in each of these host cells are also generally known in the art.

[0237] It should be noted that, with regards to animal and human cells, the term “host cell” generally refers to a cell of a cultured cell line. Animals and human beings into whom a vector as described above has been introduced are explicitly excluded from the definition of a “host cell”.

[0238] Another object of the present invention is a composition comprising, consisting essentially of or consisting of at least one antibody or antigen-binding fragment thereof, at least one fusion protein, at least one nucleic acid, or at least one expression vector as defined herein.

[0239] Another object of the present invention is a pharmaceutical composition comprising, consisting essentially of or consisting of at least one antibody or antigenbinding fragment thereof, at least one fusion protein, at least one nucleic acid, or at least one expression vector as defined herein, and at least one pharmaceutically acceptable excipient.

[0240] Examples of pharmaceutically acceptable excipients that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0241] The pharmaceutical compositions according to the present invention may comprise vehicles which are pharmaceutically acceptable for a formulation capable of being injected to a subject. 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.

[0242] Another object of the present invention is an antibody or antigen-binding fragment thereof, a fusion protein, a nucleic acid, an expression vector as defined herein for use as a medicament.

[0243] Another object of the present invention is a medicament comprising, consisting essentially of or consisting of at least one antibody or antigen-binding fragment thereof, at least one fusion protein, at least one nucleic acid, or at least one expression vector as defined herein.

[0244] For use in administration to a subject, the composition, pharmaceutical composition or medicament will be formulated for administration to the subject.

[0245] The composition, pharmaceutical composition or medicament as described herein may be administered (or to be administered) parenterally, by inhalation spray, rectally, nasally, or via an implanted reservoir.

[0246] The composition, pharmaceutical composition or medicament may be administered (or to be administered) by injection, including, without limitation, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection, or infusion techniques.

[0247] Examples of forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms, and the like.

[0248] Sterile injectable forms of the compositions, pharmaceutical compositions or medicaments of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0249] The antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament as described herein may be to be administered to the subject in need thereof in a therapeutically effective amount.

[0250] It will be however understood that the total daily usage of the antibody or antigenbinding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention will bedecided 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 disease being treated and the severity of the disease; activity of the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector employed; the duration of the treatment; drugs used in combination or coincidental with the specific antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, or expression vector employed; and like factors well known in the medical arts. For example, it is well 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. The total dose required for each treatment may be administered by multiple doses or in a single dose.

[0251] The present invention also relates to at least one antibody or antigen-binding fragment thereof, fusion protein, nucleic acid or expression vector as described herein for treating (or for use in treating) diseases, disorders or symptoms in a subject in need thereof. The present invention also relates to an antibody or antigen-binding fragment thereof, fusion protein, nucleic acid or expression vector as described herein for treating (or for use in treating) diseases, disorders or symptoms in a subject in need thereof.

[0252] Another object of the present invention is the use of at least one antibody or antigen-binding fragment thereof, at least one fusion protein, at least one nucleic acid, or at least one expression vector as defined herein in the manufacture of a medicament for treating a disease, disorder or symptoms in a subject in need thereof.

[0253] Another object of the present invention is a method for treating diseases, disorders or symptoms in a subject in need thereof, comprising administering to said subject an antibody or antigen-binding fragment thereof, a fusion protein, a nucleic acid, an expression vector, a composition, a pharmaceutical composition or a medicament as described herein, preferably in a therapeutically effective amount.

[0254] The present invention also relates to at least one fusion protein, nucleic acid encoding said fusion protein or expression vector comprising said nucleic acid as described herein for use in promoting Treg cell expansion in a subject in need thereof, thereby treating diseases, disorders or symptoms in the subject.

[0255] The present invention also relates to methods for treating diseases, disorders or symptoms or methods for promoting Treg cell expansion in a subject in need thereof, comprising administering to the subject the at least one fusion protein, nucleic acid encoding said fusion protein or expression vector comprising said nucleic acid as described herein.

[0256] Examples of diseases that may be treated in the present invention, include, but are not limited to, cancers, and inflammatory diseases.

[0257] It may be considered that a subject is successfully “treated” for a cancer or an inflammatory disease if, after receiving a therapeutic amount of at least one antibody or antigen-binding fragment thereof, fusion protein, nucleic acid or expression vector as described herein, the subject shows at least one of the following: reduction in the number of cancer cells (or tumor size) or pathogenic cells; reduction in the percent of total cells that are cancerous or pathogenic; relief to some extent of one or more of the symptoms associated with the cancer or the inflammatory disease to be treated; reduced morbidity and mortality; and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0258] In one embodiment, the disease, disorder or symptom to be treated is cancer.

[0259] Examples of cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, endometrial, pancreas or uterus.

[0260] In addition, the cancer may be selected in the following non-limiting list: malignant neoplasm; undifferentiated carcinoma; 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; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma associated with familial polyposis coli; solid carcinoma; malignant carcinoid tumor; 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; non-encapsulating 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 of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant roblastoma; Sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malign melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignantstruma ovarii; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma - small lymphocytic; malignant diffuse large cell lymphoma; malignant follicular lymphoma; 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.

[0261] In one embodiment, the disease, disorder or symptom to be treated is an inflammatory disease.

[0262] Examples of inflammatory diseases include, but are not limited to, arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, arthritis uratica, gout, chronic polyarthritis, periarthritis humeroscapularis, cervical arthritis, lumbosacral arthritis, enteropathic arthritis and ankylosing spondylitis, asthma, dermatitis, psoriasis, scleroderma, polymyositis, dermatomyositis, juvenila dermatomyositis, primary biliary cirrhosis, fibrosis, cystic fibrosis, pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, dediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic fibrosis, Keloids, scleroderma, arthrofibrosis, post transplantation late and chronic solid organ rejection,multiple sclerosis, systemic lupus erythematosus, lupus nephritis, pemphigus, Pemphigus vulgaris, Pemphigus herpetiformis, Pemphigus vegetans, IgA pemphigus, Pemphigus erythematosus, bullous pemphigoid, Pemphigoid gestationis, Mucous membrane dermatosis, Pemphigoid nodularis, Linear IgA bullous dermatosis, Bullous lichen planus, Epidermolysis bullosa acquisita, autoimmune diabetes, diabetic retinopathy, diabetic nephropathy, diabetic vasculopathy, ocular inflammation, uveitis, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), glomerulonephritis, Graves disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina, small artery disease, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, systemic sclerosis, antiphospholipid syndrome, Sjoegren's syndrome, autoimmune hemolytic anemia, colitis, Crohn's Disease, ulcerative colitis, Inflammatory Bowel Disease (IBD), embolism, pulmonary embolism, arterial embolism, venous embolism, allergic inflammation, cardiovascular disease, graft- related diseases, graft versus host disease (GVHD), disorders associated with graft transplantation rejection, chronic rejection, and tissue or cell allografts or xenografts, autoimmune diseases, degeneration after trauma, stroke, transplant rejection, allergic conditions and hypersensitivity, e.g., allergic rhinitis, allergic eczema and the like, skin diseases, dermal inflammatory disorders, or any combination thereof.

[0263] The antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament as described herein may be used alone. In other words, the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament as described herein may be used as a monotherapy.

[0264] Alternatively, the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament as described herein may be used in combination with at least one further therapeutic agent.

[0265] The antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament as describedherein may be administered simultaneously or sequentially with the at least one further therapeutic agent.

[0266] For simultaneous administration, the at least one further therapeutic agent and the antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament as described herein may be administered as one composition or as separate compositions, as appropriate.

[0267] Examples of additional therapeutic agents include, but are not limited to, chemotherapeutic agents, targeted cancer therapy, radiotherapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti-angiogenic agents, cell cycle control / apoptosis regulating agents, hormonal regulating agents, tyrosine kinase inhibitors (TIKs), vaccines, immune checkpoint inhibitors, CAR-T and CAR-NK cell therapies, CAR macrophage therapy, tumor-infiltrating lymphocytes (TIL) therapies, immune engaging biologies, steroids, JAK inhibitors, and other immunosuppressive and / or anti-inflammatory drugs including corticoids, such as, for example, glucocorticoids.

