EA043633B1 - ANTI-PD-1 ANTIBODIES AND THEIR COMPOSITIONS - Google Patents

Description

Cross reference to related applications

This application claims benefit to U.S. Provisional Patent Application No. 62/424,163, filed November 18, 2016, the entire contents of which are incorporated herein by reference.

List of sequences

The present application contains a sequence listing which is electronically adopted in ASCII format and the entire contents of which are therefore incorporated by reference. The electronic copy of the sequence list, created on November 13, 2017, is named 022675_WO056_SL.txt and has a size of 99158 bytes.

BACKGROUND OF THE INVENTION

PD-1, also known as programmed cell death protein 1 and CD279, is a 268 amino acid cell surface receptor that belongs to the immunoglobulin superfamily. PD-1 is a member of the CD28 family of T cell regulators and is expressed on T cells, B cells, and macrophages. It binds ligands PD-L1 (also known as B7 homolog) and PD-L2 (also known as B7-DC).

PD-1 is a type I membrane protein whose structure includes an extracellular IgV domain, a transmembrane region, and an intracellular tail containing two phosphorylation sites. Known as an immune checkpoint protein, PD-1 functions as an inducible immunomodulatory receptor, playing a role, for example, in the negative regulation of T cell responses to antigen stimulation.

PD-L1 is the predominant ligand for PD-1. Binding of PD-L1 to PD-1 inhibits T cell activity by reducing cytokine production and suppressing T cell proliferation. Cancer cells expressing PD-L1 are capable of using this mechanism to inactivate the antitumor activity of T cells through binding of PD-L1 to the PD-1 receptor.

Given its properties in regulating the immune response, PD-1 has been explored as a potential target for immunotherapy, including the treatment of malignancies and autoimmune diseases. Two anti-PD-1 antibodies, pembrolizumab and nivolumab, are approved in the United States and Europe for the treatment of certain malignancies.

Given the critical role of PD-1 as an immunomodulator, there is a need for new and improved immunotherapies targeting the PD-1 receptor for the treatment of malignancies and certain immune system disorders.

The essence of the invention

The present invention relates to new recombinant antibodies targeting PD-1, as well as pharmaceutical compositions containing one or more of these antibodies, and the use of antibodies and pharmaceutical compositions to enhance immunity in a patient, and for the treatment of malignant tumors originating from tissues such as skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus and pancreas. Compared to currently available treatments for such cancers, including antibody treatments, it is contemplated that the antibodies of the invention may provide superior clinical response either alone or in combination with another anticancer drug, such as an antibody directed against another immune protein. control point.

In one embodiment, the present invention provides an anti-PD-1 antibody or an antigen-binding portion thereof, wherein the antibody competes for binding to human PD-1 with any of the antibodies or binds the same human PD-1 epitope as any of the antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413 and 18483.

In one embodiment, the anti-PD-1 antibody competes for binding to human PD-1 with an antibody whose heavy chain (H) CDR1-3 and light chain (L) CDR1-3 are the same as or derived from H-CDR1-3 and L-CdRI-3 antibodies 18040, 18o49, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483.

In one embodiment, an anti-PD-1 antibody binds the same human PD-1 epitope as an antibody whose heavy chain (H) CDR1-3 and light chain (L) CDR1-3 are the same as or derived from H- CDR1-3 and L-CDR1-3 antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413 or 18483.

In one embodiment, the anti-PD-1 antibody comprises H-CDR1-3 containing the amino acid sequences of H-CDR1-3, respectively antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483.

In one embodiment, the anti-PD-1 antibody has at least 90% heavy chain variable (VH) domain (e.g., at least 92%, at least 95%, at least 98%, or at least at least 99%) identical in amino acid sequence to the VH domain of antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413 or 18483.

- 1 043633

In one embodiment, the anti-PD-1 antibody has a VH comprising the amino acid sequence of antibody VH 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483.

In one embodiment, the anti-PD-1 antibody has a heavy chain (HC) comprising the amino acid sequence of antibody VH 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483 and the amino acid sequence of the constant areas of severe chains from SEQ ID NO: 26.

In one embodiment, the anti-PD-1 antibody comprises L-CDR1-3 containing the amino acid sequences of L-CDR1-3, respectively antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483.

In one embodiment, the anti-PD-1 antibody has at least 90% light chain variable domain (VL) (e.g., at least 92%, at least 95%, at least 98%, or at least at least 99%) identical in amino acid sequence to the VL domain of antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413 or 18483.

In one embodiment, the anti-PD-1 antibody has a VL comprising the amino acid sequence of antibody VL 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483.

In one embodiment, the anti-PD-1 antibody has a light chain (LC) comprising the amino acid sequence of antibody VL 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483 and the amino acid constant sequence no area of lungs chains from SEQ ID NO: 28.

In one embodiment, the anti-PD-1 antibody comprises the H-CDR3 and L-CDR3 amino acid sequences of antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483.

In one embodiment, the anti-PD-1 antibody comprises the amino acid sequences H-CDR1-3 and L-CDR1-3 of antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483.

In one embodiment, the anti-PD-1 antibody has a VH and a VL of at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99% ) antibodies identical in amino acid sequence to VH and VL, respectively, 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413 or 18483.

In one embodiment, the anti-PD-1 antibody has a VH and a VL containing or consisting of the amino acid sequences VH and VL, respectively, antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483 .

In one embodiment, the anti-PD-1 antibody has an HC and LC containing or consisting of the amino acid sequences of HC and LC, respectively, antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, or 18483 .

In one embodiment, the anti-PD-1 antibody has (1) an HC comprising the amino acid sequence of a VH antibody selected from the group consisting of antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, and 18483, and the amino acid sequence of the heavy chain constant region of SEQ ID NO: 26; and (2) LC containing the VL amino acid sequence of the selected antibody and the light chain constant region amino acid sequence of SEQ ID NO: 28.

In specific embodiments, an anti-PD-1 antibody or antigen-binding moiety of the invention has at least one of the following properties:

a) binds to cynomolgus PD-1 with a K _D of, for example, 4x10 ^-8 M or less;

b) binds to mouse PD-1 with a K _{D of} , for example, 2x10 ^-8 M or less;

c) binds to human PD-1 with a K _D of 3x10' ⁹ M or less;

d) inhibits the interaction of PD-1 with PD-L1 at a concentration of 10 μg/ml;

e) stimulates IL-2 production in the whole blood SEB assay; And

f) stimulates IFN-γ production in a one-way mixed lymphocyte culture reaction assay.

Examples of such an antibody include, but are not limited to, antibodies 18040, 18049, 18098, 18113, 18247, 18250, 18325, 18366, 18413, and 18483 (having properties a) and c)-f)), and antibodies 18201 and 18400 (having properties a)-f )). In some embodiments, an anti-PD-1 antibody or antigen-binding moiety of the invention has all of these properties.

In some embodiments, the anti-PD-1 antibody or antigen-binding moiety of the invention competes for binding to human PD-1 with antibody 18366, 18250, 18040, 18247, 18113, and/or 18483. In some embodiments, the anti-PD-1 antibody or antigen-binding moiety according to the invention competes for binding to human PD-1 with antibody 12760 and/or 13112.

In specific embodiments, the anti-PD-1 antibody or antigen-binding moiety

- 2043633 invention does not compete for binding to PD-1 with pembrolizumab or nivolumab. In specific embodiments, the anti-PD-1 antibody or antigen-binding moiety of the invention does not bind to the same epitope as pembrolizumab or nivolumab.

In another aspect, the present invention provides pharmaceutical compositions comprising at least one anti-PD-1 antibody or antigen-binding portion thereof, as described herein, and a pharmaceutically acceptable excipient, optionally with an additional drug, such as an anti-cancer therapeutic antibody.

In addition, the present invention relates to isolated nucleic acid molecules containing a nucleotide sequence encoding a heavy chain or an antigen binding portion thereof, a nucleotide sequence encoding a light chain or an antigen binding portion thereof, or both, from an anti-PD-1 antibody as described herein .

The present invention also relates to vectors containing such an isolated nucleic acid molecule, wherein said vector may further comprise an expression control sequence.

The present invention also relates to host cells containing a nucleotide sequence encoding a heavy chain or an antigen binding portion thereof, a nucleotide sequence encoding a light chain or an antigen binding portion thereof, or both, from an anti-PD-1 antibody as described herein.

The present invention also relates to a method for producing an antibody or an antigen binding portion thereof, as described herein, comprising obtaining a host cell containing a nucleotide sequence encoding a heavy chain or an antigen binding portion thereof, and a nucleotide sequence encoding a light chain or an antigen binding portion thereof, from anti-PD-1 antibodies as described herein, culturing said host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion.

The present invention also provides a multispecific (e.g., bispecific) binding molecule having the binding specificity of an anti-PD-1 antibody described herein and the binding specificity of another, different antibody, such as another anti-PD-1 antibody (e.g., as described herein ) or an antibody that targets a distinct protein, such as another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity mediates a disease state, such as a cancer. In some embodiments, the multispecific (eg, bispecific) binding molecule comprises antigen binding portions (eg, six CDRs) of an anti-PD-1 antibody and another antibody.

The present invention also provides a method of enhancing immunity in a patient (eg, a human patient), comprising administering to said patient an anti-PD-1 antibody or antigen-binding portion thereof, a pharmaceutical composition, or a bispecific binding molecule as described herein.

In addition, the present invention provides a method of treating a cancer in a patient (eg, a human patient), comprising administering to said patient an anti-PD-1 antibody or antigen-binding portion thereof, a pharmaceutical composition, or a bispecific binding molecule as described herein. In some embodiments, the malignant tumor originates from tissue selected from skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bladder, breast, stomach , uterus and pancreas. The malignant tumor may be, for example, advanced or metastatic melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, bladder cancer, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, colorectal cancer or Hodgkin's lymphoma.

Any of the above methods may further include administering, for example, a chemotherapeutic agent, an antineoplastic agent, an antiangiogenic agent, a tyrosine kinase inhibitor, a PD-1 pathway inhibitor, or radiotherapy.

In addition, the present invention relates to the use of an antibody composition containing an anti-PD-1 antibody or an antigen-binding moiety as described herein for the manufacture of a medicament for treating cancer in a patient and/or enhancing immunity in a patient.

In addition, the present invention provides an anti-PD-1 antibody or antigen-binding moiety as described herein for use in treating a cancer in a patient and/or enhancing immunity in a patient, for example, in a method of treatment described herein.

In addition, the present invention relates to an article containing an anti-PD-1 antibody or an antigen-binding moiety as described herein, wherein said article is suitable for treating a cancer in a patient and/or enhancing immunity in a patient, e.g.

- 3 043633 measures, in the method of treatment described in the present description.

Brief description of drawings

In fig. 1A-1D show representative flow cytometry dot plots of four anti-PD-1 antibody clones having varying reactivity toward PD-1 orthologues.

In fig. 2A-2H show the blocking of PD-L1 binding to cell-expressed PD-1 for anti-PD-1 antibodies of the invention.

In fig. 3A-3F show dose response curves for twelve anti-PD-1 antibodies in a whole blood SEB assay.

In fig. 4A-4F show dose response curves for twelve anti-PD-1 antibodies in the MLR (one-way mixed lymphocyte culture response) assay.

In fig. Figure 5 shows an overview of the identified epitope groups (epitope groups) for the tested anti-PD-1 antibodies and analogues of nivolumab and pembrolizumab. Antibodies connected by black lines exhibit cross-blocking activity. Antibodies are grouped according to patterns of competition with other anti-PD-1 antibodies. Nivol. Nivo: nivolumab analogue; Pembrol. Pembro: analogue of pembrolizumab.

Detailed Description of the Invention

The present invention relates to novel anti-human PD-1 antibodies that can be used to enhance the immune system in a human patient, such as a cancer patient. Unless otherwise specified, for the purposes of the invention, PD-1 refers to human PD-1.

The human PD-1 polypeptide sequence is available from Uniprot under accession number Q15116 (PDCD1 HUMAN).

The term antibody (Ab) or immunoglobulin (Ig) as used herein refers to a tetramer containing two heavy (H) chains (approximately 50-70 kDa) and two light (L) chains (approximately 25 kDa) linked by disulfide bonds. Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs (H-CDR as used herein refers to a heavy chain CDR; and L-CDR as used herein as a light chain CDR) and four FRs arranged from the amino terminus to the carboxy terminus in the following order : FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Assignment of amino acid numbers in the heavy or light chains can be done in accordance with the IMGT® definitions (Lefranc et al., Dev. Comp. Immunol., 27(1):55-77 (2003)); or with definitions from Rabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987); or Chothia et al., Nature, 342:878-883 (1989).

The term recombinant antibody refers to an antibody expressed in a cell or cell line containing nucleotide sequence(s) encoding the antibody, wherein said nucleotide sequence(s) are not naturally associated with the cell.

The term isolated protein, isolated polypeptide or isolated antibody refers to a protein, polypeptide or antibody that, by virtue of its origin or source of production (1) is not associated with naturally associated components accompanying it in its natural state;

(2) are free from other proteins from the same species;

(3) expressed in a different form; and/or (4) do not occur in nature.

Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the cell from which it naturally occurs is isolated from its naturally associated components. The protein can also be made substantially free of its naturally associated components through isolation using protein purification methods well known in the art.

As used herein, the term germline refers to the nucleotide and amino acid sequences of antibody genes and gene fragments as they are transmitted from parents to offspring via germ cells. The germline sequences are distinct from the nucleotide sequences encoding antibodies in mature B cells, which are modified through recombination and hypermutation events during B cell maturation. An antibody that uses a particular germline sequence has a nucleotide or amino acid sequence that aligns more closely with the germline nucleotide sequence or the amino acid sequence it defines than with another germline nucleotide or amino acid sequence.

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The term affinity refers to the rate of attraction of antigen and antibody. The inherent attractiveness of an antibody to an antigen is typically expressed in the form of the equilibrium binding affinity constant (K _D ) for a particular antibody-antigen interaction. An antibody is said to specifically bind to an antigen when the K _D is <1 mM, preferably <100 nM. The binding affinity constant K _D can be measured, for example, by surface plasmon resonance (SPR) (BIAcore™) or biolayer interferometry, for example, using the IBIS MX96 SPR system from IBIS Technologies, the ProteOn™ XPR36 SPR system from Bio-Rad, or the Octet system ™ from ForteBio.

The term kof _f refers to the dissociation rate constant for a particular antibody-antigen interaction. The dissociation rate constant k _off can be measured by SPR or biolayer interferometry, for example using one of the systems listed above.

The term epitope as used herein refers to the portion (determinant) of an antigen that specifically binds to an antibody or related molecule, such as a bispecific binding molecule. Epitope determinants typically consist of reactive surface moieties of molecules, such as amino acid or carbohydrate or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. The epitope may be linear or conformational. In a linear epitope, all points of interaction between the protein (such as an antigen) and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, interaction points occur among amino acid residues on a protein that are separated from each other in the primary amino acid sequence. Once the desired epitope on an antigen has been determined, antibodies against that epitope can be produced using methods well known in the art. For example, an antibody against a linear epitope can be produced, for example, by immunizing an animal with a peptide having the amino acid residues of the linear epitope. An antibody against a conformational epitope can be produced, for example, by immunizing an animal with a mini-domain containing the corresponding amino acid residues of the conformational epitope. An antibody against a particular epitope can also be generated, for example, by immunizing an animal with a target molecule of interest (eg, PD-1) or a corresponding portion thereof, then screening for binding to the epitope.

It is possible to determine whether an antibody binds to the same epitope as an anti-PD-1 antibody of the invention, or whether the antibody competes for binding with an anti-PD-1 antibody of the invention, using methods known in the art, including, but not limited to, assays competition, epitope clustering and alanine scanning. In some embodiments, the test antibody and the anti-PD-1 antibody of the invention bind at least one common residue (eg, at least two, three, four, five or six common residues) on PD-1. In further embodiments, the contact residues on PD-1 are completely identical between the test antibody and the anti-PD-1 antibody of the invention. In one embodiment, an anti-PD-1 antibody of the invention is allowed to bind to PD-1 under saturating conditions, and the ability of the test antibody to bind to PD-1 is then measured. If the test antibody is capable of binding to PD-1 at the same time as the reference anti-PD-1 antibody, then the test antibody binds to an epitope different from that of the reference anti-PD-1 antibody. However, if the test antibody is unable to bind PD-1 at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope in close proximity to the epitope bound by the anti-PD-1 antibody of the invention. This experiment can be performed using, for example, ELISA, RIA, BIACORE™, SPR, biolayer interferometry or flow cytometry. To test whether an anti-PD-1 antibody exhibits cross-competition with another anti-PD-1 antibody, the competition method described above can be used in two directions, i.e. by determining whether a known antibody blocks a test antibody and vice versa. Such cross-competition experiments can be carried out, for example, using the IBIS MX96 SPR device or the Octet™ system.

