WO2024023246A1 - Antibody binding to pd1 - Google Patents

Abstract

The present invention relates to an antibody binding to PD-1 (Fig 8).

Description

antibody binding to PD1

FIELD OF THE INVENTION

The present application relates to an antibody binding to PD1

BACKGROUND

The PD-1/PD-L1 pathway has been shown to be an important mechanism of tumor immune evasion and high expression of PD-L1 on tumor cells has been reported to correlate with adverse patient outcomes. The antibody-based blockade of suitable epitopes on the surface of PD-1 was shown to increase T cell effector functions and reduce immune cell evasion by tumor cells. Anti -PD-1 antibodies have shown promising clinical results in a subset of patients with melanoma, non-small cell lung cancer, and other tumors. The antibody molecule of the invention may thus find application in the treatment of certain types of cancer that express, or overexpress, PD-L1.

PD-1 (Programmed Cell Death Protein 1), also referred to as CD279 was first discovered in interleukin-3 (IL-3)-deprived LyD9 (murine hematopoietic progenitor) and 2B4-11 (murine T- cell hybridoma) cell lines in 1992. PD-1 belongs to the CD28 family of receptors, that also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is 15% similar to the amino acid sequence of CD28, 20% similar to CTLA4, and 13% similar to induced T-cell co-stimulator. PD-1 is a 55-kDa transmembrane protein containing 288 amino acids with an extracellular N-terminal domain (IgV-Like), a membrane-permeating domain and a cytoplasmic tail located at the N and C ends, respectively, with two tyrosine bases. It is a surface receptor and is expressed on activated B cells, T cells, and myeloid cells.

PD-1 is an inhibitor of both adaptive and innate immune responses, and is expressed on activated T, natural killer (NK) and B lymphocytes, macrophages, dendritic cells (DCs) and monocytes. Of note, PD-1 is highly expressed on tumor-specific T cells. Transcription factors such as nuclear factor of activated T cells (NF AT), NOTCH, Forkhead box protein (FOX) 01 and interferon (IFN) regulatory factor 9 (IRF9) may trigger the transcription of PD-1. The conserved upstream regulatory regions B and C (CR-B and COR-C) are important for the expression of the PD-1 gene. There is a binding site in the CR-C region that is connected to NFATcl (NFAT2) in TCD4 and TCD8 units. Instead, c-FOS connects to sites in the CR-B region and enhances PD-1 expression when it stimulates T-cell receptors upon Ag detection in naive T cells. NFATc is activated and binds to the promoter region of the pdcdl gene. In addition, IFN-a combined with IRF9 may result in PD-1 expression via binding to the promoter of the pdcdl gene in exhausted T cells. During chronic infections, PD-1 is expressed in exhausted TCD8 cells due to its demethylated promoter, and the F0X01 transcription factor binds to the PD-1 promoter to increase its expression. Cancer cell leakage increases the expression of the c-FOS subunit of API, thereby increasing the expression of PD-1.

PD-1 plays two opposing roles, as it can be both beneficial and harmful. As regards its beneficial effects, it plays a key role in reducing the regulation of ineffective or harmful immune responses and maintaining immune tolerance. However, PD-1 causes the dilation of malignant cells by interfering with the protective immune response.

Two ligands for PD-1 have been identified, PD-L1 and PD-L2, that have been shown to downregulate T cell activation upon binding to PD-1.

Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1, but do not bind to other CD28 family members. One ligand for PD-1, PD-L1 is abundant in a variety of human cancers. The interaction between PD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well. Inhibition can be made using monoclonal antibodies. Antibodies that bind to PD-1 and uses thereof including interfering with PD-L1 signalling are common in the art.

It has yet been shown that only ~30-40% of cancer patients show a response to anti-PD-1 immunotherapy, and only a fraction of these shows a durable clinical response (Carretero- Gonzalez et al., 2018). Therefore, there is a pharmaceutical need to improve response rates of anti-PD-1 antibodies and potentially expand to more indications.

Such mAbs should boost the immune system through a potent PD-1 blockade to fight cancer with a potentially more favorable risk:benefit profile than existing anti-PD-1 antibodies. Suitably such mAbs should be fully human to eliminate potential immunogenicity and should be cross-reactive between cynomolgus monkey and man to allow translational research. Improved risk:benefit profiles could be achieved by targeting specific epitopes on the surface of PD-1.

It is hence one object of the present invention to provide treatment alternatives in cancer therapy that overcome existing imitations.

It is another object of the present invention to provide alternative anti PD-1 antibodies which have amended properties relative to the PD-1 antibodies currently on the market.

These and other objects are solved by the features of the independent claims. The dependent claims disclose embodiments of the invention which may be preferred under particular circumstances. Likewise, the specification discloses further embodiments of the invention which may be preferred under particular circumstances.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the characterization of the anti-PD-1 antibody D12 in scFv format.

Figure 1A shows the results of size exclusion chromatogram of scFv (DI 2). The monomeric form of the scFv was eluted from the S75i GL column at 11.5mL as expected. Figure IB shows the results of SDS-PAGE analysis of scFv (D12). The scFv had the expected size of 25 kDa under non-reducing (NR) and reducing (R) conditions, respectively.

Figure 2 shows the biochemical characterization of D12 in IgG format.

Figure 2A shows the results of size exclusion chromatogram of IgG (D12). The IgG was eluted from the S200i 10/300 GL column at 11.9mL as expected.

Figure 2B shows the results of SDS-PAGE analysis of IgG (D12). The IgG had the expected size of 150 kDa under non-reducing conditions and 25 kDa and 50 kDa under reducing condition.

Figure 3 shows the surface plasmon resonance (SPR) sensorgrams of D12.

Figure 3 A shows the sensorgram of monomeric scFv preparations of D12 on a sensorchip coated with human PD-1 confirming the binding of D12 scFv to human PD-1.

Figure 3B shows the sensorgram of D12 in IgG format on a sensorchip coated with cynomolgus monkey PD-1 confirming cross-reactivity of D12 with the cynomolgus monkey protein.

Figure 4 shows antibody mediated blockade of PD-1 binding to its natural receptors PD-L1 (A) and PD-L2 (B). D12 blocked binding of human PD-L1 and PD-L2 to plate-coated human PD-1 in a concentration dependent manner. The antibody KSF that is specific to hen egg lysozyme was used as a negative control.

Figure 5 shows binding of D12 to PD-1 expressed on cells, measured by flow cytometry.

Figure 5A confirms binding of D12 in IgG format to HEK cells transiently transfected with human PD-1.

Figure 5B confirms binding of D12 in IgG format to activated primary human T cells.

Figure 5C confirms that D12 in IgG format did not bind to non-activated human T cells which lack expression of PD-1. Figure 6 shows the in vitro reconstitution of PD-1 in complex with D12-Fab. Shown is a SEC- MALS analysis of free PD-1 (middle peak), D12 Fab (right peak) and their reconstituted complex (left peak). It confirms co-elution of the antigemantibody complexes with 1 :1 stoichiometry.

Figure 7 shows the structural determination of PD-1 in complex with D12 Fab. Glycosylation decorating the surface of PD-1 are shown as black sticks. The inset shows the details of the interaction surface between PD-1 and DI 2, with contacting amino acid side chains shown as sticks. Amino acid numbering is based on the sequence of the full length human PD-1 (UniProtKB QI 5116).