[0268] The at least one further therapeutic agent may be a therapeutic agent useful for treating the specific disease, disorder or condition to be treated in the present invention.

[0269] For example, for treating cancer, the at least one further therapeutic agent may be selected from the group comprising, but not limited to, chemotherapeutic agents, targeted cancer therapy, radiotherapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti-angiogenic agents, cell cycle control / apoptosis regulating agents, hormonal regulating agents, tyrosine kinase inhibitors (TIKs), vaccines, immune checkpoint inhibitors, CAR-T and CAR-NK cell therapies, CAR macrophage therapy, tumor-infiltrating lymphocytes (TIL) therapies, immune engaging biologies, and other immunosuppressive and / or anti-inflammatory drugs selected from corticoids, such as, for example, glucocorticoids.

[0270] Thus, in the context of cancer, the at least one further therapeutic agent may be selected from the group consisting of the agents described hereinabove.

[0271] In particular, the antibody or antigen-binding thereof or the fusion protein as described herein can be combined with immunotherapeutic agents (i.e., an immunotherapy), such as blocking anti-PD-1 antibodies, since the antibody or antigenbinding fragment thereof as described herein is a non-blocking anti-PD-1 antibody or fragment.

[0272] Examples of blocking anti-PD-1 antibodies include, without limitation, nivolumab, pembrolizumab, dostarlimab, sintilimab, tislelizumab, cemiplimab, cetrelimab, sasanlimab.

[0273] Thus, the antibody or antigen-binding thereof or the fusion protein as described herein may be combined with blocking anti-PD-1 antibodies, preferably, nivolumab or pembrolizumab.

[0274] Thus, the antibody or antigen-binding thereof or the fusion protein as described herein may be administered to a subject that has received, is receiving or will receive an immunotherapy, preferably a blocking anti-PD-1 antibody, more preferably, nivolumab or pembrolizumab.

[0275] For treating inflammatory diseases, the at least one further therapeutic agent may be selected from the group comprising, but not limited to, anti-inflammatory drugs, steroids, and JAK inhibitors.

[0276] Thus, in the context of inflammatory diseases, the at least one further therapeutic agent may be selected from the group consisting of the agents described hereinabove.

[0277] The present invention also relates to a method as described herein, comprising administering to said subject i) an antibody or antigen-binding fragment thereof, a fusion protein, a nucleic acid, an expression vector, a composition, a pharmaceutical composition or a medicament as described herein, and ii) a blocking anti-PD-1 antibody, preferably in a therapeutically effective amount.