In certain cases, it may also be desirable to change one or more amino acid residues of the CDR to improve binding affinity to the target epitope. This is known as affinity maturation and may optionally be carried out in combination with humanization, for example in situations where humanization of the antibody results in a decrease in binding specificity or affinity and it is not possible to sufficiently improve binding specificity or affinity through reverse mutagenesis alone. Various affinity maturation methods are known in the art, for example, the in vitro scanning saturation mutagenesis method described in Burks et al., Proc. Natl. Acad. Sci., USA, 94:412-417 (1997), and the in vitro stepwise affinity maturation method of Wu et al., Proc. Natl. Acad. Sci., USA, 95:6037-6042 (1998).

In some embodiments, the antibodies of the invention may be chimeric, humanized, or fully human. Although it is not possible to accurately predict the immunogenicity of a particular drug based on non-human antibodies

- 5 043633 antibodies tend to be more immunogenic in humans than human antibodies. Chimeric antibodies in which foreign (eg, rodent or avian) constant regions are replaced by sequences of human origin have been shown to be generally less immunogenic than antibodies of entirely foreign origin. The trend for therapeutic antibodies is toward humanized or fully human antibodies.

The term chimeric antibody refers to an antibody containing sequences from two different animal species. For example, a chimeric antibody may comprise variable domains from a mouse antibody (i.e., an antibody encoded by mouse antibody genes, such as an antibody produced from an immunized mouse using hybridoma technology) linked to constant regions from an antibody from another species (e.g., human , rabbit or rat). In the case of a chimeric antibody, the non-human portions may be further modified to humanize the antibody.

The term humanize refers to the modification of an antibody derived in whole or in part from a non-human source (for example, a mouse or chicken antibody obtained by immunizing mice or chickens, respectively, with an antigen of interest, or a chimeric antibody based on such a mouse or chicken antibody), by replacing certain amino acid sequences, particularly in framework regions (FRs) and heavy and light chain constant regions, with corresponding amino acid sequences of human FRs and constant regions, to eliminate or minimize anti-drug antibody responses in human patients. Antibodies from a non-human source can thus be humanized to reduce the risk of a human antibody response against a drug.

The term human antibody refers to an antibody in which the variable domain and constant region sequences are derived from human sequences. The term includes antibodies with sequences derived from human genes that have been modified, for example, to reduce immunogenicity, increase affinity and/or increase stability. In addition, the term includes antibodies produced recombinantly in non-human cells that may undergo glycosylation that is not typical of human cells. The term also includes antibodies produced by transgenic non-human organisms with human antibody genes (eg, OmniRat® rats).

The term antigen-binding antibody moiety (or simply antibody moiety) as used herein refers to one or more antibody moieties or fragments that retain the ability to specifically bind an antigen (eg, human PD-1 or a portion thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments covered by the term antigen-binding moiety include (i) Fab fragment: a monovalent fragment consisting of VL, VH, CL and CH1 domains;

(ii) F(ab') ₂ fragment: divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region;

(iii) Fd fragment consisting of VH and CH1 domains;

(iv) an Fv fragment consisting of the VL and VH domains of one arm of the antibody;

(v) a dAb fragment consisting of a VH domain; and (vi) a dedicated complementarity determining region (CDR) capable of specifically binding to the antigen.

In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, through a synthetic linker, allowing them to be produced in the form of a single protein chain in which the VL and VH domains are paired with formation of a monovalent molecule (known as single chain Fv (scFv)). Also included in the invention are antigen binding molecules containing VH and/or VL. In the case of VH, the molecule may also contain one or more of the CH1, hinge, CH2 or CH3 regions. Such single chain antibodies are also intended to be included within the term antigen binding portion of an antibody. Other forms of single chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on the same polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, thus forcing the domains to pair with complementary domains of another chain and form two antigen-binding sites.

Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from full-length antibodies using conventional methods, such as papain or pepsin digestion of full-length antibodies. In addition, antibodies, antibody portions, and immunoadhesion molecules can be produced using standard recombinant DNA techniques, for example, as described herein.

The class (isotype) and subclass of anti-PD-1 antibodies can be determined by any method known in the art. Typically, the class and subclass of an antibody can be determined using antibodies

- 6 043633 class and subclass specific antibodies. Such antibodies are commercially available. Class and subclass can be determined by ELISA, Western blotting, as well as other methods. Alternatively, the class and subclass can be determined by sequencing all or part of the constant regions of the heavy and/or light chains of the antibodies, comparing their amino acid sequences with the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.

When referring to specific amino acid residues at a given position in an antibody sequence, the designation, for example, 35S, refers to the position and residue, i.e. in this case indicates that a serine (S) residue is present at position 35 of the sequence. Likewise, a designation such as 13Q+35S refers to two residues at corresponding positions. Unless otherwise indicated, all antibody amino acid residue numbers referred to herein are residues in accordance with the IMGT® numbering scheme.

Antibodies against PD-1.

The present invention relates to antibodies directed against PD-1 and their antigen-binding portions. In a specific embodiment, the antibodies described herein are human antibodies, for example, obtained from transgenic rats with human antibody genes. In specific embodiments, human antibodies may contain certain mutations, for example to mutagenetically return primer-derived mutations to the germline sequence (see, e.g., Symplex-corrected sequence variant below) or to mutagenetically return framework regions to the closest germline sequence V or J (see, for example, the germline variant of the sequence below).

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies, H-CDR1-3 of which contain amino acid sequences from SEQ ID NO: 29-31, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 1;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 1;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 1 and 26;

e) antibodies, L-CDR1-3 of which contain the amino acid sequences from SEQ ID NO: 32-34, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 2;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 2;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 2 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 29-34, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1, and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 2;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 1 and whose VL contains the amino acid sequence from SEQ ID NO: 2; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 1 and 26 and whose LC contains the amino acid sequences from SEQ ID NO: 2 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 35-37, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 3;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 3;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 3 and 26;

e) antibodies, L-CDR1-3 of which contain the amino acid sequences from SEQ ID NO: 38-40, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 4;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 4;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 4 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain amino acid sequences from

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SEQ ID NO: 35-40 respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 3 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 4;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 3 and whose VL contains the amino acid sequence from SEQ ID NO: 4; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 3 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 4 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 41-43, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 5;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 5;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 5 and 26;

e) antibodies, L-CDR1-3 of which contain the amino acid sequences from SEQ ID NO: 44-46, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 6;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 6;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 6 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 41-46, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 5 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 6;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 5 and whose VL contains the amino acid sequence from SEQ ID NO: 6; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 5 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 6 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 29, 47 and 48, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 7;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 7;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 7 and 26;

e) antibodies, L-CDR1-3 of which contain the amino acid sequences from SEQ ID NO: 49-51, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 8;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 8;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 8 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 29, 47, 48 and 49-51, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 7 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 8;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 7 and whose VL contains the amino acid sequence from SEQ ID NO: 8; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 7 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 8 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 52-54, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 9;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 9;

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d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 9 and 26;

e) antibodies whose L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 38, 45 and 55, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 10;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 10;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 10 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NOs: 52-54 and 38, 45 and 55, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 9 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 10;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 9 and whose VL contains the amino acid sequence from SEQ ID NO: 10; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 9 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 10 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 29, 56 and 48, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 1;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 11;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 11 and 26;

e) antibodies whose L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 57, 33 and 51, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 12;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 12;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 12 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 29, 56 and 48 and 57, 33 and 51, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 11 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 12;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 11 and whose VL contains the amino acid sequence from SEQ ID NO: 12; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 11 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 12 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 58-60, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 13;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 13;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 13 and 26;

e) antibodies whose L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 57, 33 and 34, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 14;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 14;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 14 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NOs: 58-60 and 57, 33 and 34, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 13 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 14;

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j) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 13 and whose VL contains the amino acid sequence from SEQ ID NO: 14; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 13 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 14 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 61, 62 and 43, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 15;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 15;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 15 and 26;

e) antibodies, L-CDR1-3 of which contain the amino acid sequences from SEQ ID NO: 44-46, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 16;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 16;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 16 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 61, 62 and 43 and 44-46, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 15 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 16;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 15 and whose VL contains the amino acid sequence from SEQ ID NO: 16; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 15 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 16 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies, H-CDR1-3 of which contain amino acid sequences from SEQ ID NO: 63-65, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 17;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 17;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 17 and 26;

e) antibodies, L-CDR1-3 of which contain the amino acid sequences from SEQ ID NO: 32-34, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 18;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 18;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 18 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 63-65 and 32-34, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 17 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 18;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 17 and whose VL contains the amino acid sequence from SEQ ID NO: 18; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 17 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 18 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 66-68, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 19;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 19;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 19 and 26;

e) antibodies whose L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 38, 45 and 55, respectively;

- 10 043633

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 20;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 20;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 20 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 66-68 and 38, 45 and 55, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 19 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 20;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 19 and whose VL contains the amino acid sequence from SEQ ID NO: 20; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 19 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 20 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 69-71, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 21;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 21;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 21 and 26;

e) antibodies whose L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 72, 45 and 73, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 22;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 22;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 22 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NOs: 69-71 and 72, 45 and 73, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 21 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 22;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 21 and whose VL contains the amino acid sequence from SEQ ID NO: 22; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 21 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 22 and 28.

In one embodiment, the anti-PD-1 antibody is selected from the group consisting of

a) antibodies whose H-CDR1-3 contain the amino acid sequences from SEQ ID NO: 74-76, respectively;

b) antibodies whose heavy chain variable domain (VH) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 23;

c) an antibody whose VH contains the amino acid sequence of SEQ ID NO: 23;

d) antibodies, the heavy chain (HC) of which contains the amino acid sequences of SEQ ID NO: 23 and 26;

e) antibodies whose L-CDR1-3 contain the amino acid sequences of SEQ ID NO: 57, 33 and 34, respectively;

f) antibodies whose light chain variable domain (VL) is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 24;

g) an antibody whose VL contains the amino acid sequence of SEQ ID NO: 24;

h) antibodies, the light chain (LC) of which contains the amino acid sequences of SEQ ID NO: 24 and 28;

i) antibodies whose H-CDR1-3 and L-CDR1-3 contain the amino acid sequences of SEQ ID NOs: 74-76 and 57, 33 and 34, respectively;

j) an antibody whose VH is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 23 and whose VL is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 24;

k) an antibody whose VH contains the amino acid sequence from SEQ ID NO: 23 and whose VL contains the amino acid sequence from SEQ ID NO: 24; And

l) an antibody whose HC contains the amino acid sequences from SEQ ID NOs: 23 and 26 and whose LC contains the amino acid sequences from SEQ ID NOs: 24 and 28.

- 11 043633

In other embodiments, the anti-PD-1 antibody has a VH and VL that are at least 90% identical in amino acid sequence to the VH and VL, respectively, of any of antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413 and 18483, for example, at least 92, 95, 96, 97, 98 or 99% identical in sequence to the VH and VL of any of these antibodies. In some embodiments, the antigen binding portion of the anti-PD-1 antibody has said VH and VL.

In some embodiments, the anti-PD-1 antibody has a VH and VL comprising the amino acid sequences VH and VL, respectively, of any of antibodies 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400, 18413, and 18483. In some embodiments, the antigen binding portion of the anti-PD-1 antibody has said VH and VL.

In some embodiments, an anti-PD-1 antibody or antigen-binding moiety of the invention comprises the amino acid sequences H-CDR1-3 and L-CDR1-3 from

a) SEQ ID NO: 29, 30, 31, 32, 33 and 34 respectively;

b) SEQ ID NO: 35, 36, 37, 38, 39 and 40 respectively;

c) SEQ ID NO: 41, 42, 43, 44, 45 and 46 respectively;

d) SEQ ID NO: 29, 47, 48, 49, 50 and 51 respectively;

e) SEQ ID NO: 52, 53, 54, 38, 45 and 55 respectively;

f) SEQ ID NO: 29, 56, 48, 57, 33 and 51 respectively;

g) SEQ ID NO: 58, 59, 60, 57, 33 and 34 respectively;

h) SEQ ID NO: 61, 62, 43, 44, 45 and 46 respectively;

i) SEQ ID NO: 63, 64, 65, 32, 33 and 34 respectively;

j) SEQ ID NO: 66, 67, 68, 38, 45 and 55 respectively;

k) SEQ ID NO: 69, 70, 71, 72, 45 and 73 respectively; or

l) SEQ ID NO: 74, 75, 76, 57, 33 and 34 respectively.

In some embodiments, an anti-PD-1 antibody or antigen binding moiety of the invention comprises VH and VL that are 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequences of

a) SEQ ID NO: 1 and 2 respectively;

b) SEQ ID NO: 3 and 4 respectively;

c) SEQ ID NO: 5 and 6 respectively;

d) SEQ ID NO: 7 and 8 respectively;

e) SEQ ID NO: 9 and 10 respectively;

f) SEQ ID NO: 11 and 12 respectively;

g) SEQ ID NO: 13 and 14 respectively;

h) SEQ ID NO: 15 and 16 respectively;

i) SEQ ID NO: 17 and 18 respectively;

j) SEQ ID NO: 19 and 20 respectively;

k) SEQ ID NO: 21 and 22 respectively; or

l) SEQ ID NO: 23 and 24 respectively.

In some embodiments, an anti-PD-1 antibody or antigen-binding moiety of the invention comprises VH and VL having amino acid sequences from

a) SEQ ID NO: 1 and 2 respectively;

b) SEQ ID NO: 3 and 4 respectively;

c) SEQ ID NO: 5 and 6 respectively;

d) SEQ ID NO: 7 and 8 respectively;

e) SEQ ID NO: 9 and 10 respectively;

f) SEQ ID NO: 11 and 12 respectively;

g) SEQ ID NO: 13 and 14 respectively;

h) SEQ ID NO: 15 and 16 respectively;

i) SEQ ID NO: 17 and 18 respectively;

j) SEQ ID NO: 19 and 20 respectively;

k) SEQ ID NO: 21 and 22 respectively; or

l) SEQ ID NO: 23 and 24 respectively.

In some embodiments, the anti-PD-1 antibody comprises

a) HC with amino acid sequences from SEQ ID NO: 1 and 26 and LC with amino acid sequences from SEQ ID NO: 2 and 28;

b) HC with amino acid sequences from SEQ ID NO: 3 and 26 and LC with amino acid sequences from SEQ ID NO: 4 and 28;

c) HC with amino acid sequences from SEQ ID NO: 5 and 26 and LC with amino acid sequences from SEQ ID NO: 6 and 28;

d) HC with amino acid sequences from SEQ ID NO: 7 and 26 and LC with amino acid sequences from SEQ ID NO: 8 and 28;

- 12 043633

e) HC with amino acid sequences from SEQ ID NOs: 9 and 26 and LC with amino acid sequences from SEQ ID NOs: 10 and 28;

f) HC with amino acid sequences from SEQ ID NOs: 11 and 26 and LC with amino acid sequences from SEQ ID NOs: 12 and 28;

g) HC with amino acid sequences from SEQ ID NOs: 13 and 26 and LC with amino acid sequences from SEQ ID NOs: 14 and 28;

h) HC with amino acid sequences from SEQ ID NOs: 15 and 26 and LC with amino acid sequences from SEQ ID NOs: 16 and 28;

i) HC with amino acid sequences from SEQ ID NOs: 17 and 26 and LC with amino acid sequences from SEQ ID NOs: 18 and 28;

j) HC with amino acid sequences from SEQ ID NOs: 19 and 26 and LC with amino acid sequences from SEQ ID NOs: 20 and 28;

k) HC with amino acid sequences from SEQ ID NOs: 21 and 26 and LC with amino acid sequences from SEQ ID NOs: 22 and 28; or

l) HC with amino acid sequences from SEQ ID NOs: 23 and 26 and LC with amino acid sequences from SEQ ID NOs: 24 and 28.

In one embodiment, the anti-PD-1 antibody has a VH and VL comprising the amino acid sequences VH and VL, respectively, of antibody 18040, i.e. wherein VH contains the amino acid sequence from SEQ ID NO: 1 and VL contains the amino acid sequence from SEQ ID NO: 2. In this embodiment, VH may contain the amino acid sequence from SEQ ID NO: 1, where X at position 35 is S and/or X at position 84 is N. In certain embodiments, VL may also comprise the amino acid sequence of SEQ ID NO: 2, wherein X at position 40 is A and/or X at position 55 is Y.

In one embodiment, the anti-PD-1 antibody has a VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18049, i.e. wherein VH contains the amino acid sequence from SEQ ID NO: 3 and VL contains the amino acid sequence from SEQ ID NO: 4. In this embodiment, VL may contain the amino acid sequence from SEQ ID NO: 4, where X at position 1 is D and/or X at position 3 is Q. In some embodiments, VL contains D at position 1 and Q at position 3. In certain embodiments, VL may also contain the amino acid sequence of SEQ ID NO: 4, where X at position 53 is S.

