Bispecific anti-PD1-anti-TIM3 antibody

Abstract

The present disclosure provides means and methods for interfering with PD-1 and TIM-3-mediated inhibition in programmed cell death protein 1 (PD-1) and / or T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) positive cells. The method may include contacting the cell with an antibody or its functional part, derivative and / or analog comprising the following, thereby inhibiting PD-1 and / or TIM-3-mediated activity in the cell: a variable domain that can bind to the extracellular portion of PD-1 and a variable domain that can bind to the extracellular portion of TIM-3. The present invention also provides antibodies or variants thereof, comprising variable domains that can bind to the extracellular portion of PD-1 and variable domains that can bind to the extracellular portion of TIM-3.

Description

[0001] The present invention relates to the field of antibodies. In particular, the present invention relates to the field of therapeutic antibodies for treating diseases involving abnormal cells. More particularly, the present invention relates to antibodies that bind to the extracellular portions of two or more membrane-associated proteins and thereby modulate the biological activity expressed by the cell.

[0002] Although many advances have been made in the treatment of the disease and the understanding of the molecular events that lead to cancer has increased, cancer remains the leading cause of death in the world. For example, colorectal cancer (CRC) is the third most common cancer in the world. In 2008, 1.23 million people were diagnosed with the disease. It is the second most common cancer in Europe, with about 447,000 new cases diagnosed in 2012 (13% of the total). Colorectal cancer is the fourth most common cause of cancer death, and it is estimated that 608,000 (EU 148,000) deaths can be caused each year. Although some new treatments have been proposed in CRC, many treatments have not passed clinical testing; metastatic CRC is still largely incurable using conventional treatments. Melanoma is another example of a very frequent cancer. When it is not detected early enough, the cancer is likely to metastasize to a stage that is difficult to treat. Immunointervention therapy has been shown to be effective for at least some patients with metastatic melanoma. Non-small cell lung cancer is a type of cancer that is rarely found at an early enough stage to be operated on. These types of cancer have also been successfully treated with immunotherapy interventions.

[0003] Traditionally, most cancer drug discoveries have focused on blocking basic cell functions and killing dividing cells. However, in the case of advanced cancer, no matter how actively applied, even to the extent that the patient suffers from the life-threatening side effects of treatment, chemotherapy rarely achieves complete cure. In most cases, the tumor in the patient stops growing or temporarily shrinks (called remission) only to start proliferation again, sometimes more rapidly (called relapse), and becomes more and more difficult to treat. Recently, the focus of cancer drug development has shifted from extensive cytotoxic chemotherapy to targeted cell inhibition therapy with lower toxicity. The treatment of advanced cancer has been clinically verified in leukemia and other cancers. However, in most cancers, targeted approaches are still proven to be insufficient to effectively completely eliminate the cancer in most patients.

[0004] Targeting of cancer has been achieved using a variety of different approaches, including, for example, small molecules directed against signaling proteins that the cancer depends on for survival and / or growth; vaccines using tumor-specific proteins; cell therapy using immune cells that actively kill tumor cells and antibodies that target cytotoxic molecules to tumors; interfering with signaling and / or (re)directing the host's immune system toward tumor cells.

[0005] The present invention provides novel means and methods for (re)directing components of the immune system.The present invention also relates to means and methods for modulating biological activities expressed by cells. SUMMARY OF THE INVENTION

[0007] The present invention provides a method for interfering with PD-1 and TIM-3-mediated inhibition in programmed cell death protein 1 (PD-1) and / or T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) positive cells, the method comprising contacting the cells with an antibody or a functional part, derivative and / or analog thereof comprising the following, thereby inhibiting PD-1 and / or TIM-3-mediated activity in the cells:

[0008] - a variable domain that can bind to the extracellular portion of PD-1, and

[0009] - a variable domain that can bind to the extracellular part of TIM-3.

[0010] The present invention also provides a method for stimulating the formation, stability and / or activity of an immune synapse, comprising providing a system comprising at least two cells capable of associating with each other via an immune synapse, and providing to the system an antibody or a functional part, derivative and / or analog thereof comprising:

[0011] - a variable domain that can bind to the extracellular portion of PD-1, and

[0012] - a variable domain that can bind to the extracellular part of TIM-3.

[0013] Also provided are antibodies or variants thereof, comprising a variable domain that can bind to the extracellular portion of PD-1 and a variable domain that can bind to the extracellular portion of TIM-3.