[0278] The present invention also relates to a combination therapy comprising i) an antibody or antigen-binding fragment thereof, a fusion protein, a nucleic acid, an expression vector, a composition, a pharmaceutical composition or a medicament asdescribed herein, and ii) a blocking anti-PD-1 antibody, for use in the treatment of a disease as described herein.SEQUENCE LISTINGTable 1 - Anti-PD-1 antibodies - CDRsVH-CDR1 VH-CDR2 VH-CDR3Consensus X₄SX₅EFGX₆X₇GX₁TX₂X₃GLY(SEQ ID NO: 190)(SEQ ID NO: 189)whereinwhereinX₄ is V or IX₁ is G or Y,X₅ is A or VX2 is F or LX₆ is S or GX3 is T or SX7 is T or AClone 22 GGTFTGLY VSAEFGST(SEQ ID NO: 192) (SEQ ID NO: 193) DEGILDDSSGYYY Clone 23 GGTLTGLY VSVEFGGA KINAFDI(SEQ ID NO: 194) (SEQ ID NO: 195) (SEQ ID NO: 191) Clone 24 GYTLTGLY ISAEFGGA(SEQ ID NO: 196) (SEQ ID NO: 197)Clone 26 GYTLSGLY VSVEFGST(SEQ ID NO: 198) (SEQ ID NO: 199)Clone 27 GGTLSGLY ISVEFGGA(SEQ ID NO: 200) (SEQ ID NO: 201)Clone 28 GYTLSGLY ISAEFGST(SEQ ID NO: 198) (SEQ ID NO: 202)Clone VL-CDR1 VL-CDR2 VL-CDR3 ConsensusClone 22Clone 23 TGTSSDVGGYNYEVSNRPS SSYTSSSTYVClone 24 VS(SEQ ID NO: 204) (SEQ ID NO: 205) Clone 26 (SEQ ID NO: 203)Clone 27Clone 28Table 2 - Anti-PD-1 antibodies - VH & VLVHConsensus QVQLVQSGAEVKKPGASVKVSCKASGX₁TX₂X₃GLYMHWVRQAPGQG LEWMGIX₄SX₅EFGX₆X₇SYAQKFQGRVTMTRDTSTSTVYMELSSLRSED T AVYYC ARDEGILDD S S GYYYKIN AFDI WGQGTMVTV S S(SEQ ID NO: 206)wherein X₁ is G or Y, X2 is F or L, X3 is T or S, X₄ is V or I, X₅ is A or V, X₆ is S or G, and X7 is T or AClone 22 QVQLVQSGAEVKKPGASVKVSCKASGGTFTGLYMHWVRQAPGQGLE WMGIVSAEFGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARDEGILDDSSGYYYKINAFDIWGQGTMVTVSS(SEQ ID NO: 208)Clone 23 QVQLVQSGAEVKKPGASVKVSCKASGGTLTGLYMHWVRQAPGQGLE WMGIVSVEFGGASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV YYCARDEGILDDSSGYYYKINAFDIWGQGTMVTVSS(SEQ ID NO: 209)Clone 24 QVQLVQSGAEVKKPGASVKVSCKASGYTLTGLYMHWVRQAPGQGLE WMGIISAEFGGASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARDEGILDDSSGYYYKINAFDIWGQGTMVTVSS(SEQ ID NO: 210)Clone 26 QVQLVQSGAEVKKPGASVKVSCKASGYTLSGLYMHWVRQAPGQGLE WMGIVSVEFGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARDEGILDDSSGYYYKINAFDIWGQGTMVTVSS(SEQ ID NO: 211)Clone 27 QVQLVQSGAEVKKPGASVKVSCKASGGTLSGLYMHWVRQAPGQGLE WMGIISVEFGGASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARDEGILDDSSGYYYKINAFDIWGQGTMVTVSS(SEQ ID NO: 212)Clone 28 QVQLVQSGAEVKKPGASVKVSCKASGYTLSGLYMHWVRQAPGQGLE WMGIISAEFGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARDEGILDDSSGYYYKINAFDIWGQGTMVTVSS(SEQ ID NO: 213)VLConsensusClone 22QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLClone 23MIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTClone 24YVFGTGTKVTVLClone 26 (SEQ ID NO: 207)Clone 27Clone 28Table 3 - Tranglutaminase motif of IL-2 mutantsMotif SEQ ID NO:LEHLLQD 3LEHLQSD 4LLQLLLD 5MILQGIN 6MLLQGIN 7NNYQNP 8NNHQNP 9LLQFKF 10LLQAKF 11MLTQRF 12LLQC 13KNFQLRPRD 14KLFQLRPRD 15KLLQLRPRD 16GSQTT 17GSQST 18VIQ - LQSPGAPTSSSTKKTQ 40TQGAPTSSSTKKTQ 41LQSPTSSSTKKTQ 42TQGASSSTKKTQ 43TQGASSTKKTQ 44TQGASTKKTQ 45GGTQGA 46GGLQSP 47GLQSP 48LQSP 49NNYRQP 102NRYRQP 103MLTQGF 104LAQELK 105LAQALK 106KATEQK 107EYALQ 108DYALQ 109Table 4 - IL-2 mutantsName of Sequence SEQ ID mutant NO: Wild type APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 1 IL-2 TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFCQSIISTLTTruncated LQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM 50 IL-2 PKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFSQSIISTLT2-202 APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKL 19 TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-203 APTSSSTKKTQLQLEHLQSDLQMILNGINNYKNPKL 20 TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-204 APTSSSTKKTQLQLLQLLLDLQMILNGINNYKNPKL 21 TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-205 APTS S STKKTQLQLEHLLLDLQMILQGINNYKNPKL 22TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-206 APTSSSTKKTQLQLEHLLLDLQMLLQGINNYKNPK 23LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL NLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT-207 APTSSSTKKTQLQLEHLLLDLQMILNGINNYQNPKL 24TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-208 APTSSSTKKTQLQLEHLLLDLQMILNGINNHQNPKL 25TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-105 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 26TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT-106 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 27TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT-107 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 28TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT-209 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 29TRMLTFKFYMPKKATELKLLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-210 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 30TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-112 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 31TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKLFQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-114 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 32TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLFQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-113 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 33TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKLLQLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT-115 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 34 TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLLQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-211 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 35TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLQLKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT-212 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 36TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVILLQLKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-213 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 37TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSQTTFMCEY ADETATIVEFLNRWITFSQSIISTLT-116 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 38TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSQSTFMCEY ADETATIVEFLNRWITFSQSIISTLT-214 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 39TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSVIQTLT-120 LQSPGAPTSSSTKKTQLQLEHLLLDLQMILNGINNY 51KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPL EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF MCEY ADETATIVEFLNRWITFSQSIISTLT-215 TQGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 52PKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMC EY ADETATIVEFLNRWITFSQSIISTLT-121 LQSPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP 53KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT-216 TQGASSSTKKTQLQLEHLLLDLQMILNGINNYKNP 54KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV LNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT-122 TQGASSTKKTQLQLEHLLLDLQMILNGINNYKNPK 55LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT-217 TQGASTKKTQLQLEHLLLDLQMILNGINNYKNPKL 56TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-218 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 57TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGGTQGA-123 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 58TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLTGGLQSP-124 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 59TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLTGLQSP-125 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 60TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLTLQSP-126 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 61TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLTQGA-130 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 62TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-131 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 63TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-132 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 64TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLFQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-133 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 65TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLLQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-134 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 66TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSQSTFMCEYA DETATIVEFLNRWITFSQSIISTLT-135 LQSPGAPTSSSTKKTQLQLEHLLLDLQMILNGINNY 67KNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPL EEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT-136 LQSPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP 68 KLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEV LNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT-137 TQGASSTKKTQLQLEHLLLDLQMILNGINNYKNPK 69LTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVL NLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT-138 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 70TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGGLQSP-140 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 71TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTLQSP-141 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 72TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTQGA-142 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 73TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLFQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-143 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 74TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLLQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT-144 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 75TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSQSTFMCEYA DETATIVEFLNRWITFSQSIISTLT-145 LQSPGAPTSSSTKKTQLQLEHLLLDLQMILNGINNY 76KNPKLTRLLQAKFYMPKKATELKHLQCLEEELKPL EEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTF MCEY ADETATIVEFLNRWITFSQSIISTLT-146 LQSPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP 77KLTRLLQAKFYMPKKATELKHLQCLEEELKPLEEV LNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT-147 TQGASSTKKTQLQLEHLLLDLQMILNGINNYKNPK 78LTRLLQAKFYMPKKATELKHLQCLEEELKPLEEVL NLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT-150 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 79TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTLQSP2-151 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 80TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTQGA2-152 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 81TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLFQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-153 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 82TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLLQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-154 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 83TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSQSTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-155 LQSPGAPTSSSTKKTQLQLEHLLLDLQMILNGINNY 84KNPKLTRMLTQRFYMPKKATELKHLQCLEEELKPL EEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTF MCEYADETATIVEFLNRWITFSQSIISTLT2-156 LQSPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP 85KLTRMLTQRFYMPKKATELKHLQCLEEELKPLEEV LNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT2-157 TQGASSTKKTQLQLEHLLLDLQMILNGINNYKNPK 86LTRMLTQRFYMPKKATELKHLQCLEEELKPLEEVL NLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-158 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 87TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGGLQSP2-159 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 88TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGLQSP2-160 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 89TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTLQSP2-161 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 90TRMLTQRFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTQGA2-162 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 91 TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLFQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-163 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 92TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLLQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-164 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 93TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSQSTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-166 LQSPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP 94KLTRLLQFKFYMPKKATELKHLQCLEEELKPLEEV LNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT2-167 TQGASSTKKTQLQLEHLLLDLQMILNGINNYKNPK 95LTRLLQFKFYMPKKATELKHLQCLEEELKPLEEVL NLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-170 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 96TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTLQSP2-171 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 97TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTQGA2-102 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 99TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-103 APTSSSTKKTQLQLEHLLLDLQMILNGINNYRQPKL 110TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-104 APTSSSTKKTQLQLEHLLLDLQMILNGINRYRQPKL 111TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-108 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 112TRMLTQGFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-109 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 113TRMLTFKFYMPKLAQELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-110 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 114TRMLTFKFYMPKLAQALKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-111 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 115TRMLTFKFYMPKKATEQKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-117 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 116TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ALQTATIVEFLNRWITFSQSIISTLT2-118 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 117TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCDY ALQTATIVEFLNRWITFSQSIISTLT2-127 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 119TRLLQAKFYMPKLAQALKHLQCLEEELKPLEEVLN LAESKLLQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-128 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 120TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKLLQLRPRDLISNINVIVLELKGSETTFMCDYA LQTATIVEFLNRWITFSQSIISTLT2-129 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 121TRLLQFKFYMPKLAQALKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-139 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 122TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGLQSP2-148 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 123TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGGLQSP2-149 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 124TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGLQSP2-165 LQSPGAPTSSSTKKTQLQLEHLLLDLQMILNGINNY 125KNPKLTRLLQFKFYMPKKATELKHLQCLEEELKPL EEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-168 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 126 TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGGLQSP2-169 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 127TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGLQSP2-572 APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKL 130TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-573 APTSSSTKKTQLQLEHLQSDLQMILNGINNYKNPKL 131TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-574 APTSSSTKKTQLQLLQLLLDLQMILNGINNYKNPKL 132TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-575 APTS S STKKTQLQLEHLLLDLQMILQGINNYKNPKL 133TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-576 APTSSSTKKTQLQLEHLLLDLQMLLQGINNYKNPK 134LTRMLTFKAYMPKKATELKHLQCLEEELKPLEEVL NLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-577 APTSSSTKKTQLQLEHLLLDLQMILNGINNYQNPKL 135TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-578 APTSSSTKKTQLQLEHLLLDLQMILNGINNHQNPKL 136TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-579 APTSSSTKKTQLQLEHLLLDLQMILNGINNYRQPKL 137TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-580 APTSSSTKKTQLQLEHLLLDLQMILNGINRYRQPKL 138TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-581 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 139TRLLQAKFYMPKKATELKHLQCLEEELKPLEEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-582 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 140TRMLTAKFYMPKLAQELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-583 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 141TRMLTAKFYMPKLAQALKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-584 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 142TRMLTAKFYMPKKATEQKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-585 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 143TRMLTAKFYMPKKATELKLLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-222 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 144TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFQLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-586 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 145TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLQLKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-587 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 146TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVILLQLKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-588 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 147TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSQTTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-589 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 148TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA LQTATIVEFLNRWITFSQSIISTLT2-590 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 149TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCDY ALQTATIVEFLNRWITFSQSIISTLT2-591 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 150TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSVIQTLT2-592 TQGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 151 PKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEE VLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMC EY ADETATIVEFLNRWITFSQSIISTLT2-593 TQGASSSTKKTQLQLEHLLLDLQMILNGINNYKNP 152KLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEV LNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT2-594 TQGASTKKTQLQLEHLLLDLQMILNGINNYKNPKL 153TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-595 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 154TRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGGTQGA2-596 APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKL 155TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-597 APTSSSTKKTQLQLEHLQSDLQMILNGINNYKNPKL 156TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-598 APTSSSTKKTQLQLLQLLLDLQMILNGINNYKNPKL 157TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-599 APTS S STKKTQLQLEHLLLDLQMILQGINNYKNPKL 158TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-600 APTSSSTKKTQLQLEHLLLDLQMLLQGINNYKNPK 159LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT2-601 APTSSSTKKTQLQLEHLLLDLQMILNGINNYQNPKL 160TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-602 APTSSSTKKTQLQLEHLLLDLQMILNGINNHQNPKL 161TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-603 APTSSSTKKTQLQLEHLLLDLQMILNGINNYRQPKL 162TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-604 APTSSSTKKTQLQLEHLLLDLQMILNGINRYRQPKL 163TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-605 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 164TRLLQFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-606 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 165TRMLTQGFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-607 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 166TRMLTFKFYMPKLAQELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-608 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 167TRMLTFKFYMPKLAQALKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-609 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 168TRMLTFKFYMPKKATEQKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-610 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 169TRMLTFKFYMPKKATELKLLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-611 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 170TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFQLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-612 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 171TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLQLKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-613 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 172TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVILLQLKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-614 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 173TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSQTTFMCEYADETATIVEFLNRWITFSQSIISTLT2-615 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 174 TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSQSTFMCEYA DETATIVEFLNRWITFSQSIISTLT2-616 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 175TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA LQTATIVEFLNRWITFSQSIISTLT2-617 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 176TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCDY ALQTATIVEFLNRWITFSQSIISTLT2-618 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 177TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSVIQTLT2-619 LQSPGAPTSSSTKKTQLQLEHLLLDLQMILNGINNY 178KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPL EEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTF MCEY ADETATIVEFLNRWITFSQSIISTLT2-620 TQGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 179PKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFM CEY ADETATIVEFLNRWITFSQSIISTLT2-621 LQSPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP 180KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV LNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT2-622 TQGASSSTKKTQLQLEHLLLDLQMILNGINNYKNP 181KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT2-623 TQGASSTKKTQLQLEHLLLDLQMILNGINNYKNPK 182LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL NLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-624 TQGASTKKTQLQLEHLLLDLQMILNGINNYKNPKL 183TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT2-625 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 184TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLTGGTQGA2-626 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 185TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGGLQSP2-627 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 186TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTGLQSP2-628 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 187TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFSQSIISTLTLQSP2-629 APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKL 188TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAESKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTQGATable 5 – Fusion proteinsConstruct VH-CH1-ECD Sequencename2-229 QVQLVQSGAEVKKPGASVKVSCKASGGTFTGLYMHWVRQAPGQ GLEWMGIVSAEFGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYC ARDEGILDDS S GYYYKINAFDIWGQGTMVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLE HLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCL EEELKPLEEVLNLAESKNFQLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO: 214)2-230 QVQLVQSGAEVKKPGASVKVSCKASGYTLSGLYMHWVRQAPGQ (also GLEWMGIVSVEFGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS named EDTAVYYC ARDEGILDDS S GYYYKINAFDIWGQGTMVTVS S AST 230) KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLE HLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCL EEELKPLEEVLNLAESKNFQLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO: 215)2-231 QVQLVQSGAEVKKPGASVKVSCKASGGTLSGLYMHWVRQAPGQ GLEWMGIISVEFGGASYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYC ARDEGILDDS S GYYYKINAFDIWGQGTMVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAESKNFQLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO: 216)2-232 QVQLVQSGAEVKKPGASVKVSCKASGYTLSGLYMHWVRQAPGQ GLEWMGIISAEFGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYC ARDEGILDDS S GYYYKINAFDIWGQGTMVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLE HLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCL EEELKPLEEVLNLAESKNFQLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO: 217)2-233 QVQLVQSGAEVKKPGASVKVSCKASGGTLTGLYMHWVRQAPGQ GLEWMGIVSVEFGGASYAQKFQGRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARDEGILDDSSGYYYKINAFDIWGQGTMVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLE HLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCL EEELKPLEEVLNLAESKNFQLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO: 218)2-234 QVQLVQSGAEVKKPGASVKVSCKASGYTLTGLYMHWVRQAPGQ (also GLEWMGIISAEFGGASYAQKFQGRVTMTRDTSTSTVYMELSSLRS named EDTAVYYC ARDEGILDDS S GYYYKINAFDIWGQGTMVTVS S AST 234) KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLE HLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCL EEELKPLEEVLNLAESKNFQLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO: 219)VL-CL Sequence2-229 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKA 2-230 PKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCS 2-231 SYTS S STYVFGTGTKVTVLGQPKAAP S VTLFPP S SEELQ ANKATLV 2-232 CLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYL 2-233 SLTPEQWKSHRS YS CQVTHEGSTVEKTV APTEC S2-234 (SEQ ID NO: 220)BRIEF DESCRIPTION OF THE DRAWINGS