In one embodiment, the anti-PD-1 antibody has a VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18098, i.e. wherein VH contains the amino acid sequence of SEQ ID NO: 5 and VL contains the amino acid sequence of SEQ ID NO: 6. In this embodiment, VH may contain the amino acid sequence of SEQ ID NO: 5, where X at position 1 is E and/or X at position 5 is V. In some embodiments, VH contains E at position 1 and V at position 5. In certain embodiments, VH may also comprise the amino acid sequence of SEQ ID NO: 5, where X at position 13 is Q. X at position 35 is S, X at position 46 is E, X at position 50 is A, X at position 77 is N, X at position 80 is Y, and/or X at position 115 is M. VL may contain the amino acid sequence of SEQ ID NO: 6, where X at position 4 is M.

In one embodiment, the anti-PD-1 antibody has VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18113, i.e. wherein VH contains the amino acid sequence of SEQ ID NO: 7 and VL contains the amino acid sequence of SEQ ID NO: 8. In this embodiment, VH may contain the amino acid sequence of SEQ ID NO: 7, where X at position 64 is V. VL may comprise the amino acid sequence of SEQ ID NO: 8, wherein X at position 3 is V and/or X at position 4 is M. In some embodiments, VL contains V at position 3 and M at position 4. In certain embodiments, VL may also contain the amino acid sequence of SEQ ID NO: 8, where X at position 69 is S.

In one embodiment, the anti-PD-1 antibody has VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18201, i.e. wherein VH contains the amino acid sequence of SEQ ID NO: 9 and VL contains the amino acid sequence of SEQ ID NO: 10. In this embodiment, VH may contain the amino acid sequence of SEQ ID NO: 9, where X at position 5 is Q. In some In embodiments, VH may also comprise the amino acid sequence of SEQ ID NO: 9, wherein X at position 59 is N, X at position 76 is K, and/or X at position 83 is L. VL may comprise the amino acid sequence of SEQ ID NO: 10, where X at position 1 represents A.

- 13 043633

In one embodiment, the anti-PD-1 antibody has a VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18247, i.e. wherein VH contains the amino acid sequence from SEQ ID NO: 11 and VL contains the amino acid sequence from SEQ ID NO: 12. In this embodiment, VH may contain the amino acid sequence from SEQ ID NO: 11, where X at position 10 is G and/or X at position 16 is G. VL may comprise the amino acid sequence of SEQ ID NO: 12, wherein X at position 1 is D and/or X at position 4 is M. In some embodiments, VL contains D at position 1 and M at position 4. In certain embodiments, VL may also comprise the amino acid sequence of SEQ ID NO: 12, wherein X at position 55 is Y and/or X at position 93 is Y.

In one embodiment, the anti-PD-1 antibody has a VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18250, i.e. wherein VH contains the amino acid sequence of SEQ ID NO: 13 and VL contains the amino acid sequence of SEQ ID NO: 14. In this embodiment, VH may contain the amino acid sequence of SEQ ID NO: 13, where X at position 5 is V. In some In embodiments, VH may also comprise the amino acid sequence of SEQ ID NO: 13, wherein X at position 50 is Y, X at position 59 is Y, and/or X at position 61 is A. VL may comprise the amino acid sequence of SEQ ID NO: 14, wherein X at position 3 is V and/or X at position 4 is M. In some embodiments, VL contains V at position 3 and M at position 4. In certain embodiments, VL may also comprise the amino acid sequence of SEQ ID NO: 14 wherein X at position 55 is Y.

In one embodiment, the anti-PD-1 antibody has VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18325, i.e. wherein VH contains the amino acid sequence from SEQ ID NO: 15 and VL contains the amino acid sequence from SEQ ID NO: 16. In this embodiment, VH may contain the amino acid sequence from SEQ ID NO: 15, where X at position 1 is E, X at position 5 is V and/or X at position 6 is E. In some embodiments, VH contains E at position 1, V at position 5, and E at position 6. In certain embodiments, VH may also contain an amino acid sequence from SEQ ID NO: 15 wherein X at position 35 is S, X at position 49 is S, X at position 50 is A, X at position 73 is D and/or X at position 78 is T. VL may contain amino acid sequence from SEQ ID NO: 16, wherein X at position 1 is D, X at position 3 is Q, and/or X at position 4 is M. In some embodiments, VL contains D at position 1, Q at position 3 and M in position 4.

In one embodiment, the anti-PD-1 antibody has a VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18366, i.e. wherein VH contains the amino acid sequence from SEQ ID NO: 17 and VL contains the amino acid sequence from SEQ ID NO: 18. In this embodiment, VH may contain the amino acid sequence from SEQ ID NO: 17, where X at position 1 is E and/or X at position 6 is E. In some embodiments, VH contains E at position 1 and E at position 6. VL may comprise the amino acid sequence of SEQ ID NO: 18, where X at position 1 is D. In certain embodiments, VL may also contain the amino acid sequence of SEQ ID NO: 18, where X at position 40 is A and/or X at position 55 is Y.

In one embodiment, the anti-PD-1 antibody has VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18400, i.e. wherein VH comprises the amino acid sequence of SEQ ID NO: 19 and VL contains the amino acid sequence of SEQ ID NO: 20. In this embodiment, VH may comprise the amino acid sequence of SEQ ID NO: 19, wherein X at position 2 is V. In particular In embodiments, VH may also comprise the amino acid sequence of SEQ ID NO: 19, wherein X at position 43 is G, X at position 49 is I, X at position 59 is N, X at position 70 is I, and/or X at position 111 is Q. VL may comprise the amino acid sequence of SEQ ID NO: 20, wherein X at position 1 is A and/or X at position 3 is Q. In some embodiments, VL contains A at position 1 and Q at position 3. In certain embodiments, VL may also comprise the amino acid sequence of SEQ ID NO: 20, wherein X at position 10 is S.

In one embodiment, the anti-PD-1 antibody has VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18413, i.e. wherein VH contains the amino acid sequence of SEQ ID NO: 21 and VL contains the amino acid sequence of SEQ ID NO: 22. In this embodiment, VH may contain the amino acid sequence

- 14 043633 ity from SEQ ID NO: 21, where X at position 48 is V and/or X at position 50 is A. VL may contain the amino acid sequence from SEQ ID NO: 22, where X at position 4 is M In certain embodiments, VL may also comprise the amino acid sequence of SEQ ID NO: 22, wherein X at position 12 is S.

In one embodiment, the anti-PD-1 antibody has VH and VL comprising the amino acid sequences VH and VL, respectively, from antibody 18483, i.e. wherein VH contains the amino acid sequence of SEQ ID NO: 23 and VL contains the amino acid sequence of SEQ ID NO: 24. In this embodiment, VH may contain the amino acid sequence of SEQ ID NO: 23, where X at position 1 is E, X at position 5 is V and/or X at position 6 is E. In some embodiments, VL contains E at position 1, V at position 5, and E at position 6. In certain embodiments, VH may also contain an amino acid sequence from SEQ ID NO: 23, wherein X at position 35 is S. VL may comprise the amino acid sequence of SEQ ID NO: 24, wherein X at position 3 is V. In certain embodiments, VL may also comprise the amino acid sequence of SEQ ID NO: 24 where X at position 40 is A and/or X at position 55 is Y.

In specific embodiments, the anti-PD-1 antibody or antigen binding portion comprises the amino acid sequences H-CDR1-3 and L-CDR1-3 from an antibody selected from 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400 , 18413 and 18483, and furthermore uses the same heavy and/or light chain seed sequences as the selected antibody.

In specific embodiments, the anti-PD-1 antibody or antigen binding portion comprises the amino acid sequences H-CDR1-3 and L-CDR1-3 from an antibody selected from 18040, 18049, 18098, 18113, 18201, 18247, 18250, 18325, 18366, 18400 , 18413 and 18483, and further comprises framework regions (FRs) that are 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the FR of the selected antibody.

In some embodiments, any of the anti-PD-1 antibodies or antigen-binding moieties described herein can inhibit the binding of PD-L1 or PD-L2, or both, to PD-1.

In some embodiments, any of the anti-PD-1 antibodies or antigen binding moieties described herein may have at least one of the following properties:

a) binds to cynomolgus PD-1 with a K _D of, for example, 4x10 ⁸ M or less;

b) binds to mouse PD-1 with a KD of, for example, 2x10' ⁸ M or less;

c) binds to human PD-1 with a KD of ^3x10-9 M or less;

d) inhibits the interaction of PD-1 with PD-L1 at a concentration of 10 μg/ml;

e) stimulates IL-2 production in the whole blood SEB assay; And

f) stimulates IFN-γ production in a one-way mixed lymphocyte culture reaction assay.

In some embodiments, any of the anti-PD1 antibodies or antigen-binding moieties described herein can bind to human PD-1 with a K _{D of} ^5x10-9 M or less, ^4x10-9 M or less, ^3x10-9 M or less, 2x10 - ⁹ M or less, 1x10- ⁹ M or less, 9x10- ¹⁰ M or less, 8x10- 10 M or less, 7x10- ¹⁰ M or less, 6x10- ^{10 M} or less, 5x10- ¹⁰ M or less, 4x10- ¹⁰ M or less, ^3x10-10 M or less, ^2x10-10 M or less, or ^1x10-10 M or less. In certain embodiments, K _D is determined using surface plasmon resonance.

In some embodiments, any of the anti-PD1 antibodies or antigen-binding moieties described herein can inhibit the interaction of PD-1 with PD-L1 at a concentration of 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 µg/ml.

The class of anti-PD-1 antibody produced by the methods described herein can be changed or switched to another class or subclass. In one aspect of the invention, the nucleic acid molecule encoding VL or VH is isolated using methods well known in the art such that it does not include nucleic acid sequences encoding CL or CH. Nucleic acid molecules encoding VL or VH are then operably linked to a nucleic acid sequence encoding CL or CH, respectively, from another class of immunoglobulin molecules. This can be done using a vector or nucleic acid molecule containing a CL or CH strand as described above. For example, the class of an anti-PD-1 antibody that is originally IgM can be switched to the IgG class. In addition, class switching can be used to convert one IgG subclass to another, for example from IgG1 to IgG2. The light chain constant region k can be changed, for example, to the light chain constant region A. A preferred method of producing an antibody of the invention with a desired Ig isotype involves the steps of isolating a nucleic acid molecule encoding the anti-PD-1 antibody heavy chain and a nucleic acid molecule encoding light chain of an anti-PD-1 antibody, obtaining a heavy chain variable domain, ligating the heavy chain variable domain to a constant region

- 15 043633 heavy chain of the desired isotype, expression of the light chain and the ligated heavy chain in the cell, and collection of an anti-PD-1 antibody with the desired isotype.

The anti-PD-1 antibody of the invention may be an IgG, IgM, IgE, IgA or IgD molecule, but typically belongs to an IgG isotype, for example an IgG subclass of IgG1, IgG2a or IgG2b, IgG3 or IgG4. In one embodiment, the antibody is IgG1. In another embodiment, the antibody is IgG4.

In one embodiment, the anti-PD-1 antibody may contain at least one mutation in the Fc region. A number of different Fc mutations are known, where these mutations provide altered effector function. For example, in many cases it may be desirable to reduce or eliminate effector function, for example when ligand/receptor interactions are undesirable, or in the case of antibody-drug conjugates.

In one embodiment, the anti-PD-1 antibody contains at least one mutation in the Fc region that reduces effector function. Amino acid positions of the Fc region that may be advantageous for mutation to reduce effector function include one or more of positions 228, 233, 234 and 235, where the amino acid positions are numbered according to the IMGT numbering scheme.

In one embodiment, one or both of the amino acid residues at positions 234 and 235 can be mutagenized, for example, from Leu to Ala (L234A/L235A). These mutations reduce the effector function of the Fc region of IgG1 antibodies. Additionally or alternatively, the amino acid residue at position 228 can be mutagenized, for example, to Pro. In some embodiments, the amino acid residue at position 233 can be mutagenized, for example, to Pro, the amino acid residue at position 234 can be mutagenized, for example, to Val, and/or the amino acid residue at position 235 can be mutagenized, for example, to Ala. Amino acid positions are numbered according to the IMGT® numbering scheme.

In some embodiments, where the antibody belongs to the IgG4 subclass, it may contain the S228P mutation, i.e. have a proline at position 228, where the amino acid position is numbered according to the IMGT® numbering scheme. This mutation is known to reduce unwanted turnover of Fab arms.

In specific embodiments, an antibody or antigen-binding portion thereof of the invention may be part of a larger immunoadhesion molecule formed through the covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to produce a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas, 6:93-101 (1995)) and the use of a cysteine residue, a marker peptide, and a C-terminal polyhistidine tag to produce divalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol., 31:1047-1058 (1994)). Other examples include cases where one or more CDRs from an antibody are inserted into a molecule, either covalently or non-covalently, to produce an immunoadhesin that specifically binds to an antigen of interest. In such embodiments, the CDR(s) may be included as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be inserted non-covalently.

In another embodiment, a fusion antibody or immunoadhesin may be produced comprising all or part of an anti-PD-1 antibody of the invention linked to another polypeptide. In specific embodiments, only the variable domains of the anti-PD-1 antibody are associated with the polypeptide. In specific embodiments, the VH domain of the anti-PD-1 antibody is associated with a first polypeptide, while the VL domain of the anti-PD-1 antibody is associated with a second polypeptide associated with the first polypeptide such that the VH and VL domains can interact with each other with the formation of an antigen-binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with each other (eg, single chain antibodies). The VH-linker-VL antibody is then coupled to the polypeptide of interest. In addition, fusion antibodies can be produced in which two (or more) single chain antibodies are linked to each other. This can be used if it is desired to produce a divalent or multivalent antibody on a single polypeptide chain, or if it is desired to produce a bispecific antibody.

To produce a single-chain antibody (scFv), the VH and VL encoding DNA fragments are operably linked to another flexible linker encoding fragment, for example, encoding the amino acid sequence (Gly4 Ser)3 (SEQ ID NO: 115), such that the VH and VL sequences can be expressed as a continuous single-chain protein, with VL and VH domains connected by a flexible linker. See, for example, Bird et al., Science, 242:423, 426 (1988); Huston et al., Proc. Natl. Acad. Sci., USA, 85:5879, 5883 (1988); and McCafferty et al., Nature, 348:552,554 (1990). A single chain antibody may be monovalent if only single VH and VL are used; divalent if two VH and VL are used; or polyvalent if more than two VH and VL are used.

- 16 043633

For example, it is possible to produce bispecific or multivalent antibodies that specifically bind to human PD-1 and to another molecule.

In other embodiments, other modified antibodies can be produced using anti-PD-1 antibody-encoding nucleic acid molecules. For example, kappa antibodies (Ill et al., Protein Eng., 10:949-57 (1997)), mini-antibodies (Martin et al., EMBO J., 13:5303-9 (1994)), diabodies (Holliger et al. , Proc. Natl. Acad. Sci., USA, 90:6444-6448 (1993)) or Janus (Traunecker et al., EMBO J., 10:3655-3659 (1991); and Traunecker et al., Int. J. Cancer (Suppl.), 7:51-52 (1992)) can be prepared using standard molecular biology techniques as explained herein.

The anti-PD-1 antibody or antigen-binding portion of the invention can be derivatized or linked to another molecule (eg, another peptide or protein). Typically, antibodies or portions thereof are derivatized such that PD-1 binding is not adversely affected by derivatization or labeling. Accordingly, the antibodies and antibody portions of the invention are intended to include both intact and modified forms of human anti-PD-1 antibodies described herein. For example, an antibody or portion of an antibody of the invention may be operably linked (via chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other types of molecules, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a drug, and/or a protein or peptide that can mediate the association of an antibody or portion of an antibody with another molecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or different types, for example, to produce bispecific antibodies). Suitable cross-linking agents include those that are heterobifunctional, having two distinct reactive groups separated by a suitable spacer (eg, m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (eg, disuccinimidyl suberate). Such linkers are available, for example, from Pierce Chemical Company, Rockford, Il.

Anti-PD-1 antibody can also be derivatized using a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups can be used to improve the biological characteristics of the antibody, for example, to increase the half-life in serum.

The antibody of the present invention may also be labeled. As used herein, the terms label or labeled refer to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as the insertion of a radiolabeled amino acid or the attachment of biotinylated groups to the polypeptide that can be detected by labeled avidin (e.g., streptavidin having a fluorescent marker or enzyme activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker may be therapeutic, such as a drug conjugate or toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioactive isotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, phosphorus lanthanide), enzyme tags (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predefined polypeptide epitopes recognized by a secondary reporter (e.g., paired leucine zipper sequences, binding sites for secondary antibodies, metal binding domains, epitope tags ), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tatracaine, lidocaine, propranolol and puromycin, as well as their analogs or homologues. In some embodiments, tags are attached using spacer arms of different lengths to reduce possible steric mismatch.