Figure 8 shows a comparison of interacting surfaces between the D12 and NBOla antibodies and PD-1 ligand.

Figure 8 A shows the interaction footprint of D12 with human PD-1.

Figure 8B shows the interaction footprint of NBOla with human PD-1. The extended conformation of the PD-1 N-terminus recognized by D12 is a unique prerogative of this antibody.

Figure 8C shows the superposition of the PD-ED12 Fab complex with the structures of PD-1 in complex with PD-L1 and PD-L2. The superposition highlights the steric clashes between the ligand binding sites on PD-1 and the VH portion of the D 12 Fab recognizing the N-terminus of the receptor.

Figure 9 shows the structural comparison of the binding modes of various antibodies recognizing PD-1. The panels show a cartoon representation of the available structures of anti- PD-1 Fabs in complex with the receptor, compared to the binding mode observed in D12. It highlights the novel epitope of D12 compared to previously described anti-PD-1 antibodies.

Figure 10 shows the biochemical characterization of IL12- D12 conjugate. Figure 10A shows the results of size exclusion chromatogram of IL12- D12 conjugate. The conjugate was eluted from the S200i 10/300 GL column at 11.8mL. Figure 10B shows the results of SDS-PAGE analysis of IL12- D12 conjugate. The conjugate had the expected size of 110 kDa under both reducing and non-reducing conditions.

DETAILED DESCRIPTION

According to one aspect of the invention, a protein binder is provided which binds to human PD-1 within a discontinuous epitope comprising at least the residues Trp9, Prol 1 and Leu 115 of PD-1 according to SEQ ID NO: 1.

SEQ ID NO: 1 represents part of the extracellular domain of human PD-1. In fact, said sequence encompasses the residues 24 - 170 of full length human PD-1 (UniProtKB Q15116).

SEQ ID NO: 2 represents part of the extracellular domain of cynomolgus monkey PD-1.

The inventors have surprisingly identified a new epitope in PD-1. The closest matching epitope, based on structural comparisons, was found in the NBOla monoclonal antibody (Fenwick et al., 2019). However, NBOla does not recognize epitopes including the endmost N-terminus of PD-1, including Trp9 and Prol 1.

According to one embodiment, the protein binder binds at least a conformational epitope of human PD-1 spanning within the region of amino acid residues 7-11 and 112-116 according to SEQ ID NO 1

Preferably, the protein binder binds within a region which has at least 3 amino acids in length.

In one or more embodiments, the protein binder binds to >2, >3, >4, >5, >6, >7, >8, >9, >10, >11, >12, 13, >14, >15, >16, >17, >18, >19, >20, >21, >22, >23, >24, or >25 amino acids within the above region. The respective amino acid residues can be present in a discrete, consecutive sequence, or in two or more clusters, each of which comprising one or more amino acid residues.

According to one embodiment, the protein binder is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an antibody mimetic. As used herein, the term “monoclonal antibody (mAh)” shall refer to an antibody composition having a homogenous antibody population, i.e., a homogeneous population consisting of a whole immunoglobulin, or a fragment or derivative thereof retaining target binding capacities.

Particularly preferred, such antibody is an IgG antibody, or a fragment or derivative thereof retaining target binding capacities. Immunoglobulin G (IgG) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG has two antigen binding sites.

IgG antibodies are large molecules with a molecular weight of about 150 kDa made of four peptide chains. It contains two identical class y heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus a tetrameric quaternary structure. The two heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has two identical halves, which together form the Y-like shape. Each end of the fork contains an identical antigen binding site. The Fc regions of IgGs bear a highly conserved N-glycosylation site. The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisecting GlcNAc and a-2,6-linked sialic acid residues.

There are four IgG subclasses (IgGl, 2, 3, and 4) in humans, named in order of their abundance in serum (IgGl being the most abundant).

As used herein, the term “fragment” shall refer to fragments of such antibody retaining target binding capacities, e.g.

• a CDR (complementarity determining region)

• a hypervariable region,

• a variable domain (Fv)

• an IgG or IgM heavy chain (consisting of VH, CHI, hinge, CH2 and CH3 regions)

• an IgG or IgM light chain (consisting of VL and CL regions), and/or

• a F ab and/ or F (ab )2

• small immunoprotein (SIP)

• diabody single-chain Fv (scFv) single-chain diabody tscDb)

As used herein, the term “derivative” shall refer to protein constructs being structurally different from, but still having some structural relationship to, the common antibody concept, including the VH domains, like e.g., scFv, Fab and/or F(ab)2, Diabodies (small bivalent antibody constructs consisting of two different VH and two different VL domains), small immunoproteins (SIP, also called dimeric single-chain antibodies or minibodies, comprising the structure VL-VH-CH3), Camelid Antibodies, Nanobodies, Domain Antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures joined by a J chain and a secretory component), shark antibodies, antibodies consisting of new world primate framework plus non-new world primate CDR, dimerized constructs comprising CH3+VL+VH, and antibody conjugates (e.g. antibody or fragments or derivatives linked to a toxin, a cytokine, a radioisotope or a label). These types are well described in the literature and can be used by the skilled person on the basis of the present disclosure, without adding further inventive activity.

Methods for the production of a hybridoma cell are disclosed in Kohler & Milstein (1975).

Methods for the production and/or selection of chimeric or humanised mAbs are known in the art. For example, US6331415 by Genentech describes the production of chimeric antibodies, while US6548640 by Medical Research Council describes CDR grafting techniques and US5859205 by Celltech describes the production of humanised antibodies.

Methods for the production and/or selection of fully human mAbs are known in the art. These can involve the use of a transgenic animal which is immunized with the respective protein or peptide, or the use of a suitable display technique, like yeast display, phage display, B-cell display or ribosome display, where antibodies from a library are screened against human iRhom2 in a stationary phase.

In vitro antibody libraries are, among others, disclosed in US6300064 by MorphoSys and US6248516 by MRC/Scripps/Stratagene. Phage Display techniques are for example disclosed in US5223409 by Dyax. Transgenic mammal platforms are for example described in EP1480515A2 by Taconic Artemis. IgG, IgM, scFv, scDb, Fab and/or F(ab)2 are antibody formats well known to the skilled person.

Related enabling techniques are available from the respective textbooks.

As used herein, the term “Fab” relates to an IgG/IgM fragment comprising the antigen binding region, said fragment being composed of one constant and one variable domain from each heavy and light chain of the antibody

As used herein, the term “F(ab)2” relates to an IgG/IgM fragment consisting of two Fab fragments connected to one another by disulfide bonds.

As used herein, the term “scFv” relates to a single-chain variable fragment being a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide.

Modified antibody formats are for example bi- or trispecific antibody constructs, antibodybased fusion proteins, immunoconjugates and the like. These types are well described in the literature and can be used by the skilled person on the basis of the present disclosure, with adding further inventive activity.

As used herein, the term “antibody mimetic” relates to an organic molecule, most often a protein that specifically binds to a target protein, similar to an antibody, but is not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. The definition encompasses, inter alia, Affibody molecules, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, Monobodies, and nanoCLAMPs.