[0279] Figure 1 is a combination of histograms showing that the non-blocking anti-PD-1 antibodies of the present invention do not exhibit polyreactivity. Figure 1A: Antibodies were profiled by ELISA for binding to insulin as a measure of their poly reactivity. A polyreactivity score >20 indicates binding, 10-20 indicates low level binding and <10 indicates minimal binding (dotted lines). Human IgGl kappa and lambda isotypes were included as controls along with Bococizumab and Ustekinumab, used for experimental positive and negative controls, respectively. Figure 1B: Antibodies were profiled by ELISA for binding to double stranded DNA as a measure of their poly reactivity. Polyreactivity scores calculated as the ratio of the binding signal at 10 pg / ml to the average signal of the no antibody control signal. A polyreactivity score >20 indicates binding, 10-20 indicates low level binding and <10 indicates minimal binding (dotted lines). Human IgGl kappa and lambda isotypes were included as controls along with Bococizumab and Ustekinumab, used for experimental positive and negative controls, respectively.

[0280] Figure 2 is a graph showing the profiles of antibodies of the present invention for their self-aggregation propensity by AC-SINS. The AC-SINS ‘score’ calculated is the difference when compared to the results for PBS (PBS subtracted) and plotted as an average with the standard error of the mean (SEM). A shift of over 10 nm indicates a higher propensity to self-aggregate. AC-SINS scores for all antibodies tested are below the threshold for propensity to self-aggregate.

[0281] Figure 3 is a graph showing the binding of anti-PD- 1 IgG of the present invention (z.e. full antibodies, clones 22, 23, 24, 26, 27 and 28) at 200nM on PD1+ CHO cells and wildtype (WT) CHO cells. The data are presented here as mean fluorescence intensity (MFI). Lambda isotype (isotype) and secondary antibody alone (2ndalone) were included as negative controls along with Nivolumab (Nivo) and Pembrolizumab (Pembro), used for experimental positive controls. FB: Flow cytometry buffer assay control.

[0282] Figure 4 is a table showing the binding affinities of fusion proteins of the present invention comprising IL-2 mutant 2-222 fused to each Fab clone (2-229, 2-230, 2-231, 2-232, 2-233, 2-234) determined via single cycle kinetics. It shows that all six Fab have affinity for human PD-1 (hPD-1) in the range 6nM – 25nM.

[0283] Figure 5 is a combination of graphs showing epitope binning on Biacore with Pembrolizumab (Figure 5A) and Nivolumab (Figure 5B). Captured Pembrolizumab and Nivolumab bind human PD-1 with the expected increase in response units was observed. Subsequent injection of a fusion protein comprising IL-2 mutant 2-222 fused to Nivolumab Fab (2-222) does not result in binding to the captured PD-1 since the epitope is occupied. Injection of fusion proteins comprising IL-2 mutant 2-222 fused to a Fab derived from the non-blocking anti-PD-1 antibody clone 26 (230) or 24 (234) results in binding to Nivolumab / Pembrolizumab captured PD-1 since the Fab comprised in these fusion proteins do not share the same epitope as Nivolumab and Pembrolizumab. Data is presented as Biacore sensograms.