In specific embodiments, the antibodies of the invention may be in neutral form (including zwitterionic forms) or in the form of positively or negatively charged molecules. In some embodiments, the antibodies can be complexed with a counterion to form a pharmaceutically acceptable salt.

The term pharmaceutically acceptable salt refers to a complex containing one or more antibodies and one or more counterions, where the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.

Bispecific binding molecules.

In a further aspect, the invention relates to a bispecific binding molecule having

- 17 043633 the binding specificity of an anti-PD-1 antibody described herein and the binding specificity of another anti-PD-1 antibody (for example, another anti-PD-1 antibody described herein) or an antibody targeting a different protein, such as another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity mediates a disease state, such as a cancer. Such bispecific binding molecules are known in the art, and examples of various types of bispecific binding molecules are provided elsewhere herein.

Nucleic acid molecules and vectors.

The present invention also relates to molecules and nucleic acid sequences encoding anti-PD-1 antibodies or antigen-binding portions thereof described herein. In some embodiments, the various nucleic acid molecules encode the amino acid sequences of the heavy chain and light chain of an anti-PD-1 antibody or an antigen-binding portion thereof. In other embodiments, the same nucleic acid molecule encodes the heavy chain and light chain amino acid sequences of an anti-PD-1 antibody or antigen-binding portion thereof.

A reference to a nucleotide sequence includes its complementary sequence unless otherwise indicated. Thus, reference to a nucleic acid having a particular sequence should be understood to include its complementary strand with its complementary sequence. The term polynucleotide, as used herein, means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single- and double-stranded forms.

In any of the above embodiments, the nucleic acid molecules may be isolated.

In a further aspect, the present invention provides a vector suitable for expressing one of the antibody chains or an antigen-binding portion thereof, as described herein. The term vector, as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e. a circular double-stranded DNA fragment into which additional DNA fragments can be ligated. In some embodiments, the vector is a viral vector, where additional DNA fragments can be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal vectors for mammals). In other embodiments, vectors (eg, nonepisomal mammalian vectors) can integrate into the host cell genome upon introduction into the host cell, and thus replicate along with the host genome. In addition, specific vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors).

The invention relates to vectors containing nucleic acid molecules encoding the heavy chain of an anti-PD-1 antibody of the invention or an antigen-binding portion thereof, the light chain of an anti-PD-1 antibody of the invention or an antigen-binding portion thereof, or both the heavy and light chains of an anti-PD-1 antibody. 1 according to the invention or its antigen-binding part. The invention further relates to vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments and probes therefor.

The nucleic acid molecule encoding the heavy and/or light chains of an anti-PD-1 antibody or antigen-binding portion thereof of the invention can be isolated from any source producing such antibody or portion. In various embodiments, the nucleic acid molecules are isolated from anti-PD-1 antibody-expressing B cells isolated from an animal immunized with human PD-1 antigen or from an immortalized cell derived from such a B cell. Methods for isolating nucleic acids encoding an antibody are well known in the art. The mRNA can be isolated and used to produce cDNA for use in polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In certain embodiments, the nucleic acid molecule of the invention can be synthesized rather than isolated.

In some embodiments, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a VH domain from an anti-PD-1 antibody or antigen binding moiety of the invention linked in frame to a nucleotide sequence encoding a heavy chain constant region from any source. Likewise, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a VL domain from an anti-PD-1 antibody or antigen binding moiety of the invention linked in frame to a nucleotide sequence encoding a light chain constant region from any source.

In a further aspect of the invention, the nucleic acid molecules encoding the variable

- 18 043633 domain of heavy (VH) and/or light (VL) chains, can be converted into full-length antibody genes. In one embodiment, nucleic acid molecules encoding the VH or VL domains are converted into full-length antibody genes by insertion into an expression vector already encoding heavy chain constant regions (CH) or light chain constant regions (CL), respectively, such that the fragment VH is operably linked to the CH fragment(s) in the vector, and/or the VL fragment is operably linked to the CL fragment in the vector. In another embodiment, nucleic acid molecules encoding the VH and/or VL domains are converted into full-length antibody genes by linking, for example, ligating, a nucleic acid molecule encoding the VH and/or VL domains with a nucleic acid molecule encoding the CH and/or region CL, using standard molecular biology techniques. Nucleic acid molecules encoding full-length heavy and/or light chains can then be expressed in the cell into which they are introduced and anti-PD-1 antibody can be isolated.

Nucleic acid molecules can be used to recombinantly express large quantities of anti-PD-1 antibodies. Nucleic acid molecules can also be used to produce chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutant antibodies and antibody derivatives, as described herein.

In another embodiment, a nucleic acid molecule of the invention is used as a probe or primer for PCR for a specific antibody sequence. For example, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify regions of DNA that can be used, among other things, to isolate additional nucleic acid molecules encoding the variable domains of anti-PD-1 antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are derived from the high variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotides encode all or part of one or more of the anti-PD-1 antibody CDRs or antigen binding portions thereof of the invention, as described herein.

In another embodiment, nucleic acid molecules and vectors can be used to generate mutant anti-PD-1 antibodies. Antibodies can be subjected to mutagenesis in the variable domains of the heavy and/or light chains, for example, to change the binding properties of the antibody. For example, a mutation can be introduced into one or more of the CDRs to increase or decrease the KD of an anti-PD-1 antibody, to increase or decrease the k _off , or to change the binding specificity of the antibody. In another embodiment, one or more mutations are introduced into an amino acid residue known to be altered from the germline one in a monoclonal antibody of the invention. Mutations can be introduced into the CDR or framework region of the variable domain, or into the constant region. In a preferred embodiment, mutations are introduced into the variable domain. In some embodiments, one or more mutations are introduced into an amino acid residue known to be altered from the germline one in the CDR or framework region of a variable domain of an antibody or antigen-binding portion thereof of the invention.

In another embodiment, the framework region(s) are mutagenized such that the resulting framework(s) have the amino acid sequence of the corresponding germline gene. Mutations can be introduced into the framework region or constant region to increase the half-life of the anti-PD-1 antibody. See, for example, PCT publication WO 00/09560. A mutation can be introduced into a framework region or constant region to alter the immunogenicity of the antibody and/or to provide a site for covalent or non-covalent binding to another molecule. In accordance with the invention, a single antibody may have mutations in any one or more of the CDR or framework regions of the variable domain, or in the constant region.

In some embodiments, anti-PD-1 antibodies of the invention or antigen-binding portions thereof are expressed by inserting DNA encoding partial or full-length light and heavy chains prepared as described above into expression vectors such that the genes are operably linked to the desired sequences to control expression, such as sequences to control transcription and translation. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. An antibody coding sequence can be ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody coding sequence. The expression vector and expression control sequences can be selected to be compatible with the host cell used for expression. The antibody light chain coding sequence and the antibody heavy chain coding sequence can be inserted into separate vectors, and can be operably linked to the same or different expression control sequences (eg, promoters). In one embodiment, both coding sequences are inserted into the same expression

- 19 043633 vector and may be operably linked to the same expression control sequences (eg, a common promoter) with separate identical expression control sequences (eg, promoters) or to different expression control sequences (eg, promoters). Antibody coding sequences can be inserted into an expression vector by standard methods (eg, by ligating complementary restriction sites in the antibody gene fragment and the vector, or by blunt-end ligation if no restriction sites are present).

A convenient vector is one encoding a functionally complete human immunoglobulin CH or CL sequence, with suitable restriction sites designed such that any VH or VL sequence can be readily inserted and expressed as described above. The HC and LC genes encoding in such vectors may contain intronic sequences, which may result in improved overall antibody protein yields by stabilizing cognate mRNAs. Intronic sequences are flanked by splice donor and acceptor sites that determine where RNA splicing can occur. The location of the intronic sequences can be in either the variable or constant regions of the antibody chains, or in both the variable and constant regions when multiple introns are used. Polyadenylation and transcription termination can occur in native chromosomal regions downstream of coding regions. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In addition to antibody chain genes, the recombinant expression vectors of the invention may carry regulatory sequences that control expression of antibody chain genes in a host cell. One skilled in the art will appreciate that the design of an expression vector, including the choice of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. Preferred regulatory sequences for expression in a mammalian host cell include viral elements that drive high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from the LTR of a retrovirus, cytomegalovirus (CMV) (eg, CMV promoter/enhancer), virus monkey 40 (SV40) (eg, SV40 promoter/enhancer), adenovirus (eg, adenovirus major late promoter (AdMLP)), polyoma virus, and strong mammalian promoters such as native immunoglobulin and actin promoters. Additional description of viral regulatory elements and their sequence; see, for example, US Pat. Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants, including the description of promoters and vectors, as well as plant transformations, are known in the art. See, for example, US Pat. No. 6,517,529. Methods for expressing polypeptides in bacterial cells or fungal cells, such as yeast cells, are also well known in the art.

In addition to antibody chain genes and regulatory sequences, recombinant expression vectors are disclosed to carry additional sequences, such as sequences that regulate replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector is introduced (see, for example, US patents 4399216, 4634665 and 5179017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, to the host cell into which the vector is introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification), the peo gene (for selection with G418), and the glutamate synthetase gene.

The term expression control sequence as used herein refers to polynucleotide sequences necessary for the effect of expression and processing of the coding sequences to which they are ligated. Expression control sequences include suitable transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as spicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (ie, the Kozak consensus sequence); sequences that improve protein stability; and when desired, protein secretion enhancing sequences. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence provides advantages

- 20 043633 for example, leader sequences and binding partner sequences.

Host cells and methods for producing antibodies and antibody compositions.

A further aspect of the invention relates to methods for preparing the antibody compositions, and antibodies and antigen-binding portions thereof, of the invention. One embodiment of this aspect of the invention relates to a method for producing an antibody, as defined herein, comprising obtaining a recombinant host cell capable of expressing the antibody, culturing said host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody. Antibodies produced through such expression in such recombinant host cells are referred to herein as recombinant antibodies. The invention also relates to progeny cells of such host cells and to antibodies produced by them.

The term recombinant host cell (or simply host cell) as used herein means a cell into which a recombinant expression vector has been introduced.

The invention relates to host cells, which may contain, for example, the vector of the invention described above. The invention also relates to host cells containing, for example, a nucleotide sequence encoding a heavy chain or an antigen binding portion thereof, a nucleotide sequence encoding a light chain or an antigen binding portion thereof, or both, of an anti-PD-1 antibody or an antigen binding portion thereof of the invention. It should be understood that recombinant host cell and host cell refer not only to the particular cell in question, but also to the progeny of such cell. Since certain modifications may arise in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term host cell within the scope of the invention.

Nucleic acid molecules encoding anti-PD-1 antibodies and vectors containing these nucleic acid molecules can be used to transfect a suitable mammalian, plant, bacterial or yeast host cell. The transformation can be carried out by any known method of introducing polynucleotides into the host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells via viral vectors. Methods for transforming cells are well known in the art. See, for example, US Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines available as expression hosts are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green kidney cells marmoset cells (COS), human hepatocellular carcinoma cells (eg Hep G2), A549 cells and a number of other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high levels of expression. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium, in which host cells are grown. Antibodies can be isolated from the culture medium using standard protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

In addition, the expression of antibodies of the invention or antigen-binding portions thereof from producer cell lines can be improved using a number of known methods. For example, expression of glutamine synthetase system (GS system) genes is a common way to improve expression under certain conditions. The GS system is discussed in whole or in part in connection with patents EP 0216846, 0256055, 0323997 and 0338841.

It is likely that antibodies expressed in different cell lines or transgenic animals may have different glycosylation patterns from each other. However, all antibodies encoded by nucleic acid molecules presented in the present description or containing amino acid sequences presented in the present description form part of

- 21 043633 of the present invention, regardless of the glycosylation state of the antibodies, and more generally, regardless of the presence or absence of post-translational modification(s).

Pharmaceutical compositions.

Another aspect of the invention relates to a pharmaceutical composition containing as an active ingredient (or as the sole active ingredient) an anti-PD-1 antibody or an antigen-binding portion thereof, or an anti-PD-1 antibody composition of the invention. The pharmaceutical composition may contain any anti-PD-1 antibody composition, or an antibody or antigen-binding portion thereof, as described herein. In some embodiments, the compositions are intended to alleviate, prevent, and/or treat a PD-1-associated disorder and/or cancer. As used herein, a PD-1-related or PD-1-mediated disorder refers to a disorder, disease or condition that improves, or slows down its progression, by modulating the activity of PD-1. In some embodiments, the compositions are intended to activate the immune system. In specific embodiments, the compositions are intended to alleviate, prevent, and/or treat cancer originating from tissues such as skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head, and neck, liver, bladder, mammary gland, stomach, uterus and pancreas.

In general, the antibodies, antigen binding portions thereof, and bispecific binding molecules of the invention are suitable for administration as a formulation in admixture with one or more pharmaceutically acceptable excipient(s), for example, as described below.

The pharmaceutical compositions of the invention may contain one or more anti-PD-1 antibodies or binding moieties or bispecific binding molecules of the invention, for example one or two anti-PD-1 antibodies, binding moieties or bispecific binding molecules. In one embodiment, the composition contains one anti-PD-1 antibody of the invention or a binding portion thereof.

In another embodiment, the pharmaceutical composition may contain at least one anti-PD-1 antibody or antigen-binding portion thereof, such as one anti-PD-1 antibody or portion, and one or more additional antibodies targeting one or more corresponding cell surface receptors, e.g. one or more cancer-related receptors.

The term excipient is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient(s) depends largely on factors such as the specific route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. Within the scope of the invention, a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it is preferred to include in the composition isotonic agents, for example sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride. Additional examples of pharmaceutically acceptable substances are humectants or minor amounts of excipients, such as humectants or emulsifiers, preservatives or buffers, which improve the shelf life or potency of the antibody.

The pharmaceutical compositions of the present invention and methods for their preparation will be apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Company, 1995). Pharmaceutical compositions are preferably manufactured under GMP (Good Manufacturing Practices) conditions.

The pharmaceutical composition of the invention may be prepared, packaged or sold in bulk, in the form of a single unit dose or in the form of a plurality of single unit doses. In the context of the invention, a unit dose is a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient that can be administered to the subject, or a convenient portion of such dose, such as one second or one third of such dose.

Any method of administering peptides, proteins or antibodies accepted in the art can be suitably used for the antibodies and antigen binding portions of the invention.

The pharmaceutical compositions of the invention are generally suitable for parenteral administration. As used herein, parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical injury to tissue of the subject and administration of the pharmaceutical composition through an opening in the tissue, thereby generally resulting in direct administration into the bloodstream, muscle, or internal organ. Parenteral administration thus includes, but is not limited to, administration of the pharmaceutical composition via

- 22 043633 injection of the composition, by introducing the composition through a surgical incision, by introducing the composition through a tissue-penetrating non-surgical wound, etc. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, and intrasynovial injections or infusions; and dialytic kidney infusion methods. Regional perfusion is also provided. Specific embodiments include intravenous and subcutaneous administration methods.

Pharmaceutical composition formulations suitable for parenteral administration typically contain the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline solution. Such formulations may be prepared, packaged or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged or sold in unit dosage form, such as ampoules, or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending, stabilizing or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (ie, powder or granular) form for reconstitution using a suitable vehicle (eg, sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions, which may contain excipients such as salts, carbohydrates and buffering agents (preferably pH 3 to 9), but for some applications they may more suitably be formulated in the form of a sterile non-aqueous solution or in dry form , intended for use in conjunction with a suitable carrier such as sterile, pyrogen-free water. Exemplary forms for parenteral administration include solutions or suspensions in sterile aqueous solutions, for example, aqueous solutions of propylene glycol or dextrose. Such dosage forms can be suitably buffered, if desired. Other suitable parenteral formulations that may be used include those containing the active ingredient in microcrystalline form or in a liposomal formulation. Formulations for parenteral administration can be prepared for immediate and/or modified release. Modified release formulations include delayed, sustained, pulsatile, controlled, targeted and programmable release.

For example, in one aspect, sterile injectable solutions can be prepared by incorporating an anti-PD-1 antibody or an antigen-binding portion thereof, a bispecific binding molecule, or an anti-PD-1 antibody composition in the required amount into a suitable diluent with one or a combination of the ingredients listed above as needed. followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization to obtain a powder of the active ingredient plus any desired additional ingredient from their pre-sterilized solution by filtration. Proper fluidity of the solution can be maintained, for example, through the use of a coating such as lecithin, through maintaining the required particle size in the case of a dispersion, and through the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including an absorption delaying agent in the composition, eg, monostearate and gelatin salts, and/or by using modified release coatings (eg, sustained release coatings).