[0014] Also provided is an antibody or a variant thereof, the antibody or variant thereof comprising a variable domain that can bind to the extracellular portion of PD-1 and a variable domain that can bind to the extracellular portion of TIM-3; wherein the variable domain that can bind to PD-1 comprises a heavy chain variable region having a CDR3 region, the CDR3 region comprising Figure 3The amino acid sequence of the CDR3 of the heavy chain variable region of one of the following VHs in the embodiment: MF6076, MF6236, MF6256, MF6226, MF6930, MF6932, MF6935, MF6936, MF6972, MF6974 or MF6982, preferably MF6226, MF6256 or MF6930. In a preferred embodiment, the variable domain that can bind to PD-1 comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions comprise Figure 3 The amino acid sequence of CDR1, CDR2 and CDR3 of the heavy chain variable region of one of the following VHs shown in: MF6076, MF6236, MF6256, MF6226, MF6930, MF6932, MF6935, MF6936, MF6972, MF6974 or MF6982, preferably MF6226, MF6256 or MF6930.

[0015] Also provided is an antibody or a variant thereof, comprising a variable domain that can bind to the extracellular portion of PD-1 and a variable domain that can bind to the extracellular portion of TIM-3; wherein the variable domain that can bind to PD-1 comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence of the heavy chain variable region as shown below: MF6076, MF6256, MF6226, MF6930, MF6932, MF6935, MF6936, MF6972, MF6974 or MF6982, preferably MF6226, MF6256 or MF6930 (such as Figure 3 ), said amino acid sequence having at most 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of VH as shown in MF.

[0016] The variable domain that can bind to the extracellular portion of TIM-3 preferably comprises a heavy chain variable region having a CDR3 region, wherein the CDR3 region comprises Figure 3 The amino acid sequence of the CDR3 region of the heavy chain variable region shown below: MF7676, MF7677, MF7678 or MF7679. Preferably, the variable domain that can bind to the extracellular portion of TIM-3 comprises a heavy chain variable region having CDR1, CDR2 and CDR3 regions, wherein the CDR1, CDR2 and CDR3 regions comprise Figure 3 The amino acid sequence of CDR1, CDR2 and CDR3 of the heavy chain variable region of one of the following VHs shown in: MF7676, MF7677, MF7678 or MF7679.

[0017] The variable domain that can bind to the extracellular portion of TIM-3 preferably comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of the heavy chain variable region as shown below: MF7676, MF7677, MF7678 or MF7679 ( Figure 3 The amino acid sequence has at most 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably has 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the specified MF.

[0018] The antibody of the present invention preferably comprises a heavy chain variable region comprising Figure 3 In a preferred embodiment, the antibody further comprises a light chain variable region, wherein the light chain variable region comprises Figure 1 In a preferred embodiment, the light chain comprises the amino acid sequence of the light chain variable region shown in Figure 1 In a preferred embodiment, the heavy chain comprises the constant region of an IgG1 antibody, preferably a human IgG1 antibody. In a preferred embodiment, the CH2 region of the IgG1 constant region is modified to reduce the ADCC and / or CDC activity of the antibody. In a preferred embodiment, the CH2 region comprises Figure 2 In a preferred embodiment, the CH3 region of the antibody is modified to facilitate heterodimerization of the heavy chain. In a preferred embodiment, a heavy chain comprises Figure 2 F, and the other heavy chain comprises the sequence shown in Figure 2 The sequence shown in G.

[0019] Also provided are pharmaceutical compositions comprising one or more antibodies or variants thereof of the invention.

[0020] Also provided is a nucleic acid molecule or collection of nucleic acid molecules encoding the heavy chain or heavy chain variable region of an antibody or variant thereof of the invention.

[0021] Also provided is a nucleic acid molecule or collection of nucleic acid molecules encoding an antibody of the invention.

[0022] Also provided are cells comprising one or more nucleic acid molecules, which encode the antibodies or variants thereof of the present invention alone or together. Also provided are methods of using the cells to produce the antibodies or variants thereof of the present invention, preferably, the methods further comprising harvesting the antibodies or variants thereof from the culture of the cells.

[0023] Also provided are cellular systems comprising the antibodies or variants thereof of the invention.

[0024] Also provided is a method for treating an individual suffering from a disease involving abnormal cells, such as cancer, or suffering from a chronic viral or parasitic infection, comprising administering to an individual in need thereof an antibody or variant thereof of the invention, preferably a bispecific antibody or variant thereof of the invention.

[0025] The present invention also provides an antibody of the present invention or a variant thereof; preferably a bispecific antibody of the present invention or a variant thereof, for use in treating an individual suffering from a disease involving abnormal cells, such as cancer, or suffering from a chronic viral or parasitic infection.

[0026] In a preferred embodiment, the parasite is an intracellular parasite.