[0284] Figure 6 is a graph showing the binding of anti-PD- 1 IgG of the present invention (i.e. full antibodies, clones 22, 23, 24, 26, 27 and 28) in a concentration dependent manner on PD1+ CHO cells. The anti-PD-1 IgG EC50 of each clone are compared against Nivolumab and Pembrolizumab EC50, illustrating a difference in EC50 between the IgG 22, 23, 24, 26, 27 and 28 and both against Nivolumab and Pembrolizumab. The data are presented here as mean fluorescence intensity (MFI). Lambda isotype (isotype), secondary antibody alone (2ndalone - AF647) and Unstained were used as negative control conditions.

[0285] Figure 7 is a graph showing binding of exemplary Fabs derived from anti-PD-1 IgG of the present invention (Fab derived from clones 22, 26, 27 and 28) in a concentration dependent manner on PD1+ CHO cells. The data are presented here as mean fluorescence intensity (MFI). Unstained was used as negative control condition.

[0286] Figure 8 is a combination of graphs showing binding of the following anti-PD-1 Fab-IL2 fusion proteins of the present invention in a concentration dependent manner on PD1+ CHO cells: fusion proteins comprising the IL-2 mutant 2-222 fused with a Fab derived from clones 22, 23 and 24 (Fusion 22, Fusion-23, Fusion-24) (Figure 8A), and a Fab derived from clones 26, 27 and 28 (Fusion-26, Fusion-27, Fusion-28) (Figure 8B).The data is presented here as mean fluorescence intensity (MFI). Unstained was used as negative control condition.

[0287] Figure 9 is a graph showing the profiles of the antibodies of the present invention (z. e. full antibodies, clones 23, 24, 26, 27 and 28) for inhibition of receptor ligand complex (PD-1 / PD-L1) in a Promega Blockade Assay. The PD-1 / PD-L1 blockade shown reflects the mechanism of action (MO A) of biologies designed to block the PD-1 / PD-L1 interaction. When the PD-1 / PD-L1 interaction is disrupted (for example by nivolumab), TCR activation induces luminescence (via activation of the NF AT pathway). Data is expressed as relative light units (RLU).

[0288] Figure 10 is a graph showing STAT5 activation (%) in NK-92-PD1+ cell line treated with Nivolumab-Fab-IL2 fusion (IL-2 mutant 2-222 fused to Nivolumab Fab) alone and Nivolumab-Fab-IL2 fusion plus Pembrolizumab.

[0289] Figure 11 is a graph showing STAT5 activation (%) in NK-92-PD1+ cell line treated with exemplary molecule Fab 24-IL2 fusion of the present invention (IL-2 mutant 2-222 fused to a Fab derived from clone 24) alone and Fab 24-IL2 fusion plus Pembrolizumab.

[0290] Figure 12 is a graph showing STAT5 activation (%) in NK-92-PD1+ cell line treated with control molecule anti-HER2 Fab-IL2 fusion (IL-2 mutant 2-222 fused to an anti-HER2 Fab) alone and anti-HER2 Fab-IL2 plus Pembrolizumab.

[0291] Figure 13 is a graph showing STAT5 activation (%) in NK-92-PD1+ cell line treated with Nivolumab-Fab-IL2 fusion (IL-2 mutant 2-222 fused to Nivolumab Fab) alone and Nivolumab-Fab-IL2 fusion plus Nivolumab.

[0292] Figure 14 is a graph showing STAT5 activation (%) in NK-92-PD1+ cell line treated with exemplary molecule Fab 24-IL2 fusion (IL-2 mutant 2-222 fused to a Fab derived from clone 24) alone and Fab 24-IL2 fusion plus Nivolumab.

[0293] Figure 15 is a graph showing STAT5 activation (%) in NK-92-PD1+ cell line treated with control molecule anti-HER2 Fab-IL2 fusion (IL-2 mutant 2-222 fused to an anti-HER2 Fab) alone and anti-HER2 Fab-IL2 plus Nivolumab.

[0294] Figure 16 is a graph showing STAT5 activation (%) in CD8+-PD1+ cells treated with control molecule anti-HER2 Fab- IL2βγ fusion (IL-2 mutant 2-222 fused to an anti-HER2 Fab) and Fab 24-IL2βγ fusion (IL-2 mutant 2-222 fused to a Fab derived from clone 24)

[0295] Figure 17 is a graph showing STAT5 activation (%) in NK-92-PD1+ cell line treated with control molecule anti-HER2 Fab-IL2βγ fusion (IL-2 mutant 2-222 fused to an anti-HER2 Fab), Fab 24-IL2βγ fusion (IL-2 mutant 2-222 fused to a Fab derived from clone 24) and anti-HER2 Fab-IL2 wt fusion (IL-2 wt fused to an anti-HER2 Fab) in the presence of Pembrolizumab.

[0296] Figure 18 is a combination of graphs showing STAT5 activation (%) in NK-92 cell line treated with Nivolumab-Fab-IL2 fusion (IL-2 mutant 2-222 fused to Nivolumab Fab) and Nivolumab Fab-IL2 masked (IL-2 mutant 2-222 fused to Nivolumab Fab with a PEG of 40 kDA (PEG40) fused to the IL-2 mutant) as well as with the following panel of exemplary anti-PD-1 Fab-IL2 fusions of the present invention with or without PEG40: IL-2 mutant 2-222 fused to a Fab derived from clones 22 and 27 (Fab 22-IL2 fusion vs Fab 22-IL2 fusion masked and Fab 27-IL2 fusion vs Fab 27-IL2 fusion masked) (Figure 18A), and IL-2 mutant 2-222 fused to a Fab derived from clones 23 and 28 (Fab 23-IL2 fusion vs Fab 23-IL2 fusion masked and Fab 28-IL2 fusion vs Fab 28-IL2 fusion masked) (Figure 18B).

[0297] Figure 19 is a combination of graphs showing STAT5 activation (%) in CD4+ T cells treated with anti-HER2-IL2 wt fusion (IL-2 wt fused to an anti-HER2 Fab) and the following panel of exemplary anti-PD-1 Fab-IL2 fusions of the present invention with or without PEG40: IL-2 mutant 2-222 fused to a Fab derived from clones 22 and 26 (Fab 22-IL2 βγ fusion vs Fab 22-IL2 βγ fusion masked and Fab 26-IL2 βγ fusion vs Fab 26-IL2 βγ fusion masked) (Figure 19A), and IL-2 mutant 2-222 fused to a Fab derived from clones 27 and 28 (Fab 27-IL2 βγ fusion vs Fab 27-IL2 βγ fusion masked and Fab 28-IL2 βγ fusion vs Fab 28-IL2 βγ fusion masked) (Figure 19B).

[0298] Figure 20 is a combination of graphs showing STAT5 activation (%) in Treg cells treated with anti-HER2-IL2 wt fusion (IL-2 wt fused to an anti-HER2 Fab) and thefollowing panel of exemplary anti-PD-1 Fab-IL2 fusions of the present invention with or without PEG40: IL-2 mutant 2-222 fused to a Fab derived from clones 22 and 26 (Fab 22-IL2 βγ fusion vs Fab 22-IL2 βγ fusion masked and Fab 26-IL2 βγ fusion vs Fab 26-IL2 βγ fusion masked) (Figure 20A), and IL-2 mutant 2-222 fused to a Fab derived from clones 27 and 28 (Fab 27-IL2 βγ fusion vs Fab 27-IL2 βγ fusion masked and Fab 28-IL2 βγ fusion vs Fab 28-IL2 βγ fusion masked) (Figure 20B).