Antibodies of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, in the form of a mixture, or in the form of mixed particulate components, for example, mixed with a suitable pharmaceutically acceptable excipient) from dry powder inhalers, in the form an aerosol atomized from a pressurized container by means of a pump, spray, nebulizer (preferably an atomizer using electrohydrodynamics to produce a mist of fine particles) or nebulizer, with or without the use of a suitable propellant, or in the form of nasal drops.

The pressurized container, pump, spray, nebulizer or nebulizer typically contains a solution or suspension of the antibody of the invention containing, for example, a suitable agent for dispersing, solubilizing or enhancing the release of the active substance, propellant(s) as a solvent.

Before use in a dry powder or suspension formulation, the drug product is typically crushed to a size suitable for delivery by inhalation (typically less than 5 microns). This can be done by any suitable method, grinding, such

- 23 043633 as spiral jet milling, fluidized bed jet milling, supercritical fluid processing to produce nanoparticles, high pressure homogenization or spray drying.

Capsules, blisters and cartridges for use in inhalers or insufflators can be prepared containing a powder mixture of a compound of the invention, a suitable powder base and an activity modifier.

A suitable solution formulation for use in a nebulizer using electrohydrodynamics to produce a fine particle mist may contain a suitable dose of the antibody of the invention per trigger and the trigger volume may, for example, vary from 1 to 100 μl.

Formulations for inhalation/intranasal administration can be formulated for immediate and/or modified release. Modified release formulations include delayed, sustained, pulsatile, controlled, targeted and programmable release.

In the case of dry powder and aerosol inhalers, the unit dose is determined by means of a valve delivering a metered amount. Units in accordance with the invention are typically designed to administer a metered dose or release of an antibody of the invention. The total daily dose is usually administered in a single dose or more usually in divided doses throughout the day.

Antibodies and antibody portions of the invention can also be formulated for oral administration. Oral administration may include ingestion so that the compound enters the gastrointestinal tract and/or buccal, lingual or sublingual administration whereby the compound enters the bloodstream directly from the oral cavity.

Formulations suitable for oral administration include solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multi- or nanoparticles, liquids or powders; lozenges (including liquid-filled); chewable tablets; gels; rapidly dispersing dosage forms; films; suppositories; sprays; and buccal/mucoadhesive patches.

Liquid formulations include suspensions, solutions, syrups and elixirs. Such compositions can be used as fillers for soft or hard capsules (made, for example, from gelatin or hydroxypropyl methylcellulose), and typically contain a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or a suitable oil and one or more from emulsifiers and/or suspending agents. Liquid formulations can also be prepared by diluting a solid, for example from a sachet.

Therapeutic uses of antibodies and compositions of the invention.

In one aspect, anti-PD-1 antibodies and antigen-binding portions thereof, anti-PD-1 antibody compositions and bispecific binding molecules of the invention are used to enhance or activate the immune system in a person in need thereof. In some embodiments, the patient is immunosuppressed. For example, a physician may boost the anticancer activity of a patient's own immune system by administering an anti-PD-1 antibody, antigen binding moiety, composition, or bispecific binding molecule of the present invention, alone or in combination with other drugs (sequentially or in parallel). An anti-PD-1 antibody or part, composition or bispecific binding molecule modulates PD-1 activity in immunocytes, resulting in enhanced anti-cancer immunity. In specific embodiments, the antibody or antigen-binding moiety, composition, or bispecific binding molecule thereof is for use in the treatment of a cancer, e.g., cancers originating from tissues such as skin, lung, intestine, colon, ovary, brain, prostate gland, kidney, soft tissue, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus and pancreas, and any malignancies or other conditions that rely on PD-1 activity and/or in which the patient expresses or overexpresses PD1, PD-L1 and/or PD-L2. Cancers treated with anti-PD-1 antibodies, antigen-binding moieties thereof, anti-PD-1 antibody compositions and/or bispecific binding molecules of the invention may include, for example, melanoma (such as advanced melanoma, or unresectable or metastatic melanoma ), non-small cell lung cancer, bladder cancer, squamous cell carcinoma of the head and neck, ovarian cancer, colorectal cancer, gastric cancer, malignancy with high microsatellite instability, hepatocellular carcinoma, mesothelioma, Merkel cell carcinoma, glioma, multiple myeloma, diffuse large B cell -cell lymphoma, Hodgkin's lymphoma and renal cell carcinoma (RCC).

In some embodiments, the cancers treated with anti-PD-1 antibodies, antigen binding moieties, anti-PD-1 antibody compositions, and/or bispecific binding molecules of the invention may include, for example, advanced or metastatic melanoma, non-small cell lung cancer , squamous cell carcinoma of the head and neck, renal cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, glioblastoma,

- 24 043633 glioma, neuroendocrine tumors, squamous cell lung cancer, small cell lung cancer, hepatocellular carcinoma, bladder cancer, upper urinary tract cancer, esophageal cancer, malignant tumor of the gastroesophageal junction, stomach cancer, liver cancer, colon cancer, colorectal carcinoma , multiple myeloma, sarcomas, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, nasopharyngeal cancer, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, ovarian cancer, malignant tumor of the gastrointestinal tract, primary peritoneal cancer, malignant fallopian tumor pipes, urothelial malignancy, HTLV-associated T-cell leukemia/lymphoma, prostate cancer, genitourinary tract malignancy, meningioma, adrenal malignancy, gliosarcoma, renal cancer, breast cancer, pancreatic cancer, endometrial cancer, basal cell skin cancer, malignant tumor of the appendix, malignant tumor of the bile ducts, malignant tumor of the salivary glands, malignant tumor of Merkel cells in advanced stages, malignant tumor of the genitourinary system, bone cancer, malignant tumor of the chest, malignant tumor of the respiratory tract, adenoid cystic carcinoma, cervical cancer, astrocytoma, chordoma, hematological neoplasia, neuroblastoma, malignant tumor of the oral cavity, squamous cell carcinoma of the skin, thyroid cancer, Kaposi's sarcoma, malignant tumor of the anal canal, gall bladder cancer, malignant tumor of the thymus, uterine body cancer, diffuse large B-cell lymphoma, follicular lymphoma, mesothelioma or solid tumors. A malignant tumor may, for example, be at an early, intermediate, late or metastatic stage.

In some embodiments, anti-PD-1 antibodies, antigen binding moieties, compositions and/or bispecific binding molecules of the invention can be used to treat viral and/or parasitic infections, for example, where pathogens inhibit the host's immune response. By way of example, the pathogen may be, for example, HIV, hepatitis (A, B, or C), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), adenovirus, flavivirus, echovirus, rhinovirus, coxsackievirus, comovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-cell lymphotrophic virus (HTLV), Dengue virus, molluscum contagiosum virus, polio virus, rabies virus, John Cunningham virus (JC) , arboviral encephalitis virus, simian immunodeficiency virus (SIV), influenza virus, herpes virus, giardia virus, malaria virus, Leishmania, Staphylococcus aureus or Pseudomonas aeruginosa.

In some embodiments, anti-PD-1 antibodies, antigen binding moieties, compositions, and/or bispecific binding molecules of the invention may be used to treat a patient who has an immune deficiency or is at risk of developing an immune deficiency (eg, due to a chemotherapy agent or radiotherapy).

Treat, treatment and treatment refer to a method of alleviating or stopping a biological disorder and/or at least one of its accompanying symptoms. As used herein, alleviating a disease, disorder or condition means reducing the severity and/or frequency of symptoms of the disease, disorder or condition.

In addition, references herein to treatment include references to curative, palliative and prophylactic treatment.

A therapeutically effective amount refers to an amount of drug administered that can alleviate, to some extent, one or more symptoms of the disorder being treated. A therapeutically effective amount of an anticancer drug may, for example, result in tumor shrinkage, increased survival, killing of cancer cells, decreased disease progression, inhibition of metastasis, or other clinical endpoints desired by healthcare professionals.

Anti-PD-1 antibodies or antigen-binding portions thereof, compositions or bispecific binding molecules of the invention can be administered alone or in combination with one or more other drugs or antibodies (or in the form of any combination thereof). The pharmaceutical compositions, methods and uses of the invention thus also include embodiments of combinations (co-administration) with other active ingredients, as described in detail below.

As used herein, the terms co-administration, administered together and in combination with the antibodies and antigen binding moieties, compositions and bispecific binding molecules thereof of the invention, with one or more other drugs, mean and refer to the following and include the following:

simultaneous administration of such a combination of antibody/antigen binding moiety/antibody composition/bispecific binding molecule of the invention and drug(s) to a patient in need of treatment, where such components are formulated together in a single dosage form that releases said components at substantially the same time at the specified

- 25 043633 patients;

substantially simultaneous administration of such a combination of antibody/antigen binding moiety/antibody composition/bispecific binding molecule of the invention and drug(s) to a patient in need of treatment, where such components are formulated separately from each other in separate dosage forms that are administered in substantially the same and at the same time to said patient, whereby said components are released at substantially the same time in said patient;

sequentially administering such combination of antibody/antigen binding moiety/antibody composition/bispecific binding molecule of the invention and drug(s) to a patient in need of treatment, where such components are formulated separately from each other in separate dosage forms that are administered at successive periods of time to said patient, with significant time intervals between each administration, whereby said components are released at substantially different periods of time in said patient; and sequentially administering such combination of antibody/antigen binding moiety/antibody composition/bispecific binding molecule of the invention and drug(s) to a patient in need of treatment, where such components are formulated together in a single dosage form that releases said components in a controlled manner thereby releasing them simultaneously, sequentially and/or overlapping, at the same time and/or at different periods of time in a specified patient, where each part can be administered in either the same or a different manner.

The antibodies and antigen binding portions thereof, antibody compositions and bispecific binding molecules of the invention can be administered without additional therapeutic treatments, i.e. as independent therapy (monotherapy). Alternatively, treatment using antibodies and antigen-binding portions thereof, compositions and bispecific binding molecules of the invention may include at least one additional therapeutic treatment (combination therapy), such as another immunostimulant agent, an anticancer agent, an antiviral agent, or a vaccine (e.g., an antitumor vaccine). . In some embodiments, the antibody or antigen binding portion, composition, or bispecific binding molecule thereof may be co-administered or formulated with another medical/drug for the treatment of cancer. Additional therapeutic treatment may include, for example, a chemotherapeutic agent, an antineoplastic or antiangiogenic agent, another anticancer antibody, and/or radiotherapy.

By combining the antibodies, antigen binding moieties, compositions or bispecific binding molecules of the invention with agents known to induce terminal differentiation of cancer cells, the effect can be further improved. Such compounds may, for example, be selected from the group consisting of retinoic acid, trans-retinoic acids, cis-retinoic acids, phenylbutyrate, nerve growth factor, dimethyl sulfoxide, active form of vitamin D3, peroxisome proliferator-activated receptor gamma, 12-O-tetradecanoylphorbol13 -acetate, hexamethylene-bis-acetamide, transforming growth factor-beta, butyric acid, cyclic AMP and vesnarinone. In some embodiments, the compound is selected from the group consisting of retinoic acid, phenylbutyrate, all-trans retinoic acid, and the active form of vitamin D.

Pharmaceutical preparations comprising an anti-PD-1 antibody or antigen-binding portion thereof, a composition or bispecific binding molecule of the invention and at least one other agent (e.g., a chemotherapeutic agent, an antineoplastic agent, or an antiangiogenic agent) can be used as a combination treatment for simultaneous, separate or sequential administration in the treatment of malignant tumors. The other agent may be any agent suitable for treating the particular cancer in question, for example an agent selected from the group consisting of alkylating agents, for example, platinum derivatives such as cisplatin, carboplatin and/or oxaliplatin; plant alkaloids, for example, paclitaxel, docetaxel and/or irinotecan; antitumor antibiotics, for example, doxorubicin (Adriamycin), daunorubicin, epirubicin, idarubicin, mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin and/or mitomycin; topoisomerase inhibitors such as topotecan; and/or antimetabolites, for example, fluorouracil and/or other fluoropyrimidines.

The anti-PD-1 antibody or antigen-binding moiety, antibody composition, or bispecific binding molecule of the invention may also be used in combination with other anticancer therapeutics, such as vaccines, cytokines, enzyme inhibitors, immunostimulatory compounds, and T-cell therapies. In the case of a vaccine, it may be, for example, a protein, peptide or DNA vaccine containing one or more antigens important for the cancer being treated, or a vaccine containing dendritic cells along with the antigen. Suitable cytokines include, for example, IL-2, IFN-gamma and GM-CSF. An example of a type of enzyme inhibitor that has anticancer activity is

- 26 043633 is an indoleamine 2,3-dioxygenase (IDO) inhibitor, for example 1-methyl-D-tryptophan (1-D-MT). Adoptive T-cell therapy refers to various immunotherapy treatments that involve expanding or modifying patients' own T cells to recognize and attack their tumors.

It is also contemplated that an anti-PD-1 antibody or antigen binding portion thereof, an antibody composition, or a bispecific binding molecule of the invention may be used in adjuvant therapy in combination with tyrosine kinase inhibitors. They are synthetic, primarily quinazoline-derived, low molecular weight molecules that interact with the intracellular receptor tyrosine kinase domain and inhibit ligand-induced receptor phosphorylation by competing for the intracellular Mg-ATP binding site.

In some embodiments, an antibody or antigen-binding moiety, composition, or bispecific binding molecule thereof may be used in combination with another medical/drug that mediates immune system activation, including, but not limited to, an agent that modulates the expression or activity of A2AR, BTLA, B7-H3, B7-H4, CTLA-4, CD27, CD28, CD40, CD55, CD73, CD122, CD137, CD160, CGEN-15049, CHK1, CHK2, CTLA-3, SEACAM (for example, SEACAM-1 and/or SEACAM-5) , GAL9, GITR, HVEM, ICOS, IDO, KIR, LAIR1, LAG-3, OX40, TIGIT, TIM-3, TGFR-beta, VISTA, LILRB2, SMTM6 and/or 2B4. In specific embodiments, the agent is an antibody or antigen-binding fragment thereof that binds to one of the above molecules. In specific embodiments, an antibody or antigen binding portion thereof, composition, or bispecific binding molecule of the invention may be administered in combination with a CTLA-4 inhibitor (eg, an anti-CTLA-4 antibody such as tremelimumab or ipilimumab). In one embodiment, an antibody or antigen-binding portion thereof, composition, or bispecific binding molecule of the invention may be administered in combination with ipilimumab.

In particular aspects, the antibodies and antigen binding moieties, compositions and bispecific binding molecules of the invention can be administered in combination with another PD-1 pathway inhibitor, which can target PD-1 or one or more of its ligands. Examples of such inhibitors include other anti-PD-1 antibodies, anti-PD-L1 antibodies and anti-PD-L2 antibodies. In some embodiments, an anti-PD1 antibody or antigen-binding portion thereof, a bispecific antibody, or an antibody composition of the invention may be administered in combination with pembrolizumab and/or nivolumab.

It is understood that the antibodies and antigen-binding portions thereof, antibody compositions, and bispecific binding molecules of the invention may be used in a method of treatment as described herein, may be intended for use in a treatment as described herein, and/or may be intended to use in the manufacture of a medicinal product for treatment as described herein. The invention also relates to kits and articles containing antibodies and antigen-binding portions thereof, antibody compositions and/or bispecific binding molecules described herein.

Doses and route of administration.

The antibodies or antigen binding portions thereof, compositions and bispecific binding molecules of the invention are administered in an effective amount to treat the condition in question, i.e. in doses and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex and weight of the patient, and whether the antibodies are administered as a stand-alone treatment or in combination with one or more additional anticancer drugs.

Dosing regimens can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased as indicated by the needs of the therapeutic situation. Particular advantages are provided by the formulation of parenteral compositions in unit dose form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as single doses for patients/subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifications of the unit dosage forms of the invention are generally conditional on and are directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved; and (b) limitations inherent in the art of preparing formulations, eg, an active compound for treatment, in terms of sensitivity in individuals.

Thus, it will be understood by one skilled in the art, based on the description provided herein, that the dose and dosage regimen are adjusted in accordance with methods well

- 27 043633 well-known in the field of therapy. Those. the maximum tolerated dose can be readily determined and the effective amount to provide a detectable therapeutic benefit to a patient can also be determined, as can the timing requirements for each agent to provide a detectable therapeutic benefit to a patient can be determined. Accordingly, while specific dosages and modes of administration are illustrated herein, these examples do not in any way limit the dose and mode of administration that can be provided to a patient in the practice of the present invention.

It should be noted that the dosage level may be varied depending on the type and severity of the condition being alleviated, and dosage may include single or multiple doses. It is further understood that for any particular subject, the particular dosage regimen should be adjusted over time, in accordance with the needs of the individual and the professional judgment of the person administering or supervising the administration of the composition, and that the concentration ranges presented herein are illustrative only and are not intended to limit the scope or practical application of the claimed composition. In addition, the dosage regimen of the compositions of this invention may be based on a variety of factors, including the type of disease, age, weight, gender, medical condition of the patient, severity of the condition, route of administration, and the specific antibody used. Thus, the dosage regimen may vary widely, but can be determined in a conventional manner using standard methods. For example, dosages can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, such as toxic effects, and/or laboratory values. Thus, the present invention includes increasing the dose for the same patient, as determined by one skilled in the art. The determination of appropriate dosages and regimens is well known in the relevant field and will be understood by one skilled in the art when providing the explanations described herein.