[0027] Also provided is a method of stimulating an immune response against abnormal cells in an individual, the method comprising providing (administering) an antibody or variant thereof of the present invention, preferably a bispecific antibody or variant thereof, to the individual. The abnormal cell is preferably a cancer cell, a virally infected cell, a parasite, or a parasite-infected cell. In a preferred embodiment, the cell is a cancer cell or a neoplastic cell. DETAILED DESCRIPTION OF THE INVENTION

[0029] T cell exhaustion is mediated by several inhibitory receptors, including programmed cell death protein 1 (PD1), TIM3, and lymphocyte activation gene 3 protein (LAG3). TIM3 is an immune checkpoint inhibitor. It is a protein that can be present on Th1 cells. It is a cell surface protein that regulates macrophage activation and enhances the severity of experimental autoimmune encephalomyelitis in mice. CD4-positive T helper lymphocytes can be divided into several types (including type 1 (Th1) and type 2 (Th2)) based on their cytokine secretion patterns. Th1 cells and their associated cytokines are involved in cell-mediated immunity and delayed hypersensitivity reactions against intracellular pathogens. TIM-3 or HAVCR2 is known by many aliases, such as Hepatitis A Virus Cellular Receptor 2; T-Cell Immunoglobulin and Mucin Domain-Containing Protein 3; T-Cell Immunoglobulin Mucin Family Member 3; T-Cell Immunoglobulin Mucin Receptor 3; T-Cell Membrane Protein 3; HAVcr-2; TIMD-3; Tim-3; TIMD3; T Cell Immunoglobulin Mucin 3; T Cell Immunoglobulin Mucin-3; Kidney Injury Molecule-3; CD366; and KIM-3. The external ID of TIM-3 is HGNC: 18437; Entrez Gene: 84868; Ensembl: ENSG00000135077; OMIM: 606652; and UniProtKB: Q8TDQ0.

[0030] Galectin-9 or LGALS9 is a known ligand of TIM-3. It is known by many different names, such as Lectin, Galactoside Binding Soluble 9; Lectin, Galactoside-Binding, Soluble, 9; Tumor Antigen HOM-HD-21; Ecalectin; Gal-9; Urate Transporter / Channel Protein; LGALS9A; and HUAT. The external IDs of Galectin-9 are HGNC: 6570; Entrez Gene: 3965; Ensembl: ENSG00000168961; OMIM: 601879; and UniProtKB: O00182.

[0031] Programmed cell death protein 1 (PD-J) is a cell surface receptor belonging to the CD28 receptor family and expressed on T cells and pro-B cells. It is currently known that PD-1 binds to two ligands: PD-L1 and PD-L2. PD-1, used as an immune checkpoint, plays an important role in downregulating the immune system by inhibiting the activation of T cells, which in turn reduces autoimmunity and promotes self-tolerance. It is believed that the inhibitory effect of PD-1 is achieved by a dual mechanism that promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while reducing apoptosis of regulatory T cells (suppressor T cells). PD-1 is also known by many different aliases, such as PDCD1; Programmed Cell Death 1; Systemic Lupus Erythematosus Susceptibility Protein 2; Protein PD-1; HPD-1; PD1; Programmed Cell Death 1 Protein; CD279 Antigen; CD279; HPD-L; HSLE1; SLEB2; and PD-1. The external IDs of PD-1 are HGNC: 8760; Entrez Gene: 5133; Ensembl: ENSG00000188389; OMIM: 600244; and UniProtKB: Q15116. A new class of drugs that block PD-1 activity, PD-1 inhibitors, activate the immune system to attack tumors and are therefore successfully used to treat some types of cancer.

[0032] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing immune responses during specific events (e.g., pregnancy, tissue allografts, autoimmune diseases, and other disease states such as hepatitis). The combination of PD-L1 and PD-1 or B7.1 (CD80) transmits inhibitory signals, which reduces the proliferation of T cells expressing PD-1. It is believed that PD-1 can control the accumulation of exogenous antigen-specific T cells by apoptosis. PD-L1 is expressed by a variety of cancer cells, and its expression is believed to at least partially lead to weakened immune responses against cancer cells. PD-L1 is a member of the B7 protein family and is known by multiple other names, such as CD274 molecules; CD274 antigens; B7 homologs 1; PDCD1 ligand 1; PDCD1LG1; PDCD1L1; B7H1; PDL1; Programmed Cell Death 1 Ligand 1; Programmed Death Ligand 1; B7-H1; and B7-H. The external ID of CD274 is HGNC: 17635; Entrez Gene: 29126; Ensembl: ENSG00000120217; OMIM: 605402; UniProtKB: Q9NZQ7.