[0299] Figure 21 is a combination of graphs showing STAT5 activation (%) in CD8+ T cells treated with anti-HER2-IL2 wt fusion (IL-2 wt fused to an anti-HER2 Fab) and the following panel of exemplary anti-PD-1 Fab-IL2 fusions of the present invention with or without PEG40: IL-2 mutant 2-222 fused to a Fab derived from clones 22 and 26 (Fab 22-IL2 βγ fusion vs Fab 22-IL2 βγ fusion masked and Fab 26-IL2 βγ fusion vs Fab 26-IL2 βγ fusion masked) (Figure 21A), and IL-2 mutant 2-222 fused to a Fab derived from clones 27 and 28 (Fab 27-IL2 βγ fusion vs Fab 27-IL2 βγ fusion masked and Fab 28-IL2 βγ fusion vs Fab 28-IL2 βγ fusion masked) (Figure 21B).

[0300] Figure 22 is a combination of graphs showing STAT5 activation (%) in NK cells treated with anti-HER2-IL2 wt fusion (IL-2 wt fused to an anti-HER2 Fab) and the following panel of exemplary anti-PD-1 Fab-IL2 fusions of the present invention with or without PEG40: IL-2 mutant 2-222 fused to a Fab derived from clones 22 and 26 (Fab 22-IL2 βγ fusion vs Fab 22-IL2 βγ fusion masked and Fab 26-IL2 βγ fusion vs Fab 26-IL2 βγ fusion masked) (Figure 22A), and IL-2 mutant 2-222 fused to a Fab derived from clones 27 and 28 (Fab 27-IL2 βγ fusion vs Fab 27-IL2 βγ fusion masked and Fab 28-IL2 βγ fusion vs Fab 28-IL2 βγ fusion masked) (Figure 22B).

[0301] Figure 23 is a bar graph showing tumor volume measured after the administration of the 5th and final dose, at day 32, of PBS (vehicle) or of fusion-34, an anti-PD-1 Fab-IL2 fusion protein of the present invention comprising an IL-2 mutant 2-222 fused to i) an anti-PD-1 Fab derived from clone 24, and ii) a 40kDa branched PEG with a cleavable protease linker.

[0302] Figure 24 is Kaplan-Meier curves showing probability of survival measured after the administration of PBS (vehicle) or of fusion-34, an anti-PD-1 Fab-IL2 fusion proteinof the present invention comprising an IL-2 mutant 2-222 fused to i) an anti-PD-1 Fab derived from clone 24, and ii) a 40kDa branched PEG with a cleavable protease linker.EXAMPLES

[0303] The present invention is further illustrated by the following examples.Materials and MethodsPolyreactivity ELISA

[0304] Maxisorp plates were coated overnight (100 pl per well) at 4°C with 10 pg / ml dsDNA, 5 pg / ml insulin, 5 pg / ml human PCSK9-His (positive antigen for Bococizumab) or 5 pg / ml human IL-12 (positive antigen for Ustekinumab) in PBS. Plates were washed 5x with PBS / 0.05% Tween-20 (PBST) then blocked in PBST / lmM EDTA for 2 hours at RT. Test antibodies and controls were prepared in PBST / lmM EDTA as 8-point 2-fold dilution series (top concentration 10 pg / ml). Bococizumab positive control and Ustekinumab negative control were prepared in PBST / lmM EDTA at 10 pg / ml. Block was removed from the wells, then test antibodies and controls were added (100 pl per well) for 1 hour at RT. Plates were washed 5x with PBST and 1:5000 Goat anti-human IgG-Fc secondary antibody (in PBST / lmM EDTA (100 pl per well) was added for 1 hour at RT. Plates were washed 5x with PBST and 3x with PBS. Plates were developed with TMB (3 mins) then the reaction stopped with 0.5M sulfuric acid. Absorbance was read at 450 nm and 630 nm on the Clariostar plate reader.AC-SINS assay for self-aggregation propensity

[0305] 0.9 mg of goat anti-human Fc-specific antibody was buffer exchanged into 50 mM phosphate buffer pH8.0 with a 50 kDa MWCO. Yield was 0.736 mg from buffer exchange, sample was diluted to 0.184 mg / ml for thiolation (40-160 pg / ml recommended for assay). Traut’s reagent was added to the capture antibody to achieve a 10:1 Traut: antibody molar ratio, then split in two and incubated at RT for 1 hour to generate free thiols. The modified capture antibody was buffer exchanged into PBS 20 ml of gold nanoparticles, and centrifuged and resuspended in the same volume of milliQ water. The thiolated antibody solution was mixed with the nanoparticles and incubated on ice for 2hours. 56 mg BSA was added directly to the nanoparticles and incubated overnight at 4°C. The antibody conjugated gold particles were washed via centrifugation and resuspended in 1 ml PBS (pH 7.4). 5 pl of conjugated nanoparticles were added directly to a 384-well polystyrene plate, followed by 45 pl of each sample antibody at 0.05 mg / ml in PBS. Plate was centrifuged briefly and incubated at room temperature for 2 hours. The Absorbance was read using the ClarioStar Plus from 450 nm to 650 nm with 1 nm increments and the shift in maximum absorbance (plasmon wavelength) was analysed, curve fitting was performed for determination of the wavelength of maximum absorbance. Absorbance is read from 450 nm to 650 nm. The shift in maximum absorbance (plasmon wavelength) is analysed.Expression and purification of immunocytokine constructs

[0306] DNA coding for the amino acid sequence of the listed constructs were synthesized and cloned into the mammalian transient expression vector plasmid pETE V2. All constructs were expressed using CHO based transient expression system and the resulting antibody containing cell culture supernatants were clarified by centrifugation and filtration. All constructs were purified from cell culture supernatants via affinity chromatography. Purified antibody was buffer exchanged into phosphate buffered saline solution. The purity of the antibodies was determined by reducing and denaturing Sodium Dodecyl Sulfate Polyacrylamide gels and analyzed by semi-preparative SEC. Antibody concentrations were determined by measuring absorbance at 280nm and calculated theoretical extinction coefficient. Details of the purified constructs are detailed in Figure 4.Binding to CHO PD-1+cells by flow cytometry

[0307] CHO PD-1+were washed, resuspended in flow cytometry buffer (PBS 2% FBS), seeded at 1 xlO5cells per well and incubated at 4°C for lOmin. The plate was centrifuged at 400g for 1 min and the supernatant was removed. Different concentrations of test agents diluted in flow cytometry buffer were added (50pL / well) and incubated at 4°C for 15min. The plate was centrifuged at 400g for 1 min, the supernatant was removed, and cells were washed twice with 150 pL of flow cytometry buffer. The cells wereresuspended in flow cytometry buffer containing a labeled secondary antibody (50 pL / well) and incubated at 4°C for 15min. The plate was centrifuged at 400g for 1 min, the supernatant was removed, and cells were washed twice with 150 pL of flow cytometry buffer. Cells were transferred to a microtitre tube and read on the Navios EX.pSTAT5 activation assay on hPBMC andNK-92 cell lines

[0308] PBMCs or NK-92 cell lines were washed and counted into complete RPMI in absence of IL2. Cells were seeded at 7 xlO5cells or 5 xlO4cells, respectively, per well and incubated at 37°C for Ihr. Dilutions of the immunocytokines were prepared in PBS creating a lOx concentration of each sample. 5 µl / well of 10x immunocytokine was added to the cells and incubated at 37°C, 5% CO2for 15min. Membrane staining antibodies specific for each individual cell type were added to corresponding wells; anti-CD25-FITC (Biolegend 3561606), anti-CD127-PE (ebio 12-1278-42), anti-CD8-ECD (BC-B737660 or BC-B08467), anti-CD4-PC7 (BC-B737660), anti-CD3-Pacific Blue (BC-B49204), anti-CD56-BV510 (Biolegend 318340), and / or anti-PDl-PE (BD557946) and incubated at 37°C, 5% CO for 15min. Then, 8 pl of R1 fixative reagent was incubated in well at RT for lOmin then 65 pl FBS and 145 pl of Buffer Perm reagent were added, mixed and incubated at 37°C for 5 min. The plate was centrifuged at 400g for 2min prior to adding 50pl / well of intercellular staining antibody (pSTAT5-AF647-FL6, BD612599). The plate was incubated at RT for 30min in dark then washed once with 150pl R4 buffer wash reagent. After washing each sample was resuspended in 150pl of buffer R4 and transferred to a microtitre tube and read on the Navios EX.Target binding affinity assay