It is contemplated that a suitable dose of the antibody or antigen binding portion thereof, composition or bispecific binding molecule of the invention may be in the range of 0.1-100 mg/kg, for example about 0.5-50 mg/kg, for example about 1-20 mg/kg . The antibody, antigen binding moiety, composition or bispecific binding molecule may, for example, be administered at a dose of at least 0.25 mg/kg, such as at least 0.5 mg/kg, such as at least 1 mg/kg, such as at least at least 1.5 mg/kg, for example at least 2 mg/kg, for example at least 3 mg/kg, for example at least 4 mg/kg, for example at least 5 mg/kg; and for example up to a maximum of 50 mg/kg, for example up to a maximum of 30 mg/kg, for example up to a maximum of 20 mg/kg, for example up to a maximum of 15 mg/kg. Administration can usually be repeated at suitable intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for as long a period of time as deemed appropriate by the responsible medical professional. which may optionally increase or decrease the dose as needed.

An effective amount for tumor therapy can be measured by its ability to stabilize disease progression and/or alleviate symptoms in a patient and preferably reverse disease progression, for example, by reducing tumor volume. The ability of an antibody, antigen binding moiety, composition, or bispecific binding molecule of the invention to inhibit cancer can be assessed in in vitro assays, for example, as described in the Examples, as well as in suitable animal models that are predictive of efficacy in human tumors. Appropriate dosage regimens can be selected to provide the optimal therapeutic response in each specific situation, for example, administration in the form of a single bolus or in the form of a continuous infusion, and possible dose adjustments as dictated by the needs of each case.

Diagnostic applications and compositions.

Antibodies of the present invention can also be used in diagnostic methods (eg, in vitro, ex vivo). For example, antibodies can be used to detect and/or measure the level of PD-1 in a sample from a patient (eg, a tissue sample or a biological fluid sample such as inflammatory exudate, blood, serum, bowel fluid, saliva or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassays and immunohistology. The invention further relates to kits (eg, diagnostic kits) containing the antibodies described herein.

Unless otherwise defined herein, all scientific and technical terms used in connection with the present invention have meanings that are commonly known to one skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in practicing or testing the present invention. In case of inconsistency, the present description, including definitions, prevails.

- 28 043633

In general, the nomenclature and methods used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are well known and generally accepted in the field. Enzyme reactions and purification methods are carried out in accordance with the manufacturer's specifications, as commonly used in the art or as described herein.

In addition, unless the context otherwise requires, singular terms include the plural and plural terms include the singular. Throughout this description and embodiments, the words have and contain, or variants such as has, having, contains, or containing, are to be understood to include the specified integer or group of integers, but not to exclude any other integer or group of integers.

The entire contents of all publications and other references mentioned herein are incorporated by reference. Although a number of documents are cited herein, such citation does not constitute an admission that any of these documents constitute part of the prior art.

To enable this invention to be better understood, the following examples are provided. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Examples

Example 1. Production and screening of anti-PD-1 antibody repertoires.

Materials and methods.

OmniRat® rats (Osborn et al., J Immunol., 2013, 190(4):1481-90), an engineered rat strain from OMT (Open Monoclonal Technology, Inc.) capable of producing human antibodies, were immunized with human PD-1 antigens , cynomolgus macaque or mouse. Cloning of antibody genes from single cell sorted antibody secreting B cells (ASC) obtained from rats was performed using Symplex™ antibody detection technology (Meijer et al., J. Mol. Biol., 2006, 358(3):764- 12; US 7749697; WO 2008/104184).

The Symplex™ antibody library was prepared from single-cell sorted B cells from immunized OMT rats and contained the VH and VL cognate pair coding for each sorted B cell. Constructs for expression of the antibody repertoire encoded fully human immunoglobulins in the IgG1 format, carrying two mutations (L234A/L235A) known to reduce the effector function of the Fc region of the IgG1 antibody (Hezareh et al., J. Virol., 75 (24): 12161-8 (2001)).

CHO-S cells were transfected in a 384-well format using human, cynomolgus or mouse PD-1 expression constructs using Freestyle™ MAX reagent (Invitrogen). In addition, another population of cells was transfected with a control vector encoding the irrelevant human VEGFR2 protein and subsequently used as a negative control. To enable the development of multiplex screening conditions, control cells, intermediate-intensity carboxyfluorescein succinimidyl ester (CFSE^™™)-labeled cells, cynomolgus PD-1-transfected cells, high-intensity CFSE-labeled cells (CFSE™™*), and unlabeled PD-1 transfected cells human cells were mixed in a 1:1:1 ratio, with a density of 1x10E6 cells per ml. In 384-well plates, 40 μl of this cell mixture was mixed with 10 μl of antibody-containing supernatant, and cell-bound antibody was detected by adding goat anti-human IgG (H+L) secondary antibody AF647 (Molecular Probes, Cat. No. A21445). In parallel, antibodies were screened for binding to human (CFSE ^sex ) and mouse (CFSE ^otp ) PD-1 using a similar experimental setup. Samples were acquired using high-throughput flow cytometry (iQue® Screener, Intellicyt) and data were analyzed using ForeCyt® software by plotting CFSE versus IgG binding (AF647). The primary best PD-1-specific candidates were identified as antibody clones binding to both human (CSFE ^otp ) and cynomolgus (CFSE™™*) PD-1 transfected cells, but not to control cells (CSFE^™ ™), and plate numbers and plate coordinates were collected to select the best candidates and subsequent sequence analysis. Several primary best candidates exhibiting additional reactivity to mouse PD-1 in the second screen (CFSE ^otp ) were also selected for further analysis.

Results.

In fig. Ία-ID shows representative flow cytometry dot plots for four anti-PD-1 antibody clones having different reactivity toward PD-1 orthologues:

(a) an antibody clone that binds nonspecifically to CHO-S cells;

(b) an antibody clone that binds specifically only to cells transfected with PD-1

- 29,043,633 people;

(c) an antibody clone that binds specifically to human and cynomolgus PD-1; and (d) an antibody clone that binds to all three PD-1 species in the screen.

The top dot plots in each of (a), (b), (c) and (d) represent the first round of screening testing antibodies for binding to human PD-1 (huPD1) and cynoPD1 (cynoPD1) versus negative control cells (neg). The lower dot plots represent the second round of screening testing antibodies for binding to mouse PD-1 (moPD1) and human PD-1 (huPD1). The x-axis (horizontal) shows CFSE and the y-axis (vertical) shows human IgG binding.

Example 2. Antibody sequences.

The best screening candidates were analyzed by DNA sequencing and the antibody-encoding DNA sequences were isolated. The 488 primary best candidates cross-reactive with human and cynomolgus PD-1 were sequenced. Thus, it was revealed that the repertoire of the best candidates against PD-1 contained 254 unique antibodies, representing 140 genetic clusters. Selected antibody clones were individually expressed and functionally tested as described below. Twelve antibodies exhibiting functional activity in in vitro assays are described later herein. The numbering of the protein sequences of the VH and VL domains of these twelve antibodies is shown in table. 1. The numbering of the immunoglobulin constant region sequences (Ig heavy chain (IgHC) with mutations L234A/L235A and Ig kappa light chain (IgKV)) used for cloning the variable VH and VL genes is shown in Table. 2. The numbering of the CDR sequences of twelve functional antibodies is shown in table. 3. The CDR sequences herein are defined in accordance with the IMGT® definitions for CDR1 and CDR2. For the CDR3 heavy and light chains, the definitions herein include one additional amino acid residue to the amino terminus of IMGT-CDR3 (Cys).

Table 1

Numbering of amino acid sequences of antibody variable domains

Antibody number Protein VH, SEQ ID NO. Protein VL, SEQ ID NO. 18040 1 2 18049 3 4 18098 5 6 18113 7 8 18201 9 10 18247 eleven 12 18250 13 14 18325 15 16 18366 17 18 18400 19 20 18413 21 22 18483 23 24

table 2

Numbering of DNA sequences and amino acid sequences of antibody constant regions

Name sequences CNC, SEQ ID NO. Protein, SEQ ID NO. I dNS 25 26 IgKC 27 28

Table 3

SEQ ID NO for the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 and light chain CDR1 and CDR3 of anti-PD-1 antibodies

Antibody H-CDR1 H-CDR2 H-CDR3 L-CDR1 L-CDR2 L-CDR3 18040 29 thirty 31 32 33 34 18049 35 36 37 38 39 40 18098 41 42 43 44 45 46 18113 29 47 48 49 50 51 18201 52 53 54 38 45 55

- 30 043633

18247 29 56 48 57 33 51 18250 58 59 60 57 33 34 18325 61 62 43 44 45 46 18366 63 64 65 32 33 34 18400 66 67 68 38 45 55 18413 69 70 71 72 45 73 18483 74 75 76 57 33 34

Example 3. Mutations of framework residues in antibody sequences.

An alignment of the VH and VL amino acid sequences from Example 2 with human germline sequences was performed to identify the germline genes from which the VH and VL sequences are derived. In table 4 shows the assignment of the closest V and J germline gene for the VH and VL of each clone, as well as information about mutations of the framework residues, as described below.

Since antibody genes were isolated by RT-PCR using Symplex™ antibody detection technology (Mejier et al., J. Mol. Biol., 358:764-772 (2006); WO 2005/042774) with degenerate primers, the six initial amino acids were are susceptible to mutations that do not occur during maturation of the antibody sequence. Thus, these primer-derived mutations can be returned by mutagenesis to the germline sequence without the risk of reducing binding affinity. The numbers and specific amino acid substitutions VH and VL in these Symplex-corrected sequence variants are shown in Table 1. 4. In addition, antibodies carrying somatic hypermutations in the framework regions of the variable domains, i.e. mutations in VH or VL outside the CDR can be returned to the closest germline V- or J- sequence. The numbers and specific amino acid substitutions in the antibody framework regions that can be replaced with germline ones in these germline sequence variants are also shown in Table 1. 4. It is obvious that the variants of mutations close to germline ones, shown in Table. 4 (see columns Number of VH framework mutations in the near-germline variant and Number of mutations in the VH framework residues in the near-germline variant) include any Symplex-corrected mutations, as well as mutations outside the initial six amino acid positions.

As noted above, altering degenerate primer-derived mutations in the first amino acids of each VH or VL sequence was not expected to impair binding properties relative to the parent antibody. Thus, it is preferred that the anti-PD-1 antibodies of the invention include the Symplex-corrected mutations listed in the tables below.

As with germline variant mutations outside the initial six amino acid positions of each sequence, any one or more of these mutations can be selected. Determining whether an individual mutation has a negative effect on the antigen-binding properties of an antibody can be done by obtaining sequences of germline VH and VL variants with the mutation and comparing the antigen-binding properties of the antibody with those of the precursor with the corresponding parent or Symplex-corrected sequences. In the case where a decrease in binding affinity or other change in binding properties is observed, variants, for example, can be tested using a 2x2 VH/VL matrix using the Symplex-corrected variant and the germline-corrected heavy and light chain variant of each antibody, i.e. in this case four combinations for each antibody. This allows determination of whether one or the other or both of the proximal VH and VL germline sequences contribute to any of the observed altered binding properties. Alternatively or additionally, series of appropriate antibodies in which individual germline variant mutations have been removed can be tested to identify specific mutations that affect binding of a germline variant having all of the listed mutations.

In table 5 shows the VH and VL sequence numbers for the original sequences, as well as the corresponding Symplex-corrected and germline-near variants. From the numbers in the table. 5, it is apparent that in some cases there is no difference between the original sequence and the Symplex-corrected sequence or between the Symplex-corrected sequence and the proximate germline sequence. Mutations in germline framework residues are based on IMGT definitions. In the accompanying sequence list, the resulting amino acid substitutions are underlined and indicated in bold.

- 31 043633

Table 4-1

Mutations of framework residues in the VH sequence

Antibody The closest V— , J nucleation sequence # VH FR mutations in Symplex corrected version VH FR mutations in the Symplex corrected version # VH FR mutations close to the germline variant VH FR mutations close to the germline variant 18040 IGHV311*01, IGHJ5*02 0 2 N35S, D84N 18049 IGHV333*04, IGHJ4*02 0 0 18098 IGHV323*04, IGHJ3*02 2 Q1E, Q5V 9 Q1E, Q5V, R13Q, N35S, V46E, T50A, S77N, F80Y, T115M 18113 IGHV311*01, IGHJ5*02 0 1 A64V 18201 IGHV44*02, IGHJ3*02 1 V5Q 4 V5Q, T59N, R76K, M83L 18247 IGHV311*01, IGHJ4*02 0 2 D10G, R16G 18250 IGHV311*01, IGHJ4*03 1 Q5V 4 Q5V, H50Y, D59Y, V61A 18325 IGHV323*04, IGHJ3*02 3 Q1E, Q5V, Q6E 8 Q1E, Q5V, Q6E, N35S, A49S, T50A, G73D, M78T 18366 IGHV37*02, IGHJ5*02 2 Q1E, Q6E 2 Q1E, Q6E 18400 IGHV44*02, IGHJ3*02 1 L2V 6 L2V, K43G, V49I, S59N, M70I, P111Q 18413 IGHV323*04, IGHJ4*03 0 2 L48V, T50A 18483 IGHV37*02, IGHJ5*02 3 Q1E, Q5V, Q6E 4 Q1E, Q5V, Q6E, N35S

- 32 043633

Table 4-2

Mutations of framework residues in the VL sequence

Antibody The closest V-, J germinal sequence # VL FR mutacid in Symplexcorrected version VL FR mutation in Symplex corrected version # VL FR mutacid in approximation germinal variant VL FR mutations close to germinal variant 18040 IGKV4-l*01, IGKJl*01 1 E1D 3 EID, F40A, F55Y 18049 IGKV117*01, IGKJl*01 2 EID, V3Q 3 EID, V3Q, N53S 18098 IGKV1D12*01, IGKJl*01 1 L4M 1 L4M 18113 IGKV4-l*01, IGKJl*01 2 Q3V, L4M 3 Q3V, L4M, R69S 18201 IGKV1-6*O1, IGKJl*01 1 D1A 1 D1A 18247 IGKV4-l*01, IGKJl*01 2 EID, L4M 4 EID, L4M, F55Y, F93Y 18250 IGKV4-l*01, IGKJ2*01 2 Q3V, L4M 3 Q3V, L4M, S55Y 18325 IGKV1D12*02, IGKJl*01 3 EID, V3Q, L4M 3 EID, V3Q, L4M 18366 IGKV4-l*01, IGKJ2*01 1 E1D 3 EID, L40A, F55Y 18400 IGKV1-6*O1, IGKJl*01 2 E1A, V3Q 3 E1A, V3Q, P10S 18413 IGKV1D12*01, IGKJ4*02 1 L4M 2 L4M, P12S 18483 IGKV4-l*01, IGKJ2*01 1 Q3V 3 Q3V, L40A, F55Y

Table 5

SEQ ID NO for the VH and VL amino acid sequences of anti-PD-1 antibodies, including Symplex-corrected and germline-near variants

Antibody number Protein sequence number VH Symplex corrected version of VH VH variant close to the germline Protein sequence number VL Symplex corrected version VL VL variant close to embryonic 18040 1 1 96 2 84 106 18049 3 3 3 4 85 107 18098 5 77 97 6 86 86 18113 7 7 98 8 87 108 18201 9 78 99 10 88 88 18247 eleven eleven 100 12 89 109 18250 13 79 101 14 90 110 18325 15 80 102 16 91 91 18366 17 81 81 18 92 111 18400 19 82 103 20 93 112 18413 21 21 104 22 94 113 18483 23 83 105 24 95 114

Example 4 Flow cytometry analysis of anti-PD-1 antibodies for PD-L1 blocking activity.

This example describes testing of anti-PD-1 antibodies for PD-L1 blocking activity by flow cytometry.

Ways.

PD-L1 ligand blocking activity was examined in a cell-based assay in which human PD-1 was recombinantly expressed on CHO-S cells and binding of R-PE (R-phycoerythrin)-labeled human PD-L1-Fc chimeric protein was analyzed by flow cytometry. A commercially available recombinant PD-L1-Fc chimeric protein (R&D Systems, USA) was conjugated to R-PE using the Lightning-Link® R-phycoerythrin conjugation kit (Innova Biosciences, UK). CHO-S cells were transiently transfected to express human PD-1. Then

- 33 043633 cells were incubated with 50 μl of anti-PD-1 antibody at 20 μg/ml on ice followed by the addition of 50 μl of R-PE labeled PD-L1-Fc at approximately 3.4 μg/ml (final concentration 16.4 nM) followed by further incubation for an additional 20 min (final concentration of anti-PD-1 antibody: 10 μg/ml). Bound antibody was detected using APC (allophycocyanin) conjugated to anti-human IgG light chain antibody. The binding of PD-L1 and anti-PD-1 antibody was quantified by flow cytometry detecting R-PE and APC fluorescence, respectively.