[0033] PD-L2 is the second ligand of PD-1. Binding of PD-L2 to PD-1 inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2 / PD-1 binding inhibits B7-CD28 signaling. At high antigen concentrations, PD-L2 / PD-1 binding reduces cytokine production. PD-L expression is upregulated on antigen presenting cells by interferon gamma treatment. It is expressed in some normal tissues and a variety of tumors. PD-L1 and PD-L2 are thought to have overlapping functions and regulate T cell responses. This protein is known by many other names, such as Programmed Cell Death 1 Ligand 2; B7 dendritic cell molecule; Programmed Death Ligand 2; Butyrophilin B7-DC; PDCD1 ligand 2; PD-1 ligand 2; PDCD1L2; B7-DC; CD273; B7DC; PDL2; PD-1-ligand 2; CD273 antigen; BA574F11.2; and Btdc. The external IDs of PD-L2 are HGNC: 18731; Entrez gene: 80380; Ensembl: ENSG00000197646; OMIM: 605723; and UniProtKB: Q9BQ51.

[0034] Reference sequence identifiers are used to identify which protein is targeted. The antibodies of the present invention also recognize at least some of its variants, such as its allelic variants, splice variants and mutant variants, as long as the epitope recognized by the corresponding variable domains of the antibody is not affected. Some alternative names may also be used or may not be used to refer to other proteins. These names are provided for reference purposes only. The antibodies of the present invention are combined with proteins such as those expressed on cells. It may also be combined with variants of proteins, as long as the epitope combined with the antibody is available. Therefore, splice variants or mutant proteins (if any) may also be combined, as long as the epitope is available. The fact that an antibody is combined with a specified protein means that it can be combined with the protein in nature, and does not imply that the antibody is actually combined with the target, although it can be combined. This does not mean that the antibody is not combined with other proteins.

[0035] The present invention discloses an antibody or variant thereof that binds to the extracellular portion of a PD-1 (first membrane protein) member and the extracellular portion of a TIM-3 (second membrane protein), which is preferably a bispecific antibody or variant thereof. Such (bispecific) antibodies are also referred to as "antibodies or bispecific antibodies of the present invention". Compositions and kits (kit of parts) are also provided, which include two or more (bispecific) antibodies as described herein.

[0036] Antibodies typically bind to their targets through a so-called antigen binding site. Unmodified antigen binding sites are typically formed by and present in the variable domains of antibodies. The variable domains contain the antigen binding site. An antigen-binding variable domain is a variable domain that contains an antigen-binding site that binds to an antigen.

[0037] In one embodiment, the antibody variable domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). The antigen binding site may be present in the combined VH / VL variable domain, or only in the VH region or only in the VL region. When the antigen binding site is present in one of the two regions of the variable domain, the corresponding variable region may promote the folding and / or stability of the bound variable region, but does not significantly promote the binding of the antigen itself.

[0038] Antigen binding as used herein refers to the typical binding capacity of an antibody to its antigen. The binding of an antibody to an antigen can be assessed in a variety of ways. One way is to incubate the antibody with an antigen (preferably a cell expressing the antigen), remove unbound antibody (preferably by a washing step), and detect the bound antibody by a labeled antibody bound to the bound antibody.

[0039] Antigen binding of antibodies is usually mediated by the complementarity determining region (CDR) of the antibody and the specific three-dimensional structure of both the antigen and the variable domain, thereby allowing the two structures to be precisely combined together (similar to the interaction of a lock and a key), which is in contrast to random, non-specific protein adhesion. Since antibodies usually recognize the part of the antigen called the antigenic epitope, and since such an epitope may also be present in other compounds, the antibodies according to the present invention may also recognize other proteins, if such other compounds contain the same epitope. Therefore, the term "binding" does not exclude the binding of antibodies to another protein or more proteins containing the same epitope. Such other proteins are preferably not human proteins.

[0040] The proteins, such as antibodies, of the present invention generally do not bind to other proteins than the specific target protein on the cell membrane in postnatal humans, preferably adults.

[0041] The term "antibody" as used herein means a protein molecule, which preferably belongs to the immunoglobulin class of protein, comprising one or more variable domains that bind to an epitope on an antigen, wherein such a domain is derived from the variable domains of an antibody or has sequence homology with the variable domains of an antibody. Antibodies for therapeutic use are preferably as close as possible to the natural antibodies of the object to be treated (e.g., human antibodies for human objects). Antibody binding can be represented according to specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by the binding domain. Affinity is a measure of the intensity of binding to a specific antigen or epitope. Preferably, the affinity of the separate arm of an antibody according to the present invention is within the nanomolar range. Antibodies of the present invention, such as bispecific antibodies, generally comprise the constant domains (Fc parts) of natural antibodies, which can be transformed as described elsewhere herein, for example, to facilitate heterodimerization or reduce ADCC and / or CDC activity, or other reasons. Antibodies of the present invention are generally bispecific full-length antibodies, which are preferably human IgG subclasses.