As used herein, the term “single chain diabody” relates to a construct of two single chain Fv (scFv) antibodies with a short linker, preferably 3 - 10 amino acids long, more preferably 5 amino acid long (also known as “diabodies”), joined to one another by a longer linker, preferably 5 - 20 amino acids long, more preferably 15 amino acid long, according to the following scheme (N->C orientation): In a single-chain diabody, two sets of VH and VL domains are connected together in sequence on the same polypeptide chain. For example, the two sets of VH and VL domains may be assembled in a single-chain sequence as follows: (VH-VL)— (VH-VL), where the brackets indicate a set. The two sets of VH and VL domains are connected as a single-chain by a long or ‘flexible’ peptide linker. This type of peptide linker sequence is long enough to allow pairing of the VH and VL domains of the first set with the complementary VH and VL domains of the second set. Generally, a long or ‘flexible’ linker is 15 to 20 amino acids.

In one or more embodiments, the protein binder is an isolated antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an isolated antibody mimetic

In one or more embodiments, the antibody is an engineered or recombinant antibody, or a target binding fragment or derivative thereof retaining target binding capacities, or an engineered or recombinant antibody mimetic.

According to one embodiment of the invention, the protein binder is an antibody in at least one of the formats selected from the group consisting of: IgG, scFv, Fab, (Fab)2 or scDb.

According to one embodiment, an antibody or a target-binding fragment or derivative thereof retaining target binding capacity to human PD-1 is provided, which a) comprises a set of six heavy chain/light chain complementarity determining regions (CDR) comprised in the heavy chain/light variable domain sequences of SEQ ID NOs 9 and 10: b) comprises a set of six heavy chain/light chain complementarity determining regions (CDR) in the order HCDR1; HCDR2; HCDR3; LCDR1; LCDR2 and LCDR3, as set forth in SEQ ID NOs 3, 4, 5, 6, 7, and 8 c) comprises a set of heavy chain/light chain complementarity determining regions (CDR) as set forth in option b), with the proviso that at least one of the CDRs has up to 3 amino acid substitutions relative to the CDRs comprised in the respective SEQ ID NOs, and/or d) comprises a set of heavy chain/light chain complementarity determining regions (CDR) as set forth in option b) or c), with the proviso that at least one of the CDRs has a sequence identity of > 66 % to the CDRs comprised in the respective SEQ ID NOs,

The CDRs are embedded in a suitable protein framework so as to be capable to bind to human PD-1.

The inventors have surprisingly shown that this new antibody nicknamed “D12”, binds to a previously undiscovered epitope on the surface of PD-1 involving a broad surface, shaped by N-terminal PD-1 residues 7-11 and residues 112-116 (example 4, figure 8).

This is an important finding. Several anti-PD-1 and anti-PD-Ll antibodies targeting different epitopes have been described in the art. They have shown slight differences in their safety profile regarding autoimmune reactions as well as in their efficacy data (Chen et al., 2021; Ma et al. 2021, Fenwick et al 2019, Park et al., 2022). Targeting novel epitopes on PD-1 with monoclonal antibodies is therefore of pharmaceutical interest to explore differences in therapeutic efficacy and safety.

In particular, the key recognition element is represented by the side chains of PD-1 Trp9 and Prol 1, which lie in between VH and VL loops of D12 in a hydrophobic network generated by VH residues Tyr59, AsnlOl, Hisl02, Metl04 and VL residues Leu94 and Phe96. A tight electrostatic interaction involving PD-1 residue Glut 13 and VH residue Ser54 stabilizes the antigemantibody complex. A second key hydrophobic interaction involves PD-1 Leul l5, forming close van der Waals contacts with the side chains of VH residues Tyr32, TyrlOO, and AsnlOl. Comparison with available structural data of PD-1 in complex with other antibodies revealed that D12 binds in an unprecedented mode, particularly regarding the N-terminus of the molecule, which adopts an elongated conformation when bound to the antibody exposing the side chain of Trp9. As discussed above, the closest matching epitope, based on structural comparisons, was found in the NBOla monoclonal antibody (Fenwick et al. 2019).

The antibody is cross-reactive with the corresponding extracellular domain of cynomolgus PD-

1 as set forth e.g. in SEQ ID NO: 2. This is surprising because, though the two proteins are highly similar (95.8% sequence identity), one of the two motifs the antibody binds to, residues 7-11, is dissimilar, with RPWNP in human PD-1 and RPWNA in cynomolgus PD-1.

As shown herein, the antibody D12 has an affinity to PD-1 in the two-digit nanomolar range as shown in Example 1.6., Fig. 3. Without being bound to theory, it maybe that such medium affinity to the target, PD-1, reduces trapping of the antibody in secondary lymphoid organs, and thus increases bioavailability of the antibody in the tumors to be treated.

As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described inter alia by Kabat et al. (1977), Kabat et al. (1991 and Chothia et al. (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other.

Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein.

Preferably, the CDRs as set forth in this specification are hence determined according to the Kabat or Chothia numbering set forth in table 1.

Table 1: CDR definitions

As used herein, the term “framework” when used in reference to an antibody variable region is entered to mean all amino acid residues outside the CDR regions within the variable region of an antibody. Therefore, a variable region framework is between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs. In one embodiment, the term “capable to bind to target X” has to be understood as meaning that respective binding domain binds the target with a KD of 10'⁴ or smaller. KD is the equilibrium dissociation constant, a ratio of k_off/k_on, between the antibody or fragment and its antigen. KD and affinity are inversely related. The KD value relates to the concentration of antibody or fragment (the amount of antibody or fragment needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the binding domain. The following table shows typical KD ranges of monoclonal antibodies

Table 2: K_Dand Molar Values

Preferably, the antibody or fragment has up to 2 amino acid substitutions, and more preferably up to 1 amino acid substitution.

Preferably, at least one of the CDRs of the antibody or fragment has a sequence identity of >

67 %; > 68 %; > 69 %; > 70 %; > 71 %; > 72 %; > 73 %; > 74 %; > 75 %; > 76 %; > 77 %; >

78 %; > 79 %; > 80 %; > 81 %; > 82 %; > 83 %; > 84 %; > 85 %; > 86 %; > 87 %; > 88 %; >

89 %; > 90 %; > 91 %; > 92 %; > 93 %; > 94 %; > 95 %; > 96 %; > 97 %; > 98 %; > 99 %, and most preferably 100 % to the respective SEQ ID NO.

“Percentage of sequence identity” as used herein, is determined by comparing two optimally aligned biosequences (amino acid sequences or polynucleotide sequences) over a comparison window, wherein the portion of the corresponding sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (z.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The disclosure provides polypeptides that are substantially identical to the polypeptides exemplified herein. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.

Preferably, at least one of the CDRs has been subject to CDR sequence modification, including

• affinity maturation

• reduction of immunogenicity

Affinity maturation in the process by which the affinity of a given antibody is increased in vitro. Like the natural counterpart, in vitro affinity maturation is based on the principles of mutation and selection. It has successfully been used to optimize antibodies, antibody fragments or other peptide molecules like antibody mimetics. Random mutations inside the CDRs are introduced using radiation, chemical mutagens or error-prone PCR. In addition, the genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods like phage display usually results in antibody fragments with affinities in the low nanomolar range. For principles see Eylenstein et al. (2016) or US20050169925A1, the content of which is incorporated herein by reference for enablement purposes.

Engineered antibodies contain murine-sequence derived CDR regions that have been engrafted, along with any necessary framework back-mutations, into sequence-derived V regions. Hence, the CDRs themselves can cause immunogenic reactions when the humanized antibody is administered to a patient. Methods of reducing immunogenicity caused by CDRs are disclosed in Harding et al. (2010), or US2014227251A1, the content of which is incorporated herein by reference for enablement purposes.