Results.

The results of the competition experiment are presented in Fig. 2A-2H, where the X-axis (horizontal) shows PD-L1 binding and the Y-axis (vertical) shows human IgG binding. All anti-PD-1 antibodies tested were capable of inhibiting the interaction of PD-1 with PD-L1 at a final antibody concentration of 10 μg/ml. PD-L1 binding to PD-1-expressing cells in the presence of a nonspecific antibody was used as a negative control to block PD-L1. In addition, a representative graph of PD-L1 binding in the presence of a non-blocking antibody is shown.

Example 5: Measurement of affinity of anti-PD-1 antibody for human and cynomolgus monkey ECD PD-1 antigen.

This example shows how twelve functional anti-PD-1 antibodies exhibit strong binding affinities for human PD-1, with K _D ranging from low nM to intermediate pM. The same antibodies also cross-react with cynomolgus PD-1 with a K _D in the intermediate to low nM range. Antibodies 18201 and 18400 also cross-react with mouse PD-1 with intermediate to low nM KD.

Ways.

Binding kinetics analysis was performed by surface plasmon resonance (SPR) using a Continuous Flow Microspotter (CFM, Wasatch Microfluidics, US) coupled with an Ibis MX96 SPR device (IBIS Technologies, The Netherlands). Surface plasmon resonance imaging analysis was performed on G-ahu-IgG Fc SensEye® SPR sensors (Ssens BV, The Netherlands). Anti-PD-1 antibodies expressed in the IgG1 LALA format (i.e., having the L234A/L235A mutations) were diluted to 2.5 nM in PBS-T (1x PBS with 0.05% Tween 20, pH 7.4). Antibodies were spotted onto Ga-hu-IgG Fc SensEye® for 15 min using a Continuous Flow Microspotter (CFM, Wasatch Microfluidics, Salt Lake City, US). After spotting, SensEye® was positioned in the IBIS MX96 biosensor and antibodies were chemically fixed to the sensor surface using a Fix It kit (Ssens BV, The Netherlands). Kinetics analysis was performed using kinetic titration series (Karlsson et. al., Anal. Biochem., 349(1):136-47 (2006)), where monomeric PD-1 antigen was injected in increasing concentrations from 2 nM to 100 nM without using surface regeneration stages after each antigen injection. Binding to human PD-1 ECD (Acro Biosystems, cat. no. PD1-H52219), mouse PD-1 ECD (Sino Biological, cat. no. 50124-M08H), and cynomolgus ECD PD-1 (Acro Biosystems, cat. no. 50124-M08H) was tested. No. PD1-C5223) in three separate experiments. Antigen binding was carried out for 15 min and antigen dissociation was carried out for 60 min. Kinetics analysis was performed at 25°C. Once completed, the recorded binding responses were fit to a simple 1:1 Langmuir binding model using Scrubber 2 software to calculate binding rate constants (k _on or k _a ), dissociation rate (ko _ff or k _d ), and affinity (KD).

Results.

The surface plasmon resonance (SPR) results are shown in the table below. 6. Antibodies were generally of high affinity and all antibodies tested cross-reacted with cynomolgus PD-1. Antibodies 18201 and 18400 also cross-reacted with mouse PD-1, indicating that the epitopes recognized by these two antibodies were unique compared to other anti-PD-1 antibodies, including the reference antibodies pembrolizumab and nivolumab, which do not cross-reacted with mouse PD-1. These properties provide advantages because they allow antibodies to be directly tested in non-human primate or mouse models prior to testing in human subjects.

- 34 043633

Table 6

Binding kinetics of anti-PD-1 antibodies to human, cynomolgus or mouse PD-1 ECD as measured by surface plasmon resonance (SPR)

Antibody ECD PD-1 k _on (M^s- ¹ ) _kOff (s ^-1 ) K _D (M) 18040 Human 2.6E+05 1.1E-04 4.1E-10 18040 Cynomolgus macaque 3.4E+05 9.7E-04 2.9E-09 18040 Mouse N.B. N.B. N.B. 18049 Human 3.4E+04 6.0E-05 1.8E-09 18049 Cynomolgus macaque 3.8E+04 8.0E-05 2.1E-09 18049 Mouse N.B. N.B. N.B. 18098 Human 1.0E+05 2,ZE-04 2,ZE-09 18098 Cynomolgus macaque 3.9E+04 1,ZE-OZ 3.4E-08 18098 Mouse N.B. N.B. N.B. 18113 Human 1.7E+05 1.0E-04 6.0E-10 18113 Cynomolgus macaque 1.4E+05 7.6E-04 5,ZE-09 18113 Mouse N.B. N.B. N.B. 18201 Human 2.9E+05 1,ZE-04 4,ZE-10 18201 Cynomolgus macaque 1.9E+05 2,ZE-04 1.2E-09 18201 Mouse 3.8E+04 7,ZE-05 1.9E-09 18247 Human 1.1E+05 1.5E-04 1.4E-09 18247 Cynomolgus macaque 9.8E+04 2.7E-03 2.8E-08 18247 Mouse N.B. N.B. N.B. 18250 Human 3.5E+05 2.9E-04 8,ZE-10 18250 Cynomolgus macaque 2.8E+05 1.8E-03 6.4E-09 18250 Mouse N.B. N.B. N.B. 18325 Human 1,ЗЭ+05 1.1E-04 8.2E-10 18325 Cynomolgus macaque 1.4E+05 6.2E-04 4,ZE-09 18325 Mouse N.B. N.B. N.B. 18366 Human 3.0E+05 4.0E-04 1,ZE-09 18366 Cynomolgus macaque 2.2E+05 6.4E-03 2.9E-08 18366 Mouse N.B. N.B. N.B. 18400 Human 2,ЗЭ+05 3.5E-04 1.6E-09 18400 Cynomolgus macaque 2,ЗЭ+05 8.7E-04 3.7E-09 18400 Mouse 4.7E+03 5.0E-05 1.1E-08 18413 Human 7.7E+04 1.1E-04 1.5E-09 18413 Cynomolgus macaque 9.7E+04 6.2E-04 6.4E-09 18413 Mouse N.B. N.B. N.B. 18483 Human 5.8E+05 1.7E-04 2.9E-10 18483 Cynomolgus macaque 2.6E+05 8.0E-04 3.1E-09 18483 Mouse N.B. N.B. N.B. nivolumab analogue Human 2.4E+05 1.9E-04 8.0E-10 nivolumab analogue Cynomolgus macaque 4.1E+05 6.2E-04 1.5E-09 nivolumab analogue Mouse N.B. N.B. N.B. pembrolizumab/keytruda Human 4.2E+05 1.4E-03 3.4E-09 pembrolizumab/keytruda Cynomolgus macaque 3.5E+05 1,ZE-OZ 3.6E-09 pembrolizumab/keytruda Mouse N.B. N.B. N.B.

N.B.=no binding.

Example 6: In vitro functional evaluation of anti-PD-1 monoclonal antibodies.

This example shows how twelve anti-PD-1 antibodies function in two different functional assays: a Staphylococcal Enterotoxin B (SEB) whole blood assay and one

- 35 043633 third-party mixed lymphocyte culture reaction (MLR). The ability of these twelve anti-PD-1 mAbs to stimulate IL-2 production in the SEB-treated whole blood assay or interferon-gamma (IFN-γ) production in the unilateral MLR assay was assessed as described below.

Ways.

SEB is a superantigen that binds to MHC class II molecules and specific regions of Ve T cell receptors (TCRs) and drives nonspecific T cell stimulation. This leads to the activation/proliferation of polyclonal T cells and the release of cytokines, including IL-2 and IFN-γ. SEB was added at 1 μg/ml to whole blood, and after two days of culture, supernatants were collected and IL-2 levels were determined by conventional ELISA.

In a unilateral MLR assay, dendritic cells (DCs) and CD4 positive (CD4+) T cells isolated from two different healthy donors were cocultured to induce an alloantigen-specific response leading to cytokine production and T cell activation/proliferation. Dendritic cells were differentiated from CD14+ monocytes by six days of culture with 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/ml interleukin-4 (IL-4), and mixed at a ratio of 1:10 with CD4+ T -cells isolated from peripheral blood mononuclear cells (PBMC) from material from a healthy donor. After 5 days of culture, supernatants were collected and IFN-γ levels were determined using the Meso Scale electrochemiluminescent cytokine assay (MSD).

Dose response curves were obtained by 2-fold dilutions of the antibody with an initial concentration of 50 μg/ml.

Results.

Dose response curves for the twelve antibodies in the whole blood SEB assay and the one-way MLR assay are shown in FIG. 3A-3F and 4A-4F respectively. Each point in the graph represents the mean of three replicates, where error bars represent SEM. All of these antibodies were found to induce a dose-dependent increase in IL-2 production in the SEB whole blood assay and IFN-γ production in the MLR assay.

Example 7. Grouping of epitopes of monoclonal antibodies against PD-1.

This example illustrates how anti-PD-1 antibodies were grouped into epitope groups based on pairwise competition patterns. Antibodies belonging to different epitope groups recognize different epitopes on ECD PD-1.

Ways.

Pairwise antibody competition studies were performed by surface plasmon resonance (SPR) analysis using a Continuous Flow Microspotter (CFM) (Wasatch Microfluidics, US) coupled with an IBIS MX96 SPR device (IBIS Technologies, The Netherlands). Surface plasmon resonance imaging analysis was performed on a G-a-hu-IgG Fc SensEye® SPR sensor (Ssens BV, The Netherlands). A total of eighteen anti-PD-1 antibodies (human, IgG1) were diluted to 10 μg/ml in PBS buffer containing 0.05% Tween 20 (PBS-T), pH 7.0. Antibodies were fixed to the surface of the anti-Fc sensor by spotting for 15 min using a Continuous Flow Microspotter. After spotting, SensEye® was positioned in the IBIS MX96 biosensor and the remaining anti-Fc sites were blocked by injection of 30 μg/ml non-specific human IgG1. Bound antibodies were conjugated to the surface using the FixIt kit (Ssens BV, The Netherlands). After sensor preparation, antibody competition assays were performed using a classic sandwich assay. Monovalent ECD PD-1 antigen (Sino Biological, China) was diluted in HBS-EP running buffer and injected at a concentration of 50 nM, and coupled to a conjugated anti-PD-1 antibody array. Individual injections of each of eighteen anti-PD-1 antibodies diluted to 100 nM in HBS-EP running buffer were then performed to establish patterns of antibody competition. After each competition cycle, the sensor surface was regenerated using 10 mM Glycine HCl buffer, pH 2.0.

Results.

The competition pattern of eighteen anti-PD-1 antibodies is shown in FIG. 5. Antibodies 12866 and 12807 were not found to have functional activity in cell-based assays (data not shown), but are included because they recognize individual epitopes that can be used to characterize other epitope groups. The anti-PD-1 antibodies tested can be attributed to two non-overlapping epitope groups. Functional antibodies belonging to epitope group 1 all cross-blocked each other and could be further divided into subgroups based on the blocking of antibodies 12866 and 12807. For example, antibodies blocking both mAbs 12866 and 12807 are assigned to epitope group 1C. Epitope group 1C includes antibodies 18366, 18483, 18113, 18247, 18040 and 18250. Antibodies in epitope group 1D include the nivolumab analogue (Nivol.) and 18049, and are characterized by blocking mAb 12866, but not 12807. Antibodies belonging to the epitope no group 1E , are characterized only by blocking mAb 12807. Epitope group 1E includes the pembrolizumab analogue (Pembrol) and antibodies 18098, 18201, 18400, 18413 and 18325.

- 36 043633

Antibodies 12760 and 13112 do not block ligand PD-L1 and PD-L2, and are assigned to a separate epitope group 2 because they cross-blocked each other but did not block the binding of any of the antibodies from epitope group 1. Antibodies 12760 and 13112 likely bind with a site on PD-1 that does not overlap with the ligand binding site of PD-L1 and PD-L2.

List of sequences. SEQ ID NO:1

QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMXWIRQAPGKGLEWVSYISSTGSTIY

YADSVKGRFTISRDNAKNSLYLQMXSLRAEDTAVYYCARATNWGSDYWGQGTLVTVSS

X at position 35 represents N or S

X at position 84 represents D or N

SEQ ID NO:2

XIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLXWYQQKPGQPPKLLIXWAST

RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKVEIK

X in position 1 represents E or D

X at position 40 represents F or A

X at position 55 represents E or Y

SEQ ID NO:3

QVQLVESGGGWQPGRSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWYDGSDKY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGGNYYGDFWGQGTLVTVSS

SEQ ID NO:4

XIXMTQSPSSLSSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYVASXLQSGVP

SRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNSYPWTFGQGTKVEIK

X V position 1 is is E or D X V position 3 is is V or Q X V position 53 ; is is N or S SEQ ID NO:5

XVQLXESGGGLVXPGGSLRLSCAASGFTFSSFAMXWVRQAPGKGLXWVSXITGGGTTSY

YADSVKGRFTISRDNSKXTLXLQMNSLRAEDTAVYYCAKWGSWSAGAFDIWGQGTXVTVSS

X in position 1 represents Q or E X in position 5 represents Q or V X in position 13 represents R or Q X in position 35 represents N or S X in position 46 represents V or E X in position 50 represents T or A X in position 77 represents S or N X in position 80 represents F or Y X in position SEQ ID NO:6 115 represents T or M

DIQXTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK

X at position 4 represents L or M

SEQ ID NO:7

QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIY

YADSXKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWAFDYWGQGTLVTVSS

X at position 64 represents A or V

SEQ ID NO:8

DIXXTOSPDSLAVSLGERATINCKSSOSVFYSANNKNYLAWYOOKPGOPPKLLIYWTST RESGVPDRFXGSGSGTDFTLTISSLQAEDVAVYYCQQFYSTPRTFGQGTKVEIK

X in position 3 is is Q or V X in position 4 is itself L or M X in position 69 SEQ ID NO:9 is by yourself: R or S

QVQLXESGPGLVKPSGTLSLTCAVSGGSISSNNWWSWVRQPPGKGLEWIGEIYHDGTTX

YNPSLKSRVTISVDKSXNQFSLKXSSVTAADTAVYYCARGDWGSGAFDIWGQGTMVTVSS

X at position 5 represents V or Q

X at position 59 represents T or N

- 37 043633

X at position 76 represents R or K

X at position 83 represents M or L

SEQ ID NO:10

XIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIK

X in position 1 represents D or A SEQ ID NO:11

QVQLVE S GGXLVKPGXS LRL S CAAS G FT FS DYYMSWIRQAPGKGLEWVS YISSSSSTIY YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWAFDYWGQGTLVTVSS

X at position 10 represents D or G

X at position 16 represents R or G

SEQ ID NO:12

XIVXTQSPDSLAVSLGERATINCKSSQSVFYSSNNKNYLAWYQQKPGQPPKLLIXWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYXCQQFYSTPRTFGQGTKVEIK

X V position 1 represents E or D X V position 4 represents L or M X V position 55 represents F or Y X V position 93 represents F or Y SEQ ID NO:13

QVQLXESGGGLVKPGGSLRLSCAASGFTFRDYYMSWIRQAPGKGLEWVSXISSSGSIIX

YXDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWALDYWGQGTLVTVSS

X at position 5 represents Q or V

X at position 50 represents H or Y

X at position 59 represents D or Y

X at position 61 represents V or A

SEQ ID NO:14

DIXXTOSPDSLAVSLGERATINCKSSOSVFYSSNNKNYLAWYOQKPGOPPKLLIXWAST

RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK

X in position 3 represents Q or V

X in position 4 represents L or M

X at position 55 represents S or Y

SEQ ID NO:15

XVQLXXSGGGLVQPGGSLRLSCAASGTFSSHVMXWVRQAPGKGLEWVXXISGSGVDTY

YADSVKGRFTISRXNSKNXLYLQMNSLRAEDTAVYYCAKWGSWSAGAFDIWGQGTMVTVSS

X V position 1 is is Q or E X V position 5 is is Q or V X V position 6 is is Q or E X V position 35 is is N or S X V position 49 is is A or S X V position 50 is is T or A

- 38 043633

X at position 73 represents G or D

X at position 78 represents M or T

SEQ ID NO:16

XIXXTOSPSSVSASVGDRVTITCRASOGISSWLAWYOOKPGKAPKLLIYAASSLOSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK

X in position 1 represents E or D

X in position 3 represents V or Q

X in position 4 represents L or M

SEQ ID NO:17

XVQLVXSGGGLVQPGGSLRLSCAASGFFSSYWMSWVRQAPGKGLEWVANIKQDGSEKY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWGFDNWGQGTLVTVSS

X in position 1 represents Q or E

X at position 6 is Q or E SEQ ID NO:18

XIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLXWYQQKPGQPPKLLIXWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK

X in position 1 represents E or D

X at position 40 represents L or A

X at position 55 represents F or Y

SEQ ID NO:19

QXQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPXKGLEWXGEIFHDGTTX YNPSLKSRVTXSVDKSKNQFSLKLSSVTAADTAVYYCARGNWGSGALDIWGXGTMVTVSS

X in position 2 represents is L or V X in position 43 represents is K or G X in position 49 represents is V or I X in position 59 represents is S or N X in position 70 represents is M or I X in SEQ position ID NO:20 111 represents P or Q

XIXMTQSPSXLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIK

X in position 1 is itself E or A X in position 3 is is V or Q X at position 10 SEQ ID NO:21 । is by yourself: P or S

EVQLVESGGGLVQPGGSLRLSCAASGTFSSFVMSWVRQAPGKGLEWXSXISGGGGSTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDWDLYYFDYWGQGTLVTVSS

X in position 48 is yourself L or V X in position 50 is yourself T or A SEQ ID NO:22

- 39 043633

DIQXTQSPSSVXASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK

X in position 4 represents L or M

X at position 12 represents P or S

SEQ ID NO:23

XVOLXXSGGGLVOPGGSLRLSCAASGFTFSDYWMXWVROAPGKGLEWVANIKEDGNEKY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWGSDYWGQGTLVTVSS

X V position 1 is is Q or E X V position 5 is is Q or V X V position 6 is is Q or E X V position 33 > represents ¹ is N or S SEQ ID NO:24

DIXMTQSPDSLAVSLGERATINCKSSQSVFYSSNNKNYLXWYQQKPGQPPKLLIXWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK

X in position 3 represents Q or V

X at position 40 represents L or A

The X at position 55 represents F or Y Antibody constant sequences.