[0042] The variable domain is composed of the variable region of the heavy chain and the variable region of the light chain. The variable region of the heavy chain is usually formed by the VDJ region of rearrangement. The variable region of the light chain is usually formed by the VJ region of rearrangement. The VDJ / VJ region can also be artificially generated using a large amount of sequence information available for example for functional antibodies.

[0043] The antibody of the present invention is preferably a "full-length" antibody. The term "full-length" according to the present invention is defined as comprising a substantially complete antibody, and there is no one or more artificially added parts greater than 20 amino acid residues, such as additional antigen binding sites or additional activation sites or additional ligands or additional ligand binding moieties. However, a full-length antibody does not necessarily have all the functions of a complete antibody. In order to avoid doubt, a full-length antibody comprises two heavy chains and two light chains. Each chain comprises a constant (C) region and a variable (V) region, which can be decomposed into domains called CH1, CH2, CH3, and VH for the heavy chain, and can be decomposed into domains called CL and VL for the light chain. The domains of the heavy chain preferably exist in the order of natural antibodies (VH-CH1-CH2-CH3, meaning that the VH domain is adjacent to the CH1 domain, followed by the CH2 domain, and then followed by the CH3 domain). The domains of the light chain also preferably exist in the order of natural antibodies (VL-CL: meaning that the VL domain is adjacent to the CL domain). The antibody binds to the antigen through the variable domains contained in the Fab fragment portion. Antibodies can interact with molecules and cells of the immune system through their constant domains, primarily through the Fc portion.

[0044] In some embodiments, antibody of the present invention is IgG, preferably full-length IgG. Full-length IgG antibody is preferred because it usually has a favorable half-life, and for immunogenic reasons it is desirable to keep close to complete autologous (human) molecules. In some embodiments, antibody of the present invention is full-length IgG1, full-length IgG2, full-length IgG3 or full-length IgG4 antibody.

[0045] Full-length antibodies according to the present invention encompass antibodies in which there may be a mutation providing the desired characteristics or only those that are substituted in the original chain. Such a mutation should not be a deletion of the substantial part of any region. However, the term "full-length antibody" includes antibodies in which one or more amino acid residues are inserted, deleted, replaced, or a combination thereof and the antigen-binding properties of the resulting antibody are not substantially changed. For example, an IgG antibody may have 1 to 20 amino acid residues inserted, replaced, deleted, or a combination thereof in the constant region.

[0046] The antibody of the present invention or its functional part, derivative and / or analog is preferably a bispecific antibody or its functional part, derivative and / or analog. In a preferred embodiment, it is a bispecific IgG antibody with reduced effector function. In a preferred embodiment, the antibody of the present invention is a bispecific full-length antibody. The antibody of the present invention is preferably a bispecific full-length IgG antibody, which is preferably mutated in the CH2 / lower hinge region to reduce effector function. The IgG1 that mutates in the CH2 / lower hinge region to reduce effector function is favored based on its long circulation half-life in people. In order to prevent any immunogenicity in people, the bispecific antibody according to the present invention is preferably a human antibody.

[0047] The term "bispecific" (bispecific, bs) means that one part of an antibody (as defined above) binds to an epitope on an antigen, while the second part binds to a different epitope on the same antigen or a different antigen. Different epitopes are usually present on different antigens. However, different epitopes can also be present on the same antigen. According to the present invention, the first and second antigens are actually two different proteins. Preferred bispecific antibodies are antibodies that contain parts of two different monoclonal antibodies and can therefore bind to two different epitopes, preferably two different epitopes on two different antigens. Depending on the expression level, (sub) cellular localization and stoichiometry of the two antigens recognized by the bispecific antibody, the two Fab arms of the antibody may or may not bind to their epitopes at the same time. One arm of a bispecific antibody usually contains the variable domain of one antibody, and the other arm contains the variable domain of another antibody (i.e., one arm of a bispecific antibody is formed by pairing a heavy chain with a light chain, while the other arm is formed by pairing different heavy chains with light chains). The heavy chain variable regions of the bispecific antibodies of the present invention are usually different from each other, while in the bispecific antibodies of the present invention, the light chain variable regions are preferably the same. Bispecific antibodies in which different heavy chain variable regions are associated with the same or common light chain variable regions are also referred to as bispecific antibodies with a common light chain variable region (cLcv). Preferably, the light chain constant regions are also the same. Such bispecific antibodies are referred to as having a common light chain (cLc). Therefore, a bispecific antibody according to the present invention in which the two arms comprise a common light chain is also provided.