According to one embodiment, the antibody or fragment or derivative comprises a) the heavy chain/light chain variable domain (HCVD/LCVD) pairs set forth in of SEQ ID NOs 9 and 10 b) the heavy chain/light chain variable domains (HCVD/LCVD) of a), with the proviso that

• the HCVD has a sequence identity of > 80 % to the HCVD comprised in the respective SEQ ID NO, and/or

• the LCVD has a sequence identity of > 80 % to the LCVD comprised in the respective SEQ ID NO, c) the heavy chain/light chain variable domains (HCVD/LCVD) as set forth in option a) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to the HCVD or LCVD comprised in the respective SEQ ID NO, said antibody or fragment being capable to bind to human PD-1.

A “variable domain” when used in reference to an antibody or a heavy or light chain thereof is intended to mean the portion of an antibody which confers antigen binding onto the molecule and which is not the constant region. The term is intended to include functional fragments thereof which maintain some of all of the binding function of the whole variable region. Variable region binding fragments include, for example, functional fragments such as Fab, F(ab)2, Fv, single chain Fv (scFv) and the like. Such functional fragments are well known to those skilled in the art. Accordingly, the use of these terms in describing functional fragments of a heteromeric variable region is intended to correspond to the definitions well known to those skilled in the art. Such terms are described in, for example, Huston et al., (1993) or Pliickthun and Skerra (1990).

Preferably, the HCVD and/or LCVD has a sequence identity of > 81 %; > 82 %; > 83 %; > 84 %; > 85 %; > 86 %; > 87 %; > 88 %; > 89 %; > 90 %; > 91 %; > 92 %; > 93 %; > 94 %; > 95 %; > 96 %; > 97 %; > 98 %; > 99 %; or most preferably 100 % to the respective SEQ ID NO.

The antibody characterized by the HCVD/LCVD is fully human.

According to one embodiment of the antibody or fragment or derivative according to the above description, at least one amino acid substitution is a conservative amino acid substitution.

A “conservative amino acid substitution”, as used herein, has a smaller effect on antibody function than a non-conservative substitution. Although there are many ways to classify amino acids, they are often sorted into six main groups on the basis of their structure and the general chemical characteristics of their R groups.

In some embodiments, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with

• basic side chains (e.g., lysine, arginine, histidine),

• acidic side chains (e.g., aspartic acid, glutamic acid),

• uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),

• nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),

• beta-branched side chains (e.g., threonine, valine, isoleucine) and ^ aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Other conserved amino acid substitutions can also occur across amino acid side chain families, such as when substituting an asparagine for aspartic acid in order to modify the charge of a peptide. Conservative changes can further include substitution of chemically homologous non- natural amino acids (i.e. a synthetic non-natural hydrophobic amino acid in place of leucine, a synthetic non-natural aromatic amino acid in place of tryptophan). According to another aspect of the invention, an antibody or fragment or derivative is provided which has a target binding affinity of ≥ 50 % to human PD-1 compared to that of the antibody or fragment or derivative according to the above description. As used herein the term “binding affinity” is intended to mean the strength of a binding interaction and therefore includes both the actual binding affinity as well as the apparent binding affinity. The actual binding affinity is a ratio of the association rate over the disassociation rate. Therefore, conferring or optimizing binding affinity includes altering either or both of these components to achieve the desired level of binding affinity. The apparent affinity can include, for example, the avidity of the interaction. For example, a bivalent heteromeric variable region binding fragment can exhibit altered or optimized binding affinity due to its valency. A suitable method for measuring the affinity of a binding agent is through surface plasmon resonance (SPR). This method is based on the phenomenon which occurs when surface plasmon waves are excited at a metal/liquid interface. Light is directed at, and reflected from, the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. Biomolecular binding events cause changes in the refractive index at the surface layer, which are detected as changes in the SPR signal. The binding event can be either binding association or disassociation between a receptor-ligand pair. The changes in refractive index can be measured essentially instantaneously and therefore allows for determination of the individual components of an affinity constant. More specifically, the method enables accurate measurements of association rates (k on) and disassociation rates (koff). Measurements of k _on and kotr values can be advantageous because they can identify altered variable regions or optimized variable regions that are therapeutically more efficacious. For example, an altered variable region, or heteromeric binding fragment thereof, can be more efficacious because it has, for example, a higher k_on valued compared to variable regions and heteromeric binding fragments that exhibit similar binding affinity. Increased efficacy is conferred because molecules with higher k_on values can specifically bind and inhibit their target at a faster rate. Similarly, a molecule of the invention can be more efficacious because it exhibits a lower k_oir value compared to molecules having similar binding affinity. Increased efficacy observed with molecules having lower k_oir rates can be observed because, once bound, the molecules are slower to dissociate from their target. Although described with reference to the altered variable regions and optimized variable regions of the invention including, heteromeric variable region binding fragments thereof, the methods described above for measuring associating and disassociation rates are applicable to essentially any antibody or fragment or fragment thereof for identifying more effective binders for therapeutic or diagnostic purposes.

Another suitable method for measuring the affinity of a binding agent is through surface is by FACS/scatchard analysis. Methods for measuring the affinity, including association and disassociation rates using surface plasmon resonance are well known in the arts and can be found described in, for example, Jonsson and Malmquist, (1992) and Wu et al. (1998). Moreover, one apparatus well known in the art for measuring binding interactions is a BIAcore 2000 instrument which is commercially available through Pharmacia Biosensor, (Uppsala, Sweden).

Preferably said target binding affinity is > 51%, > 52%, > 53%, > 54%, > 55%, > 56%, > 57%,

> 58%, > 59%, > 60%, > 61%, > 62%, > 63%, > 64%, > 65%, > 66%, > 67%, > 68%, > 69%,

> 70%, > 71%, > 72%, > 73%, > 74%, > 75%, > 76%, > 77%, > 78%, > 79%, > 80%, > 81%,

> 82%, > 83%, > 84%, > 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%,

> 94%, > 95%, > 96%, > 97%, > 98%, and most preferably > 99 % compared to that of the reference binding agent.

According to another aspect of the invention, an antibody is provided that binds to human PD- 1, and competes for binding to human PD-1 with the antibody according to the above description. According to another aspect of the invention, an antibody is provided that binds to essentially the same, or the same, region on human PD-1 as the antibody according to the above description.

As regards the format or structure of such antibody or fragment, the same preferred embodiments as set forth above apply. In one embodiment, said antibody or fragment is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an antibody mimetic.

As used herein, the term "competes for binding" is used in reference to one of the antibodies defined by the sequences as above, meaning that the actual antibody or fragment as an activity which binds to the same target, or target epitope or domain or subdomain, as does said sequence defined antibody or fragment, and is a variant of the latter. The efficiency (e.g., kinetics or thermodynamics) of binding may be the same as or greater than or less than the efficiency of the latter. For example, the equilibrium binding constant for binding to the substrate may be different for the two antibodies.

Such competition for binding can be suitably measured with a competitive binding assay. Such assays are disclosed in Finco et al. 2011, the content of which is incorporated herein by reference for enablement purposes, and their meaning for interpretation of a patent claim is disclosed in Deng et al 2018, the content of which is incorporated herein by reference for enablement purposes.