[0309] A capture assay set up was utilised for receptor binding analysis. Anti-human IgG (Cat No. BR100839) was coupled to CM5 chips (GE Healthcare) using standard amine chemistry to ~ 9000RU. Human PD1 at Ipg / ml in HBS-P+ running buffer was captured to -90RU on one flow cell. A four point 3-fold dilution series for each analyte (Immunocytokine) was used for single cycle kinetic analysis. An analyte concentration range of 2.22nM - 60nM and 66.6nM - 180nM was used. Between each run the chip was regenerated with 3M MgCh. A single cycle kinetic protocol was run on Biacore T200and 1: 1 kinetic analysis or steady state affinity measurement following double referencesubtraction was performed using Biacore T200 evaluation software V 2.0.1.Target binding competition assay

[0310] A capture assay set up was utilized for receptor binding analysis. Anti -human IgG (Cat No. BR100839) was coupled to CM5 chips (GE Healthcare) using standard amine chemistry to ~ 9000RU. Pembrolizumab captured to -2000 RU, Nivolumab captured to -1600 RU Each diluted in running buffer. Human PD1 was diluted in running buffer to 200 nM and injected for 180 seconds association. Nivolumab Fab was diluted in running buffer to 180 nM and injected for 180 s association. Non-blocking Fab candidates were diluted in running buffer to 180 nM and injected for 180 s association. Between each run the chip was regenerated with 3M MgCl₂. A single cycle kinetic protocol was run on Biacore T200 and 1:1 kinetic analysis or steady state affinity measurement following double reference-subtraction was performed using Biacore T200 evaluation software V 2.0.1.PD1 PD-L1 blockade assay

[0311] PD-1 Jurkat effector cells were cultured in RPMI 1640 + L-GlutaMAx)-10%FBS (Gibco 10270)- 10 mM HEPES 1 mM Sodium Pyruvate IX MEM NAA- P / S (Gibcol5140) plus 200 pg / mL Hygromycin B (100 pL of 100 mg / ml stock); 0.5 mg / mL (500 pL of 50 mg / mL stock) was added just before culturing. PD-L1 APC CHO-K1 cells were cultured in Ham's F-12 med-10%FBS. 95 pL of medium was removed from wells of 96-well assay plate containing PD-L1 aAPC / CHO-Kl cells. 40 pL of the appropriate antibody dilution was immediately added to the pre-plated PD-L1 aAPC / CHO-Kl cells. 80 pL of assay buffer was added to the outside wells. The stimulated PD-1 effector cells were transferred via dispensing 40 pL (50'000 cells) of PD-1 effector cells into the wells containing PD-L1 aAPC / CHO-Kl cells. The serially diluted antibody samples were prepared in RPMI 1640 / 1% FBS and added to the plate. The assay plate was covered with a lid and incubated at 37°C / 5% CO2 for 6 hours. The assay plates were removed from the incubator and equilibrate to RT for 5-10 min. The Bio-Gio reagent was prepared according to manufacturer’s protocol and 80 pL of reagent wasadded to the inner 60 wells of the assay plate. A further 80 pL was added to control wells of each assay plate to measure background signal. The plate was incubated at room temperature for 5-30 minutes before measuring luminescence using a luminometer or luminescence plate reader.In vivo tumor efficacy study

[0312] C57BL / 6-B-hPDl mice were implanted subcutaneously with 0.5 x 106MC38 cells / mouse. Mice were randomized into treatment groups, and treatments were initiated when tumors reached 50-150 mm3. Treatment schedule included five intravenous injections of Img / kg Fusion-34 (N=12), or vehicle (N=12) over a 15 - day period. Tumor volume (TV) was measured at least 3 times a week using a callipers. When measurable, tumor volume was calculated using the formula: Length x (Width)2x 0.52. Starting from TV > 1000 mm3, TV was measured daily. Euthanasia of individual mice was performed when TV > 1500 mm3.

[0313] Survival study

[0314] C57BL / 6-B-hPDl mice were implanted subcutaneously with 0.5 x 106MC38 cells / mouse. Mice were randomized into treatment groups, and treatments were initiated when tumors reached 50-150 mm3. Treatment schedule included five intravenous injections of Img / kg Fusion-34 (N=12), or vehicle (N=12) over a 15 - day period. Survival was measured over time and calculated as either when mice were found dead or euthanasia of individual mice was performed when TV > 1500 mm3.Results

[0315] The 6 non-blocking anti-PD-1 antibodies clones we obtained (clones 22, 23, 24, 26, 27 and 28) were tested for poly-reactivity with ELISA assays for binding to insulin and double strand DNA, and for self-aggregation propensity by AC-SINS. As shown in Figures 1 and 2, the 6 clones have a minimal binding with insulin and double strand DNA, and all are below the threshold for propensity to self-aggregate.

[0316] Next, the binding properties of anti-PD-1 antibodies (i.e. full antibodies, clones 22, 23, 24, 26, 27 and 28), PD-1 Fab (i.e. Fab derived from clones 22, 26, 27 and 28) andfusion proteins comprising PD-1 Fab fused to an IL-2 mutant (i.e. fusion-22, fusion -23, fusion-24, fusion-26, fusion-27 and fusion-28) for PD-1 were evaluated. The data show that the anti-PD-1 antibodies, Fab and fusion protein of the present invention bind to PD-1, meaning that the antibodies are still active in the Fab format and when fused to interleukins (Figures 3, 6-8). Of note, there is a difference in EC50 between the IgG 22, 23, 24, 26, 27 and 28 and both against Nivolumab and Pembrolizumab (Figure 6).Regarding the affinity for PD-1, the Fab of the present invention exhibit an affinity in the range of 6nM – 25nM, for human PD-1 (Figure 4).

[0317] Epitope binning and PD1 / PD-L1 assays were also performed to confirm that the antibodies of the present invention do not share the same epitope as known blocking anti-PD-1 antibodies and do not block PD-1 / PD-L1 interaction. Figure 5 shows that injection of fusion proteins comprising an IL-2 mutant fused to a Fab derived from antibodies of the present invention results in binding to Nivolumab / Pembrolizumab captured PD-1, indicating that the Fab comprised in these fusion proteins do not share the same epitope as Nivolumab and Pembrolizumab. Figure 9 shows that the anti-PD-1 antibodies do not induce PD1 / PD-L1 blockade compared to Nivolumab.

[0318] The capacity of fusion proteins of the present invention and control molecules to activate NK-92 PD-1 cell line was next assessed with pSTAT5 activation assays.

[0319] Figure 10 confirms the presence of a shift in functional activity of a fusion protein comprising an IL-2 mutant and Nivolumab Fab when Pembrolizumab is added (due to competition for PD-1 binding). By contrast, there is no shift in functional activity of a fusion protein of the present invention or of a control molecule (fusion protein comprising an IL-2 mutant and a Fab directed to HER-2), when Pembrolizumab is added and no competition for PD-1 binding is detected (Figures 11-12).

[0320] Similarly, Figure 13 confirms a shift in functional activity of Nivolumab Fab-IL2 when Nivolumab is added to compete for PD-1 binding. By contrast, there is no shift in functional activity of a fusion protein of the present invention or of a control molecule (fusion protein comprising an IL-2 mutant and a Fab directed to HER-2), when Nivolumab is added and no competition for PD-1 binding is detected (Figures 14-15).

[0321] In addition, Figure 16 shows that the potency of a fusion protein comprising an IL-2 mutant (with a reduced binding for IL-2 receptors comprising α chain) fused to aFab of the present invention is increased through the CIS binding effect of the Fab of thepresent invention, resulting in increased potency to that of a fusion protein comprising ananti-HER2 Fab and the same IL-2 mutant (i.e. with a reduced binding for IL-2 receptorscomprising α chain).