[0048] As described herein, bispecific antibodies preferably include a common light chain variable domain, preferably a common light chain. According to the term "common light chain" of the present invention, it refers to a light chain that may be identical or have some amino acid sequence differences but does not affect the binding specificity of a full-length antibody. For example, within the scope of the definition of a common light chain as used herein, light chains that are not identical but still functionally equivalent may be prepared or found, such as by introducing and testing conservative amino acid changes, which are amino acid changes that do not or only partially promote binding specificity in the region when paired with a heavy chain, etc. The terms "common light chain", "common LC", "cLC", "single light chain" are used interchangeably herein whether or not the term "rearranged" is added. The terms "common light chain variable region", "common VL", "common LCv", "cLCv", "single VL" are used interchangeably herein whether or not the term "rearranged" is added. A preferred aspect of the present invention is that the bispecific antibody has a common light chain (variable region) that can be combined with at least two and preferably multiple heavy chains (variable regions) with different binding specificities to form an antibody (WO2009 / 157771) with a functional antigen binding domain. The common light chain (variable region) is preferably a human light chain (variable region). The common light chain (variable region) preferably has a germline sequence. The preferred germline sequence is a light chain variable region that is frequently used in the human library and has good thermodynamic stability, yield and solubility. A preferred germline light chain is O12. The common light chain is preferably a rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01 ( Figure 1 A). The common light chain variable region is preferably the variable region of a rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01. The common light chain preferably comprises a sequence having 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. Figure 1 The common light chain preferably further comprises a light chain constant region, preferably a kappa light chain constant region. The nucleic acid encoding the common light chain may be codon optimized for a cell system for expressing the common light chain protein. The encoding nucleic acid may deviate from the germline nucleic acid sequence.

[0049] In a preferred embodiment, the light chain comprises a light chain region comprising a light chain region having 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. Figure 1 The amino acid sequence of the O12 / IgVκ1-39*01 gene segment shown in A. Throughout the specification, the phrase "O12 light chain" is used as "comprising a light chain having 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof". Figure 1The light chain variable region of the amino acid sequence of the O12 / IgVκ1-39*01 gene segment shown in A is an abbreviation of "light chain". IgVκ1-39 is an abbreviation of the immunoglobulin variable κ1-39 gene. This gene is also called immunoglobulin κ variable 1-39; IGKV139; IGKV1-39; O12a or O12. The external ID of this gene is HGNC: 5740; Entrez gene: 28930; Ensembl: ENSG00000242371. The preferred amino acid sequence of IgVκ1-39 is in Figure 1 E. It lists the sequence of the V region. The V region can be combined with one of the five J regions. Figure 1 B and 1D depict two preferred sequences of IgVκ1-39 in combination with the J region. The linked sequences are denoted IGKV1-39 / jk1 and IGKV1-39 / jk5; alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (according to the nomenclature of the IMGT database World Wide Web at imgt.org).

[0050] Preferably, the light chain variable region comprising O12 / IgVκ1-39*01 is a germline sequence. Also preferably, the light chain variable region comprising IGJκ1*01 or / IGJκ5*01 is a germline sequence. In a preferred embodiment, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0051] In a preferred embodiment, the light chain variable region comprises germline O12 / IgVκ1-39*01. In a preferred embodiment, the light chain variable region comprises a κ light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In a preferred embodiment, IgVκ1-39*01 / IGJκ1*01. The light chain variable region preferably comprises a germline κ light chain IgVκ1-39*01 / IGJκ1*01 or a germline κ light chain IgVκ1-39*01 / IGJκ5*01, preferably a germline IgVκ1-39*01 / IGJκ1*01.

[0052] Mature B cells that produce antibodies with O12 light chains typically produce light chains that have undergone one or more mutations relative to the germline sequence (i.e., the normal sequence in the non-lymphoid cells of an organism). The process that leads to these mutations is generally called somatic (hyper) mutation. The resulting light chain is called an affinity matured light chain. Such a light chain is an O12-derived light chain when derived from an O12 germline sequence. In this specification, the phrase "O12 light chain" will include O12-derived light chains, and mutations introduced by somatic hypermutation can of course also be introduced artificially in the laboratory. In the laboratory, other mutations can also be introduced without affecting the properties of the light chain (type, not necessarily in quantity). If the light chain contains 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof, such as Figure 1 A. Figure 1 B. Figure 1 D or Figure 1 E, then it is at least an O12 light chain. In a preferred embodiment, the O12 light chain is a light chain comprising 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4 amino acid insertions, deletions, substitutions, additions, or a combination thereof. Figure 1 A, 1B, 1D or 1E. In a preferred embodiment, the O12 light chain is a light chain comprising 0 to 5, preferably 0 to 4, more preferably 0 to 3 amino acid insertions, deletions, substitutions, additions, or a combination thereof. Figure 1 A. Figure 1 B. Figure 1 D or Figure 1 In a preferred embodiment, the O12 light chain is a light chain comprising 0 to 2, more preferably 0 to 1, and most preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof. Figure 1 A. Figure 1 B. Figure 1 D or Figure 1 In a preferred embodiment, the O12 light chain is a light chain comprising the amino acid insertion, deletion, substitution, addition, or a combination thereof as mentioned above. Figure 1 A or Figure 1 A light chain having the sequence shown in B. In a preferred embodiment, the light chain comprises Figure 1 In a preferred embodiment, the light chain variable region comprises Figure 1 The sequence of B.