In order to test for this characteristic, suitable epitope mapping technologies are available, including, inter alia,

• X-ray co-crystallography and cryogenic electron microscopy (cryo-EM)

• Array-based oligo-peptide scanning

• Site-directed mutagenesis mapping

• High-throughput shotgun mutagenesis epitope mapping

• Hydrogen-deuterium exchange

• Cross-linking-coupled mass spectrometry These methods are, inter alia, disclosed and discussed in Banik et al (2010), and DeLisser (1999), the content of which is herein incorporated by reference for enablement purposes.

According to one embodiment, the human PD-1 to which the antibody or fragment or derivative binds, comprises a) the amino acid sequence set forth in SEQ ID NO: 1 or b) an amino acid sequence that has at least 80 % sequence identity with SEQ ID NO:

1

As disclosed elsewhere herein, SEQ ID NO: 1 is the sequence of part of the extracellular domain of human PD-1. SEQ ID NO: 2 is the sequence of part of the extracellular domain of PD-1 of cynomolgus monkey (Macaca fascicular is).

According to several embodiments of the invention, the antibody or fragment or derivative is in at least one of the formats selected from the group consisting of: IgG, scFv, Fab, (Fab)2, small immunoprotein (SIP), diabody, or a single-chain diabody. These formats are explained elsewhere herein.

According to another aspect of the invention, a nucleic acid is provided that encodes for at least one chain of the protein binder, antibody or fragment or derivative thereof according to the above description.

In one embodiment, a nucleic acid, or a pair of nucleic acids, is provided which encodes e.g. for the heavy chain and the light chain, respectively, of the binding agent, in case the latter is a monoclonal antibody having a heteromeric structure of at least one light chain and one heavy chain.

Such nucleic acid can be used for recombinant production of the protein binder, antibody or fragment or derivative thereof in a suitable expression system, like e,g, CHO cells or E. coli.

Such nucleic acid can also be used for pharmaceutic purposes. The nucleic acid can be an RNA molecule, or an RNA derivative comprising, e.g., modified nucleotides, like pseudouridine (T) orN-1 Methyl Pseudouridine (ml'P) to provide stability and reduce immunogenicity (see, e.g., US8278036 and US9428535, the contents of which are incorporated herein for enablement purposes). In another embodiment, the RNA comprises the most GC-rich codon is selected to provide stability and reduce immunogenicity (see e.g. EP1392341 the content of which is incorporated herein for enablement purposes). The mRNA can for example be delivered in suitable liposomes and comprises either specific sequences or modified uridine nucleosides to avoid immune responses and/or improve folding and translation efficiency, sometimes comprising cap modifications at the 5’- and/or 3 ’ terminus to target them to specific cell types.

The nucleic acid can likewise be a DNA molecule. In such case, the molecule can be a cDNA that is optionally integrated into a suitable vector, e.g., an attenuated, non pathogenic virus, or is provided as one or more plasmids. Such plasmids can for example be administered to a patient by means of an electroporation device as e.g. disclosed in patent EP3397337B1, the content of which is incorporated herein for enablement purposes.

Generally, due to the degeneracy of the genetic code, there is a large number of different nucleic acids that have the capacity to encode for such chain. The skilled person is perfectly able to determine if a given nucleic acid satisfies the above criterion. On the other hand, the skilled person is perfectly able to reverse engineer, from a given amino acid sequence, based on codon usage tables, a suitable nucleic acid encoding therefore. For this purpose, software tools such as “reverse translate” provided by the online tool “sequence manipulation suite”, (https://www.bioinformatics.org/sms2/rev_trans.html) can be used. Hence, there are plenty of alternative DNA and RNA sequences that encode for the protein sequences as claimed. These alternative sequences shall be deemed to fall under the scope of the present invention.

According to another aspect of the invention, an immunocytokine comprising the protein binder, antibody or fragment or derivative according to the above description is provided.

As used herein, the term “immunocytokine” relates to a fusion construct which comprises at least (i) one antibody or fragment or derivative according to the above description and (ii) an immunomodulatory cytokine fused thereto. Such recombinant immunocytokine can be produced in suitable recombinant expression systems without subsequent need to conjugate the cytokine to the antibody or fragment. Immunocytokines can be used to improve site-specific delivery and prolong the cytokine halflife. Immunocytokines are delivered systemically but can specifically target via specific tumor antigens. Suitable cytokines to be fused to the antibody are, inter alia, TNF alpha, IL2 and IL12. In such way, the maximum tolerated dose can be increased, and in IL- 12 was found to be 30 times higher than the maximum tolerated dose of IL- 12 alone.

According to one embodiment, the immunocytokine comprises a cytokine selected from the group consisting of TNF alpha, IL2, IL12, IL15 or IL15 sushi-domain (Xu et al, 2021).

According to another aspect of the invention, a pharmaceutical composition is provided, comprising the protein binder according to the above description, the antibody or fragment or derivative according to the above description, the nucleic acid according to the above description or the immunocytokine according to the above description, and optionally one or more pharmaceutically acceptable excipients.

According to another aspect of the invention, a combination is provided, comprising (i) the protein binder according to the above description, the antibody or fragment or derivative according to the above description, the nucleic acid according to the above description, the immunocytokine according to the above description, or the composition according to the above description, and (ii) one or more therapeutically active compounds.

According to another aspect of the invention, the use of the protein binder according to the above description, the antibody or fragment or derivative according to the above description, the nucleic acid according to the above description, the immunocytokine according to the above description, the composition according to the above description or the combination according to the above description is provided (for the manufacture of a medicament) in the treatment of a human or animal subject

• being diagnosed for,

• suffering from or

• being at risk of developing a neoplastic disease, or for the prevention of such condition.

T1 This language is deemed to encompass both the swiss type claim language accepted in some countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets is deemed absent).

According to another aspect of the invention, a method for treating or preventing a neoplastic disease is provided, which method comprises administration, to a human or animal subject, the protein binder according to the above description, the antibody or fragment or derivative according to the above description, the nucleic acid according to the above description, the immunocytokine according to the above description, the composition according to the above description, or the combination according to the above description, in a therapeutically sufficient dose.

According to another aspect of the invention, a therapeutic kit of parts is provided, comprising: a) the protein binder according to the above description, the antibody or fragment or derivative according to the above description, the nucleic acid according to the above description, the immunocytokine according to the above description, the composition according to the above description, or the combination according to the above description, b) an apparatus for administering the protein binder, antibody or fragment or derivative, nucleic acid, immunocytokine, composition or combination, and c) instructions for use.

EXAMPLES

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

Example 1: Cloning of Programmed Cell Death Protein 1 (PD-1) including characterization, phage display selection against antigen and isolation of Dll antibody in scFv format

1.1 Expression of the antigen

A human PD-1 -ECD (extracellular domain) recombinant fragment containing a C-terminal 6xHis tag and a Bir A target sequence (GLNDIFEAQKIEWHE, SEQ ID NO: 28)) was expressed using transient gene expression (TGE) in CHO-S cells using Polyethyleneimine (PEI). The protein fragment was purified from the cell culture medium by nickel affinity chromatography and dialyzed into phosphate buffered saline (PBS, pH7.4).

1.2 Antigen biotinylation

The purified PD-1 protein was site specifically biotinylated using BirA (E. coli biotin ligase) following the protocol of Fairhead et al. The protein was first dialyzed in BirA buffer (lOOmM Tris PH 7.5, 200mM Nacl, 5mM MgCl). Biotinylation reaction was performed for 24 hours by adding Img of protein, 28uL of 40mM Biotin, 43ug of BirA, 70uL of 0.5M ATP and protease inhibitors. After 24 hours, the protein was purified by size exclusion chromatography and dialyzed back in PBS buffer, pH 7.4.