SEQ ID NO:25 (IgHC DNA sequence)

GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAA CGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA CGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC AGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC CTGCCCCCATCCCGGGAG GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT ACACG CAGAAGAGCCTCTCCCTGTCCCCGGGTAAA

SEQ ID NO:26 (IgHC protein sequence)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL

- 40 043633

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

SEQ ID NO:27 (IgKC DNA sequence)

CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC TGGAACTGCCCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTAC GAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT

SEQ ID NO:28 (IgKC protein sequence)

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:29 (18040/18113/18247 H-CDR1) GFTFSDYY SEQ ID NO:30 (18040 H-CDR2) ISSTGSTI SEQ ID NO:31 (18040 H-CDR3) CARATNWGSDY SEQ ID NO:32 (18040/18366 L-CDR1) QSVLYSSNNNKNY SEQ ID NO:33 (18040/18247/18250/18366/18483 L-CDR2) W.A.S. SEQ ID NO:34 (18040/18250/18366/18483 L-CDR3) CQQYYSTPYT SEQ ID NO:35 (18049 H-CDR1) GFTFSNYG SEQ ID NO:36 (18049 H-CDR2) IWYDGSDK SEQ ID NO:37 (18049 H-CDR3) CAGGGNYYGDF SEQ ID NO:38 (18049/18201/18400 L-CDR1) QGIRND SEQ ID NO:39 (18049 L-CDR2) VAS SEQ ID NO:40 (18049 L-CDR3) CLQYNSYPWT SEQ ID NO:41 (18098 H-CDR1) GETESSFA SEQ ID NO:42 (18098 H-CDR2) ITGGGTTS

- 41 043633

SEQ ID NO:43 (18098/18325 H-CDR3)

CAKWGSWSAGAFDI

SEQ ID NO:44 (18098/18325 L-CDR1) QGISSW SEQ ID NO:45 (18098/18201/18325/18400/18413 L-CDR2) A.A.S. SEQ ID NO:46 (18098/18325 L-CDR3) CQQANSFPWT SEQ ID NO:47 (18113 H-CDR2) ISSSGSTI SEQ ID NO:48 (18113/18247 H-CDR3) CARDTNWAFDY SEQ ID NO:49 (18113 L-CDR1) QSVFYSANNKNY SEQ ID NO:50 (18113 L-CDR2) WTS SEQ ID NO:51 (18113/18247 L-CDR3) CQQFYSTPRT SEQ ID NO:52 (18201 H-CDR1) GGSISSNNW SEQ ID NO:53 (18201 H-CDR2) IYHDGTT SEQ ID NO:54 (18201 H-CDR3) CARGDWGSGAFDI SEQ ID NO:55 (18201/18400 L-CDR3) CLQDYNYPRT SEQ ID NO:56 (18247 H-CDR2) ISSSSSTI SEQ ID NO:57 (18247/18250/18483 L-CDR1) QSVFYSSNNNKNY SEQ ID NO:58 (18250 H-CDR1) GFTFRDYY SEQ ID NO:59 (18250 H-CDR2) ISSSGSII SEQ ID NO:60 (18250 H-CDR3) CARDTNWALDY SEQ ID NO:61 (18325 H-CDR1) GFTFSSHV SEQ ID NO:62 (18325 H-CDR2) ISGSGVDT

- 42 043633

SEQ ID NO:63 (18366 H-CDR1) GFTFSSYW SEQ ID NO:64 (18366 H-CDR2) IKQDGSEK SEQ ID NO:65 (18366 H-CDR3) CARDTNWGFDN SEQ ID NO:66 (18400 H-CDR1) GGSISSSNW SEQ ID NO:67 (18400 H-CDR2) IFHDGTT SEQ ID NO:68 (18400 H-CDR3) CARGNWGSGALDI SEQ ID NO:69 (18413 H-CDR1) GETESSEV SEQ ID NO:70 (18413 H-CDR2) ISGGGGST SEQ ID NO:71 (18413 H-CDR3) CAKDWDLYYFDY SEQ ID NO:72 (18413 L-CDR1) QGISNW SEQ ID NO:73 (18413 L-CDR3) CQQANSFPLT SEQ ID NO:74 (18483 H-CDR1) GFTFSDYW SEQ ID NO:75 (18483 H-CDR2) IKEDGNEK SEQ ID NO:76 (18483 H-CDR3) CARDTNWGSDY SEQ ID NO:77

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFAMSWVRQAPGKGLVWVSTITGGGTTSYY ADSVKGRFTISRDNSKSTLFLQMNSLRAEDTAVYYCAKWGSWSAGAFDIWGQGTTVTVSS SEQ ID NO:78

QVQLQESGPGLVKPSGTLSLTCAVSGGSISSNNWWSWVRQPPGKGLEWIGEIYHDGTTTY NPSLKSRVTISVDKSRNQFSLKMSSVTAADTAVYYCARGDWGSGAFDIWGQGTMVTVSS SEQ ID NO:79

QVQLVESGGGLVKPGGSLRLSCAASGFTFRDYYMSWIRQAPGKGLEWVSHISSSGSIIDY

VDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWALDYWGQGTLVTVSS SEQ ID NO:80

EVOLVESGGGLVOPGGSLRLSCAASGFTFSSHVMNWVROAPGKGLEWVATISGSGVDTYY ADSVKGRFTISRGNSKNMLYLQMNSLRAEDTAVYYCAKWGSWSAGAFDIWGQGTMVTVSS

- 43 043633

SEQ ID NO:81

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYY VDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWGFDNWGQGTLVTVSS SEQ ID NO:82

QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPKKGLEWVGEIFHDGTTSY NPSLKSRVTMSVDKSKNQFSLKLSSVTAADTAVYYCARGNWGSGALDIWGPGTMVTVSS SEQ ID NO:83

EVOLVESGGGLVOPGGSLRLSCAASGFTFSDYWMNWVROAPGKGLEWVANIKEDGNEKYY VDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWGSDYWGQGTLVTVSS SEQ ID NO:84 piVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLFWYQQKPGQPPKLLIFWASTR ESGVPDRFS GSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKVEIK SEQ ID NO:85

DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYVASNLQSGVPS RFSGSGSGTEFTLTISSLQPEDFATYYCLQYNSYPWTFGQGTKVEIK SEQ ID NO:86

DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK SEQ ID NO:87

DIVMTOSPDSLAVSLGERATINCKSSOSVFYSANNKNYLAWYOOKPGOPPKLLIYWTSTR ESGVPDRFRGSGSGTDFTLTISSLQAEDVAVYYCQQFYSTPRTFGQGTKVEIK SEQ ID NO:88

AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIK SEQ ID NO:89

DIVMTQSPDSLAVSLGERATINCKSSQSVFYSSNNKNYLAWYQQKPGQPPKLLIEWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQFYSTPRTFGQGTKVEIK

SEQ ID NO:90

DIVMTOSPDSLAVSLGERATINCKSSOSVFYSSNNKNYLAWYOOKPGOPPKLLISWASTR

ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK SEQ ID NO:91

DIQMTOSPSSVSASVGDRVTITCRASOGISSWLAWYOOKPGKAPKLLIYAASSLOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK SEQ ID NO:92

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLLWYQQKPGQPPKLLIFWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK

SEQ ID NO:93

AIQMTQSPSPLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIK SEQ ID NO:94

- 44 043633

DIQLTQSPSSVPASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYAASSLQSGVPS

RFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPLTFGGGTKVEIK

SEQ ID NO:95

DIVMTQSPDSLAVSLGERATINCKSSQSVFYSSNNKNYLLWYQQKPGQPPKLLIFWASTR

ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK

SEQ ID NO:96

QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSTGSTIYY

ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARATNWGSDYWGQGTLVTVSS SEQ ID NO:97

E VQLVE S GGGLVQPGGS LRL S CAAS G FT FS S FAMSWVRQAPGKGLEWVSAITGGGTTSYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGSWSAGAFDIWGQGTMVTVSS SEQ ID NO:98

QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYY

ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWAFDYWGQGTLVTVSS SEQ ID NO:99

QVQLQESGPGLVKPSGTLSLTCAVSGGSISSNNWWSWVRQPPGKGLEWIGEIYHDGTTNY

NPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGDWGSGAFDIWGQGTMVTVSS SEQ ID NO:100

QVQLVE S GGGLVKPGGS LRL S CAAS G FT FS DYYMSWIRQAPGKGLEWVS YISSSSSTIYY

ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWAFDYWGQGTLVTVSS SEQ ID NO:101

QVQLVESGGGLVKPGGSLRLSCAASGFTFRDYYMSWIRQAPGKGLEWVSYISSSGSIIYY

ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWALDYWGQGTLVTVSS SEQ ID NO:102

EVOLVESGGGLVOPGGSLRLSCAASGFTFSSHVMNWVROAPGKGLEWVSAISGSGVDTYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGSWSAGAFDIWGQGTMVTVSS SEQ ID NO:103

QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIFHDGTTNY

NPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGNWGSGALDIWGQGTMVTVSS SEQ ID NO:104

EVQLVESGGGLVQPGGSLRLSCAASGTFSSFVMSWVRQAPGKGLEWVSAISGGGGSTYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDWDLYYFDYWGQGTLVTVSS SEQ ID NO:105

EVOLVESGGGLVOPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVANIKEDGNEKYY

VDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTNWGSDYWGQGTLVTVSS SEQ ID NO:106

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTR

ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKVEIK

SEQ ID NO:107

DIQMTQSPSSLSSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYVASSLQSGVPS

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Claims (15)

RFSGSGSGTEFTLTISSLQPEDFATYYCLQYNSYPWTFGQGTKVEIK SEQ ID NO:108 DIVMTOSPDSLAVSLGERATINCKSSOSVFYSANNKNYLAWYOOKPGOPPKLLIYWTSTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYSTPRTFGQGTKVEIK SEQ ID NO:109 DIVMTQSPDSLAVSLGERATINCKSSQSVFYSSNNKNYLAWYQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYSTPRTFGQGTKVEIK SEQ ID NO:110 DIVMTOSPDSLAVSLGERATINCKSSOSVFYSSNNKNYLAWYOOKPGOPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK SEQ ID NO:111 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK SEQ ID NO:112 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIK SEQ ID NO:113 DIQLTQSPSSVPASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK SEQ ID NO:114 DIVMTQSPDSLAVSLGERATINCKSSQSVFYSSNNKNYLAWYQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK SEQ ID NO:115 GGGGSGGGGSGGGGS CLAIM 1. An anti-PD-1 antibody or an antigen-binding portion thereof, wherein said antibody comprises the amino acid sequences H-CDR1-3 and L-CDR1-3: SEQ ID NO: 52, 53, 54, 38, 45 and 55, respectively. 2. An anti-PD-1 antibody or an antigen-binding portion thereof, wherein said antibody comprises a heavy chain variable domain and a light chain variable domain, wherein said heavy and light chain variable domains have amino acid sequences selected from a) SEQ ID NO: 9 and 10 respectively; b) SEQ ID NO: 78 and 10 respectively; c) SEQ ID NO: 99 and 10 respectively; d) SEQ ID NO: 9 and 88 respectively; e) SEQ ID NO: 78 and 88 respectively; or f) SEQ ID NO: 99 and 88 respectively. 3. Antibody against PD-1 according to claim 1 or 2, where a) the antibody belongs to the IgG isotype of the IgG1 subclass, where one or both amino acid residues at positions 234 and 235 are mutagenized to Ala; or b) the antibody belongs to the IgG isotype of the IgG1 subclass, where the amino acid residue at position 228 has been mutagenized to Pro, where the residues are numbered according to the IMGT® numbering scheme. 4. An anti-PD-1 antibody or an antigen-binding portion thereof according to any one of claims 1 to 3, wherein the antibody or portion has at least one of the following properties: a) binds to cynomolgus PD-1 with a K _D of 4x10 ⁸ M or less; b) binds to mouse PD-1 with a K _D of 2x10 ^-8 M or less; c) binds to human PD-1 with a K _D of 3x10 ^-9 M or less; d) inhibits the interaction of PD-1 with PD-L1 at a concentration of 10 μg/ml; e) stimulates IL-2 production in the whole blood SEB assay; And f) stimulates IFN-γ production in a one-way mixed lymphocyte culture reaction assay. 5. Antibody against PD-1, which contains a) HC with amino acid sequences from SEQ ID NOs: 9 and 26 and LC with amino acid sequences from SEQ ID NOs: 10 and 28; b) HC with amino acid sequences SEQ ID NO: 78 and 26 and LC with amino acid sequence SEQ ID NO: 10 and 28; - 46 043633 c) HC with amino acid sequences SEQ ID NOs: 99 and 26 and LC with amino acid sequences SEQ ID NOs: 10 and 28; d) HC with amino acid sequences SEQ ID NO: 9 and 26 and LC with amino acid sequence SEQ ID NO: 88 and 28; e) HC with amino acid sequences SEQ ID NO: 78 and 26 and LC with amino acid sequence SEQ ID NO: 88 and 28; or f) HC with amino acid sequences SEQ ID NO: 99 and 26 and LC with amino acid sequence SEQ ID NO: 88 and 28. 6. A pharmaceutical composition containing an anti-PD-1 antibody or an antigen-binding portion thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient. 7. An isolated nucleic acid molecule containing a nucleotide sequence encoding a heavy chain and a nucleotide sequence encoding a light chain, antibodies against PD-1 or an antigen-binding portion thereof according to any one of claims 1 to 5. 8. An expression vector containing an isolated nucleic acid molecule according to claim 7, wherein said vector further comprises an expression control sequence. 9. A host cell containing a nucleotide sequence encoding a heavy chain and a nucleotide sequence encoding a light chain, antibodies against PD-1 or an antigen-binding portion thereof according to any one of claims. 1-5. 10. A method for producing an antibody or an antigen-binding portion thereof according to any one of claims 1 to 5, comprising obtaining a host cell according to claim 9, culturing said host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion. 11. Bispecific binding molecule, characterized in that it contains the binding domain of the antibody according to claim 1. 12. A method of enhancing immunity in a patient, comprising administering to said patient an anti-PD-1 antibody or antigen-binding moiety according to any one of claims 1 to 5, a pharmaceutical composition according to claim 6, or a bispecific binding molecule according to claim 11. 13. A method of treating a malignant tumor in a patient, comprising administering to said patient an anti-PD-1 antibody or antigen-binding moiety according to any one of claims 1 to 5, a pharmaceutical composition according to claim 6, or a bispecific binding molecule according to claim 11. 14. The method of claim 13, wherein the malignant tumor is selected from the group consisting of advanced or metastatic melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, bladder cancer, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, colorectal cancer and Hodgkin's lymphoma. 15. The method according to any one of claims 12 to 14, further comprising administering to the patient an immunostimulant agent, a vaccine, a chemotherapeutic agent, an antineoplastic agent, an antiangiogenic agent, a tyrosine kinase inhibitor, a PD-1 pathway inhibitor, or radiotherapy. -