[0053] The common light chain (variable region) may be a lambda light chain, and therefore this is also provided in the context of the present invention, however a kappa light chain is preferred. The constant portion of the common light chain of the present invention may be a constant region of a kappa or lambda light chain. It is preferably a constant region of a kappa light chain, preferably wherein the common light chain is a germline light chain, preferably a rearranged germline human kappa light chain comprising an IgVK1-39 gene segment, most preferably a rearranged germline human kappa light chain IgVK1-39*01 / IGJK1*01 ( Figure 1 ). The terms rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01, IGKV1-39 / IGKJ1, huVκ1-39 light chain or simply huVκ1-39, or simply 1-39 are used interchangeably throughout this application. Those skilled in the art will recognize that "common" also refers to functional equivalents where the amino acid sequence of the light chain is not identical. There are many variants of the light chain in which there are mutations (deletions, substitutions, additions) that do not affect the formation of a functional binding region.

[0054] IgVκ1-39 is the abbreviation of immunoglobulin variable κ1-39 gene. This gene is also called immunoglobulin κ variable 1-39; IGKV139; IGKV1-39; O12a or O12. The external ID of this gene is HGNC: 5740; Entrez gene: 28930; Ensembl: ENSG00000242371. The preferred amino acid sequence of IgVκ1-39 is Figure 1 . It lists the sequences of the V regions. The V regions can be combined with one of the five J regions. Figure 1 Two preferred sequences of IgVκ1-39 in combination with the J region are described. The linked sequences are denoted IGKV1-39 / jk1 and IGKV1-39 / jk5; alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (according to the nomenclature of the IMGT database World Wide Web at imgt.org).

[0055] The common light chain variable region is preferably linked to a kappa light chain constant region. In a preferred embodiment, the light chain comprises a kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In a preferred embodiment, IgVκ1-39*01 / IGJκ1*01.

[0056] Common light chain producing cells can produce, for example, the rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01, as well as a light chain comprising the variable region of the mentioned light chain fused to a lambda constant region.

[0057] As described herein, the bispecific antibody or variant thereof preferably has a heavy chain variable region / light chain variable region (VH / VL) combination that binds to the extracellular portion of PD-1 and a second VH / VL combination that binds to the extracellular portion of TIM-3. In a preferred embodiment, the VL in the first VH / VL combination is similar to the VL in the second VH / VL combination. In a more preferred embodiment, the VL in the first and second VH / VL combinations are the same. In a preferred embodiment, the bispecific antibody is a full-length antibody having a heavy chain / light chain (H / L) combination that binds to the extracellular portion of PD-1 and a H / L chain combination that binds to the extracellular portion of TIM-3. In a preferred embodiment, the light chain in the first H / L chain combination is similar to the light chain in the second H / L chain combination. In a more preferred embodiment, the light chains in the first and second H / L chain combinations are the same.

[0058] Several methods have been published to facilitate the production of bispecific antibodies or, conversely, monospecific antibodies. In the present invention, preferably, relative to the production of corresponding monospecific antibodies, cells are more conducive to the production of bispecific antibodies. This is usually achieved by modifying the constant region of the heavy chain so that it is more conducive to heterodimerization (i.e., heavy chain dimerization with another heavy chain / light chain combination) relative to homodimerization. In a preferred embodiment, the bispecific antibody of the present invention comprises two different immunoglobulin heavy chains with a compatibility heterodimerization domain. Various compatibility heterodimerization domains have been described in the art. The compatibility heterodimerization domain is preferably a compatibility immunoglobulin heavy chain CH3 heterodimerization domain. When using a wild-type CH3 domain, the co-expression of two different heavy chains (A and B) and a common light chain will produce three different antibody species: AA, AB and BB. AA and BB are representations of two monospecific bivalent antibodies, and AB is a representation of a bispecific antibody. In order to increase the percentage of the desired bispecific product (AB), CH3 modification can be used, or in other words, a heavy chain with a compatible heterodimerization domain can be used, as defined below. The art describes a variety of ways in which such heterodimerization of heavy chains can be achieved. One way is to produce a "knob into hole" bispecific antibody. See U.S. Patent Application 20030078385 (Arathoon et al.).