1.3 Antigen characterization

The recombinantly produced PD-1 protein was analyzed by SDS-PAGE and by size exclusion chromatography using a Superdex 200 increase 10/300 GL column on an AKTA FPLC.

1.4 Isolation of scFv antibody fragments from phage display library

Fully human monoclonal antibodies specific to the extracellular domain of human PD-1 were isolated from a scFv phage library, following a previously described protocol (Viti et al., (2000). Briefly, 120 pmol of biotinylated huPD-1 were immobilized on 60 pL streptavidin- coated magnetic beads. After blocking in 4% milk-phosphate buffered saline (PBS), 800 pL of scFv displaying phage were added (10¹² transforming units of phage/ml) and incubated for 1 ^x/i hours. Beads were washed six times with 0.1% Tween 20 in PBS and subsequently six times with PBS. Bound phage was eluted with triethylamine and amplified in E. coli TG-1 using VCS-M13 Interference-Resistant Helper Phage. Phage particles were precipitated from culture supernatant using 20% polyethylene glycol/2.5 M NaCl. Individual colonies were inoculated in 2YT media supplemented with 100 pg/mL ampicillin.

After two rounds of bio-panning, the clone D12 was selected for further exploration based on its ELISA signal.

1.5 Expression and purification of scFv antibody fragments

D12 was produced as a scFv antibody fragment in E. Coli strain TG-1 by inoculating a single fresh colony in 10 mL 2YT medium (100 pg/ml ampicillin, and 1% glucose). The pre-culture was grown overnight at 37°C, then diluted 1 : 100 in 800ml 2YTmedium (100 pg/ml ampicillin, 0.1% glucose) and grown at 37°C until reaching the exponential growth phase. scFv production was induced by the addition of ImM IPTG and grown at 30°C overnight. The scFv fragments were purified from the bacterial supernatant by affinity chromatography using Protein A Sepharose and dialyzed into PBS, pH7.4. The D12 scFv was then characterized by size exclusion chromatography using a Superdex 75 increase 10/300 GL column on an AKTA FPLC. SDS-PAGE analysis was also performed with 4-12% Bis-Tris gel under reducing and non-reducing conditions. Results are shown in Figure 1.

1.6 Affinity measurement

The affinity of D12 in monomeric scFv format was measured by SPR on a BIAcore X100 system. PD-1 -coated chips were prepared by coupling biotinylated antigen to a streptavidin coated sensor chip, yielding a density of 800 RU. Monomeric preparations of scFv fragments were prepared by gel filtration on a Superdex 75 increase 10/300G1 column and analyzed in serial 2-fold dilutions at a flow rate of 10 pl/min. As negative control, KSF (scFv), an antibody specific to hen egg lysozyme, was used. The binding curves were analyzed with the BIAevaluation 3.2 software (Figure 3 A).

1.7 Reformatting of D12 scFv into IgGl format

The D 12 scFv fragments were used as template to reformat it into IgG format. First, the leader sequence for secretion was added 5’ of the variable heavy chain by PCR and the construct was then inserted into the mammalian expression vector pMM137 via double digestion using Hindlll and Xhol as restriction enzymes. In a second step, the same leader sequence was added 5’ of the variable light chain via PCR and the construct inserted into the pMM137 vector already carrying the heavy chain, by first digesting it with BsiWI and then Spel. The amino acid sequence of the D12 antibody in IgGl format is shown in SEQ ID NOs 13 and 14.

1.8 Characterization of the D12 antibody in IgGl format

Antibody D12 in IgGl format was expressed by transient gene expression in CHO-S cells using Polyethyleneimine (PEI) and purified by protein A affinity chromatography after 6 days. The antibody was dialyzed into PBS pH7.4 and characterized by SDS-PAGE and size-exclusion chromatography using a Superdex 200 increase 10/300 GL column on an AKTA FPLC as described above. Results are shown in Figure 2.

1.9 Cross-reactivity of D12 with cynomolgus monkey PD-1

Cross-reactivity of D12 with cynomolgus monkey PD-1 (cmPD-1) was investigated by surface plasmon resonance. cmPD-1 was recombinantly expressed in CHO-S cells by TGE using PEI and purified by affinity chromatography after 6 days. The protein was site-specifically biotinylated as described above and coated on a streptavidin sensor chip yielding a final density of 800RU. D12 in IgGl format was run in 2-fold serial dilutions on a BIAcore X100 as described above. Results are shown in Figure 3B.

Example 2: Competition ELISA

Blockage of PD-1 binding to its natural receptors PD-L1 and PD-L2 by D12 was investigated by a competition ELISA. Briefly, antibodies were mixed in decreasing concentrations with 3xl0'⁸ M of either huPD-LlmFc or huPD-L2mFc and added to a 96 well plate coated with biotinylated huPD-1 (IxlO^-8 M). Binding of huPD-LlmFc or huPD-L2mFc was detected using a horseradish peroxidase (HRP)-conjugated goat anti-mouse Fc-specific polyclonal antibody and developed using a POD colorimetric substrate. Absorbance at 420 vs. 620 nm was detected on a microplate reader and plotted as a function of the anti-PD-1 antibody concentrations using Prism 7.0. The KSF antibody that binds to hen-egg-lysozyme was used as a negative control antibody. Results are shown in Figure 4.

Example 3: Binding to PD-1 expressed on cells 3.1 Binding to transfected HEK cells

Binding of D12 to PD-1 expressed on cells was investigated by flow cytometry. As a first step, HEK cells were transiently transfected, using jetPRIME and a pcDNA3.1(+) plasmid encoding full length human PD-1. Flow cytometry was performed 24 h to 48 h after transfection. Briefly, cells were stained with anti-PD-1 antibody for 30 min on ice. Binding of anti-PD-1 antibodies was detected using 5 pg/mL Protein A-Alexa488 and analyzed using a Cytoflex flow cytometer. (Figure 5).

3.2 Binding to primary human T cells

Blood was collected from healthy human donors in 10 mL heparin tubes and diluted 1 : 1 with PBS pH 7.4 (2mM EDTA). PBMCs were isolated by density centrifugation using Ficoll paque (Cytiva). T cells were separated from the PBMC mix by negative magnetic cell sorting, using the Pan T cell Isolation Kit, following the manufacturer’s instructions. T cells were cultivated in Advanced RPMI media supplemented with 10% FBS, 2mM Ultraglutamine and 1% Anti- Anti. To increase PD-1 expression levels, half of the cells were activated using CD3/CD28 dynabeads. After 72h, dynabeads were removed from the cells by magnetic separation and activated as well as non-activated T cells analyzed by flow cytometry. T cell were stained with fluorescently labeled antibodies (fluorescein isothiocyanater). Fluorescent signal was amplified by incubation with a rabbit anti-FITC antibody, followed by a goat anti-rabbit Alexa- 488 conjugated antibody. Flow cytometric analysis was performed as described above (Figure 5).