[0322] Similarly, Figure 17 shows that the potency of a fusion protein comprising the same IL-2 mutant (with a reduced binding for IL-2 receptors comprising α chain) fusedto a Fab of the present invention is restored through the CIS binding effect of the Fab of the present invention, resulting in similar potency to that of a fusion protein comprising an IL-2 wild type fused to an anti-HER2 Fab. Figure 17 also demonstrates that the EC50 of a fusion protein comprising an IL-2 mutant fused to a Fab of the present invention is not impacted by the presence of Pembrolizumab (as there is no competition occurring).

[0323] Fusion proteins comprising an IL-2 mutant, a Fab of the present invention and a masking moiety altering the binding of the IL-2 mutant to its receptor (i.e. a PEG of 40 kDa) were also generated and tested for their capacity to active cells. To generate those masked fusion proteins, IL-2 protein was mutated to introduce a transglutaminase site and the masking moiety was fused at the transglutaminase site of the IL-2 by transglutamination.

[0324] As shown in Figure 18, the presence of mask reduces the activation of NK-92 cells. Figure 19 shows that the presence of mask strongly reduces the activation of CD4+ T cells while the non-masked counterpart is capable of activation of CD4+ T cells. Figure 20 shows that the presence of mask reduces the activation of Treg cells. The non-masked counterpart is capable of activating Treg cells in line with the activation potential of an alpha attenuated IL-2 mutant. Figure 21 shows that the presence of mask strongly reduces the activation of CD8+ T cells while the non-masked counterpart is capable of full activation of CD8+ T cells. Figure 22 shows that the presence of mask strongly reduces the activation of NK cells while the non-masked counterpart is capable of full activation of NK cells.

[0325] In addition, the ability of a fusion protein comprising an IL-2 mutant, a Fab of the present invention and a masking moiety altering the binding of the IL-2 mutant to its receptor (i.e. a PEG of 40 kDa) to decrease tumor volume was tested in vivo, in a tumor model.

[0326] Figure 23 shows a reduction of the tumor volume with the fusion protein as compared to the vehicle, demonstrating the anti-tumor activity of a fusion protein of the present invention in an anti-PDl insensitive MC38 syngeneic tumor model.

[0327] In addition, Figure 24 shows an increase in the probability of survival with the fusion protein as compared to the vehicle, demonstrating the anti-tumor activity of a fusion protein of the present invention in an anti-PDl insensitive MC38 syngeneic tumor model.

[0328] Altogether, the obtained data show that the anti-PD-1 antibodies of the present invention bind PD-1 without blocking PD-1 / PD-L1 interaction, and that they are still functional in the Fab format and fused to interleukins. These data support the use of anti-PD-1 antibodies of the present invention in the generation of fusion proteins, and in combination with blocking anti-PD-1 antibodies.

[0329] In addition, the in vivo data obtained in the mice tumor model support the use of the anti-PD-1 antibodies of the present invention and fusion proteins comprising the same for treating cancer.

Claims

CLAIMS1. An isolated anti-human PD-1 antibody or antigen-binding fragment thereof, wherein the variable region of the heavy chain (VH) comprises at least one of, preferably two of, and more preferably the three following complementary- determining regions (CDRs):- CDR1: GX₁TX₂X₃GLY (SEQ ID NO: 189), wherein X₁ is G or Y, X2is F or L, and X3 is T or S;- CDR2: X₄SX₅EFGX₆X₇ (SEQ ID NO: 190), wherein X4is V or I, X5is A or V, X₆ is S or G and X7 is T or A; and / or- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191), andwherein the variable region of the light chain (VL) comprises at least one of, preferably two of, and more preferably the three following CDRs:- CDR1: TGTSSDVGGYNYVS (SEQ ID NO: 203);- CDR2: EVSNRPS (SEQ ID NO: 204); and / or- CDR3: SSYTSSSTYV (SEQ ID NO: 205).

2. The isolated anti -human PD-1 antibody or antigen-binding fragment thereof according to claim 1, wherein the VL comprises the three CDRs with SEQ ID NOs: 203-205, and wherein the VH comprises:(a) The following CDRS:- VH-CDR1: GGTFTGLY (SEQ ID NO: 192);- VH-CDR2: VSAEFGST (SEQ ID NO: 193); and- VH-CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(b) The following CDRS- VH-CDR1: GGTLTGLY (SEQ ID NO: 194);- VH-CDR2: VSVEFGGA (SEQ ID NO: 195); and- VH-CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(c) The following CDRS- VH-CDR1: GYTLTGLY (SEQ ID NO: 196);VH-CDR2: ISAEFGGA (SEQ ID NO: 197); andVH-CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(d) The following CDRS- CDR1: GYTLSGLY (SEQ ID NO: 198);- CDR2: VSVEFGST (SEQ ID NO: 199); and- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(e) The following CDRS- CDR1: GGTLSGLY (SEQ ID NO: 200);- CDR2: ISVEFGGA (SEQ ID NO: 201); and- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191);or,(I) The following CDRS- CDR1: GYTLSGLY (SEQ ID NO: 198);- CDR2: ISAEFGST (SEQ ID NO: 202); and- CDR3: DEGILDDSSGYYYKINAFDI (SEQ ID NO: 191).

3. The isolated anti -human PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein said antibody or fragment comprises: a VH comprising or consisting of the sequence SEQ ID NO: 206, wherein X₁ is G or Y, X2 is F or L, X3 is T or S, X₄ is V or I, X₅ is A or V, X₆ is S or G, and X7 is T or A, anda VL comprising or consisting of the sequence SEQ ID NO: 207.

4. The isolated anti -human PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein said antibody or fragment comprises: a VH comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 208-213; anda VL comprising or consisting of SEQ ID NO: 207.

5. A fusion protein comprising at least one isolated anti -human PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The fusion protein according to claim 5, wherein the fusion protein comprises at least one anti -human PD-1 antibody or antigen-binding fragment thereof and at least one interleukin-2 (IL-2) or a mutant thereof.

7. The fusion protein according to claim 6, wherein the IL-2 mutant comprises or consists of one of the sequences selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 130-143, SEQ ID NO: 145-188, SEQ ID NOs: 19-39, SEQ ID NOs: 51-97, SEQ ID NO: 99, SEQ ID NOs: 110-117, SEQ ID NOs: 119-127.

8. The fusion protein according to any one of claims 6 to 7, wherein the mutant IL-2 comprises or consists of one of the sequences selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 30, SEQ ID NO: 170, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 64, SEQ ID NO: 58, SEQ ID NO: 185, and SEQ ID NO: 70, preferably wherein the mutant IL-2 comprises or consists of one of the sequences selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 64, and SEQ ID NO: 70.

9. The fusion protein according to any one of claims 5 to 8, wherein the fusion protein further comprises at least one masking moiety selected from the group consisting of glycans, lipids, proteins, peptides, small organic molecules and polymers.

10. The fusion protein according to claim 9, wherein the masking moiety is a water- soluble polymer, preferably a polyethylene glycol (PEG).

11. An isolated nucleic acid encoding the isolated anti -human PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the fusion protein according to any one of claims 5 to 10.

12. The isolated anti -human PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 10, for use as a medicament.

13. The isolated anti -human PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the fusion protein according to any one ofclaims 5 to 10, for use in treating a cancer or an inflammatory disease in a subject in need thereof.

14. The isolated anti -human PD-1 antibody or antigen-binding fragment thereof, or the fusion protein for use according to claim 13, wherein the subject has received, is receiving or will receive an immunotherapy.

15. The isolated anti -human PD-1 antibody or antigen-binding fragment thereof, or the fusion protein for use according to claim 14, wherein the immunotherapy is a blocking anti-PD-1 antibody, preferably nivolumab or pembrolizumab.

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