Example 4: Structural determination of Dll Fab bound to PD-1

4.1 In vitro reconstitution and size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis

Purified extracellular PD-1 and D12 Fab were mixed at a 1 : 1.2 molar concentration ratio, incubated for 2 hours and subjected to analytical and/or preparative size exclusion chromatography (SEC) to optimize the in vitro reconstitution of antigemantibody complexes. Preparative reconstitutions were carried out by loading samples onto a Superdex 200 Increase 10/300 GL connected to an Akta Purifier Fast Protein Liquid Chromatography (FPLC). SEC runs were performed at a flow rate of 0.75 mL/min in PBS, pH 7.4. For analytical SEC-MALS, samples were loaded onto a Protein KW.802.5 column mounted on a Prominence high-pressure liquid chromatography (HPLC) system connected to a miniDAWN MALS detector, a differential refractive index detector for quantitation of the total mass and to a UV detector for evaluation of the sole protein content. SEC-MALS runs were carried out at a flow rate of 1 mL/min in PBS. Chromatograms from PD-1 :D12 reconstituted complexes were compared with runs performed using individual PD-1 and D12 Fab components. Results were analyzed using the protein conjugate module of the Astra software, using an estimated dn/dc value of 0.185 ml/g for proteins and 0.140 ml/g for glycans. The calibration of the instrument was verified by injection of 10 pl of 3 mg/1 monomeric BSA. Results are shown in Figure 6.

4.2 Crystallization and structure determination

Reconstituted PD-ED12 Fab complexes were concentrated to 11.6 mg/mL and were used to set up crystallization experiments using commercial sparse-matrix crystallization screens using the sitting-drop method using a Gryphon robotic nanodispenser. Initial hits were optimized manually using the hanging drop method by mixing 0.1 mL of PD-ED12 complex with an equal volume of a reservoir composed of 22% PEG 3350, 250 mM sodium malonate, pH 4.5. Crystals were harvested using litholoops, flash-cooled in liquid nitrogen and sent to the European Synchrotron Radiation Facility (ESRF). X-ray diffraction data were collected at ESRF ID30A-1 (MASSIF-1) from a single PD-1 :D12 crystal. Data processing and scaling were carried out using XDS and AIMLESS. The structures were solved by molecular replacement (MR) using PHASER using the crystal structure of PD-1 from its complex with nivolumab Fab (PDB ID 5WT9) and a computational model of D12 obtained using Alphafold2. Structure refinement was performed using phenix. refine aided by extensive non-crystallographic symmetry density averaging using PARROT and manual model (re)building using COOT. Molprobity and the PDB validation tools were used for structure validation. Final data collection and refinement statistics are shown in Table 3. Structural figures were prepared using Pymol (Figures 7-9).

Table 3: X-ray data collection and refinement statistics

Data Collection⁰

X-ray source ESRF ID30A-1 (MASSIF-1) Processing programs XDS, AIMLESS Space group P2i a = 81.2 A; a = 90.0°

Cell parameters b = 187.1 A; P = 90.2° c = 190.8 A; y = 90.0°

Wavelength (A) 0.965 Resolution (A) 190.84-3.5 (3.58-3.50) Total reflections 117360 (8267) Unique reflections 68755 (4708)

CCl/2^b 0.940 (0.431)

Redundancy 1.7 (1.8) Mean l/o(l) 3.9 (1.1) Completeness (%) 96.1 (97.6)

Rsym^C 0.141 (0.642) . d fApim 0.100 (431)

Refinement

Rwork/Rfree^e 0.2432/0.2929

Average B-factor (A)² 99.6

Number of non-H atoms: 33524

Protein 32856

Ligands (Glycosylations) 668

Structure quality

RMS bond lengths (A) 0.004 RMS bond angles (°) 0.70 Ramachandran Favored (%) 95.7 Ramachandran allowed (%) 4.1 0.2

Ramachandran outliers (%) ^a Values in parentheses are for reflections in the highest resolution shell. ^b Resolution limits were determined by applying a cut-off based on the mean intensity correlation coefficient of half-datasets (CCI/2) approximately of 0.5. ^c Rsym = [ ShkiSj | Ihkij - <lhki> | ] / [ ShkiSj Ihkij ] , where I is the observed intensity for a reflection and <l> is the average intensity obtained from multiple observations of symmetry-related reflections. d Rpim ⁼ [ £hkl (l/(n-l))^1/2 | Ihkij ^— < I hki> I ] / [ £hk£j Ihkij ] where I is the observed intensity for a reflection and <l> is the average intensity obtained from multiple observations of symmetry-related reflections. ^e Rfree values are calculated based on 5% randomly selected reflections.

Table 4: amino acid numbering in Figure 7: transposition to SEQ ID NO:1.

Q15116).

Example 5: Generation of IL12-D12 conjugate.

The IL12-D12 conjugate contained the human IL12 fused to the N-terminus of the D12 antibody in scDb format (SEQ ID NO 18). The gene encoding for the D12 scDb (SEQ ID NO 16) was PCR amplified and fused to a genestrand encoding for human IL 12 (SEQ ID NO: 20) by PCR assembly. The resulting PCR product was double digested using Sall and Notl restriction enzymes and cloned into the mammalian expression vector pcDNA3.1(+). The conjugate was expressed by transient gene expression in CHO cells as described previously and purified from the cell culture medium to homogeneity by protein A affinity chromatography. IL12-D12 conjugate was analyzed by size-exclusion chromatography using a Superdex 200 increase 10/300 GL column on an Akta Pure FPLC (Figure 10A). Correct size of the conjugate was investigated by SDS-PAGE using 4-12% Bis-Tris gels under reducing and non-reducing conditions (Figure 10B).

References

• Kohler, G. & Milstein, C. (1975): Continuous cultures of fused cells secreting antibody of predefined specificity. In: Nature. Bd. 256, S. 495-497.

• Jonsson and Malmquist, Advances in Biosensors, 2:291-336 (1992)

• Wu et al. Proc. Natl. Acad. Sci. USA, 95:6037-6042 (1998)

• Banik, SSR; Doranz, BJ (2010). "Mapping complex antibody epitopes". Genetic Engineering & Biotechnology News. 3 (2): 25-8

• DeLisser, HM (1999). Epitope mapping. Methods Mol Biol. 96. pp. 11-20

• Finco et al, Comparison of competitive ligand-binding assay and bioassay formats for the measurement of neutralizing antibodies to protein therapeutics. J Pharm Biomed Anal. 2011 Jan 25;54(2):351-8.

• Deng et al., Enhancing antibody patent protection using epitope mapping information MAbs. 2018 Feb-Mar; 10(2): 204-209

• Huston et al., Cell Biophysics, 22: 189-224 (1993); • Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990)

• Harding, The immunogenicity of humanized and fully human antibodies. MAbs. 2010 May-Jun; 2(3): 256-265.

• Eylenstein, et al, Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti -human GM-CSF antibody, mAbs, 8: 1, 176-186 (2016)

• Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991)

• Chothia et al., J. Mol. Biol. 196:901-917 (1987)

• Chen, J. et al., Medicine (Baltimore), 2021. 100(15): p. e25180

• Ma M, Qi H, Hu C, Xu Z, Wu F, Wang N, Lai D, Li Y, Zhang H, Jiang H, Meng Q, Guo S, Kang Y, Zhao X, Li H, Tao SC. The binding epitope of sintilimab on PD-1 revealed by AbMap. Acta Biochim Biophys Sin (Shanghai). 2021 Apr 15;53(5):628- 635.

• Park UB, Jeong TJ, Gu N, Lee HT, Heo YS. Molecular basis of PD-1 blockade by dostarlimab, the FDA-approved antibody for cancer immunotherapy. Biochem Biophys Res Commun. 2022 Apr 9;599:31-37. doi: 10.1016/j.bbrc.2022.02.026. Epub 2022 Feb 9. PMID: 35168061

• Xu, H., Shi, M., Shao, C. et al. Development of IL-15/IL-15Ra sushi domain-IgG4 Fc complexes in Pichia pastoris with potent activities and prolonged half-lives. Microb Cell Fact 20, 115 (2021).

• Fairhead et al., Methods Mol Biol. 2015;1266: 171-84. doi: 10.1007/978-1-4939-2272- 7_12

• Viti et al., Methods Enzymol, (2000), 326, 480-505)

• Carretero-Gonzalez A, Lora D, Ghanem I, Zugazagoitia J, Castellano D, Sepulveda JM, Lopez-Martin JA, Paz-Ares L, de Velasco G. Analysis of response rate with ANTI PD1/PD-L1 monoclonal antibodies in advanced solid tumors: a meta-analysis of randomized clinical trials. Oncotarget. 2018 Jan 20;9(9):8706-8715.

• Fenwick C, Loredo-Varela JL, Joo V, Pellaton C, Farina A, Rajah N, Esteves- Leuenberger L, Decaillon T, Suffiotti M, Noto A, Ohmiti K, Gottardo R, Weissenhorn W, Pantaleo G. Tumor suppression of novel anti -PD-1 antibodies mediated through CD28 costimulatory pathway. J Exp Med. 2019 Jul 1 ;216(7): 1525-1541. SEQUENCES

The following sequences form part of the disclosure of the present application. A WIPO ST 26 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones. In some cases, the signal peptides may be encompassed in the reproduced sequences. In such case, the sequences shall be deemed disclosed with and without signal peptides. A readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https://services.healthtech.dtu.dk/service.php7SignalP

Table 4: Sequences

Claims

What is claimed is:

1. A protein binder which binds to human PD-1 within a discontinuous epitope comprising at least the residues Trp9, Prol 1 and Leul 15 of PD-1 according to SEQ ID NO: 1.

2. The protein binder according to claim 1, which binds at least a conformational epitope of human PD-1 spanning within the region of amino acid residues 7-11 and 112-116 according to SEQ ID NO: 1.

3. The protein binder according to claim 1, which is a monoclonal antibody, or a targetbinding fragment or derivative thereof retaining target binding capacities, or an antibody mimetic.

4. An antibody or a target-binding fragment or derivative thereof retaining target binding capacity to human PD-1 which a) comprises a set of six heavy chain/light chain complementarity determining regions (CDR) comprised in the heavy chain/light variable domain sequences of SEQ ID NOs 9 and 10 b) comprises a set of six heavy chain/light chain complementarity determining regions (CDR) in the order HCDR1; HCDR2; HCDR3; LCDR1; LCDR2 and LCDR3, as set forth in SEQ ID NOs 3, 4, 5, 6, 7, and 8 c) comprises a set of heavy chain/light chain complementarity determining regions (CDR) as set forth in option b), with the proviso that at least one of the CDRs has up to 3 amino acid substitutions relative to the CDRs comprised in the respective SEQ ID NOs, and/or d) comprises a set of heavy chain/light chain complementarity determining regions (CDR) as set forth in option b) or c), with the proviso that at least one of the CDRs has a sequence identity of > 66 % to the CDRs comprised in the respective SEQ ID NOs, wherein the CDRs are embedded in a suitable protein framework so as to be capable to bind to human PD-1. The antibody or fragment or derivative according to claim 4, which comprises a) the heavy chain/light chain variable domain (HCVD/LCVD) pairs set forth in of SEQ ID NOs 9 and 10 b) the heavy chain/light chain variable domains (HCVD/LCVD) of a), with the proviso that

• the HCVD has a sequence identity of > 80 % to the HCVD comprised in the respective SEQ ID NO, and/or

• the LCVD has a sequence identity of > 80 % to the LCVD comprised in the respective SEQ ID NO, c) the heavy chain/light chain variable domains (HCVD/LCVD) as set forth in option a) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to the HCVD or LCVD comprised in the respective SEQ ID NO, said antibody or fragment being capable to bind to human PD-1. The antibody or fragment or derivative according to any one of claims 4 - 5, wherein at least one amino acid substitution is a conservative amino acid substitution. An antibody or fragment or derivative which has a target binding affinity of > 50 % to human PD-1 compared to that of the antibody or fragment or derivative according to any one of claims 4 - 6. An antibody that binds to human PD-1, and competes for binding to human PD-1 with the antibody according to any one of claims 4 - 6. An antibody that binds to essentially the same, or the same, region on human PD-1 as the antibody according to any one of claims 4 - 6. The antibody or fragment or derivative according to any one of claims 4 - 9, wherein the human PD-1 to which the antibody or fragment or derivative binds, comprises a) the amino acid sequence set forth in SEQ ID NO: 1 or b) an amino acid sequence that has at least 80 % sequence identity with SEQ ID NO: 1 The antibody or fragment or derivative according to any one of claims 4 - 10, which is in at least one of the formats selected from the group consisting of: IgG, scFv, Fab, (Fab)2, small immunoprotein (SIP), diabody, a single-chain diabody. A nucleic acid that encodes for at least one chain of the protein binder, antibody or fragment or derivative thereof according to any one of the aforementioned claims. An immunocytokine comprising the protein binder, antibody or fragment or derivative according to any one of the aforementioned claims. The immunocytokine according to claim 13, which comprises a cytokine selected from the group consisting of TNF alpha, IL2, IL12, IL15 or IL15 sushi-domain. A pharmaceutical composition comprising the protein binder according to any one of claims 1 - 3, the antibody or fragment or derivative according to any one of claims 4 - 11, the nucleic acid according to claim 12 or the immunocytokine according to claim 13 or 14, and optionally one or more pharmaceutically acceptable excipients. A combination comprising (i) the protein binder according to any one of claims 1 - 3, the antibody or fragment or derivative according to any one of claims 4 - 11, the nucleic acid according to claim 12, the immunocytokine according to claim 13 or 14, or the composition according to claim 15, and (ii) one or more therapeutically active compounds. Use of the protein binder according to any one of claims 1 - 3, the antibody or fragment or derivative according to any one of claims 4 - 11, the nucleic acid according to claim 12, the immunocytokine according to claim 13 or 14, the composition according to claim 15 or the combination according to claim 16 (for the manufacture of a medicament) in the treatment of a human or animal subject

• being diagnosed for,

• suffering from or

• being at risk of developing a neoplastic disease, or for the prevention of such condition. A method for treating or preventing a neoplastic disease, which method comprises administration, to a human or animal subject, the protein binder according to any one of claims 1 - 3, the antibody or fragment or derivative according to any one of claims 4 - 11, the nucleic acid according to claim 12, the immunocytokine according to claim 13 or 14, the composition according to claim 15, or the combination according to claim 16, in a therapeutically sufficient dose. A therapeutic kit of parts comprising: a) the protein binder according to any one of claims 1 - 3, the antibody or fragment or derivative according to any one of claims 4 - 11, the nucleic acid according to claim 12, the immunocytokine according to claim 13 or 14, the composition according to claim 15, or the combination according to claim 16, b) an apparatus for administering the protein binder, antibody or fragment or derivative, nucleic acid, immunocytokine, composition or combination, and c) instructions for use.