[0059] As used herein, the term "compatible heterodimerization domain" refers to a protein domain that is engineered such that the engineered domain A' will preferentially form a heterodimer with the engineered domain B', and vice versa, homodimerization between A'-A' and B'-B' is reduced.

[0060] In US13 / 866,747 (now authorized as US 9,248,181), US14 / 081,848 (now authorized as US 9,358,286) and PCT / NL2013 / 050294 (published as WO2013 / 157954), which are incorporated herein by reference, methods and means for producing bispecific antibodies using compatible heterodimerization domains are disclosed. These means and methods may also be advantageously used in the present invention. Specifically, the bispecific antibodies of the present invention preferably comprise mutations to substantially only produce bispecific full-length IgG molecules. Preferred mutations are amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK variant" heavy chain) and amino acid substitutions L351D and L368E ("DE variant" heavy chain) in the second domain, or vice versa. Previously, it was demonstrated in our US 9,248,181 and US 9,358,286 patents and WO2013 / 157954 PCT application that the DE variants preferentially pair with the KK variants to form heterodimers (so-called "DEKK" bispecific molecules). Due to the repulsion between the charged residues in the CH3-CH3 interface between the same heavy chains, homodimerization of the DE variant heavy chains (DEDE homodimers) hardly occurs.

[0061] Bispecific antibodies can be produced by (transient) transfection of a plasmid encoding a light chain and two different heavy chains, and the heavy chain is transformed through CH3 to ensure effective heterodimerization and the formation of bispecific antibodies. The generation of these chains in a single cell results in a formation that is more conducive to bispecific antibodies relative to the formation of monospecific antibodies. The preferred mutation that substantially only produces bispecific full-length IgG1 molecules is an amino acid replacement at position 351 and position 366 in the first CH3 domain, such as L351K and T366K (numbered according to EU numbering) ("KK variant" heavy chain), and an amino acid replacement at position 351 and position 368 in the second CH3 domain, such as, L351D and L368E ("DE variant" heavy chain), or vice versa.

[0062] The effector function of Fc region-mediated antibody, for example complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). According to the application of therapeutic antibodies or Fc fusion proteins, it is expected to reduce or improve the effector function. The effector function reduced is preferred in the present invention. As in some embodiments of the present invention, when it is necessary to activate, enhance or stimulate an immune response, it is expected that the effector function reduced. Antibodies with reduced effector function can be used for cell surface molecules of targeted immune cells, etc.

[0063] It was found that IgG binding to FcγR or C1q requires residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain ( Figure 2 D) is associated with FcγR and C1q binding. It has been shown that substitution of IgG2 residues at positions 233 to 236 and IgG4 residues at positions 327, 330 and 331 into human IgG1 greatly reduces ADCC and CDC (Armouret al., 1999. Eur J Immunol. 29(8): 2613-24; Shields et al., 2001. J Biol Chem. 276(9): 6591-604). In addition, Idusogie et al. demonstrated that alanine substitutions at various positions including K322 significantly reduced complement activation (Idusogie et al., 2000. J Immunol. 164(8): 4178-84).

[0064] Due to their reduced effector function, IgG4 antibodies represent an IgG subclass for receptor blocking without cell depletion. IgG4 molecules can exchange half molecules in a dynamic process called Fab arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG4. The S228P mutation is an example of a mutation that ensures reduced Fab arm exchange capacity (Labrijn. et al., 2009. Nat Biotechnol. 27(8): 767-71).

[0065] The antibody with reduced effector function is preferably an IgG antibody comprising a modified CH2 / lower hinge region, for example to reduce Fc-receptor interactions or reduce C1q binding. In some embodiments, the antibody of the present invention is an IgG antibody with a mutated CH2 and / or lower hinge domain, such that the interaction of the bispecific IgG antibody with the Fc-γ receptor is reduced. The antibody comprising a mutated CH2 region is preferably an IgG1 antibody. Such a mutated IgG1 CH2 and / or lower hinge domain preferably comprises an amino acid substitution at position 235 and / or position 236 (numbered according to EU numbering), preferably L235G and / or G236R substitutions ( Figure 2 E).

[0066] Variants of antibodies or bispecific antibodies as described herein include functional parts, derivatives and / or analogs of the antibodies or bispecific antibodies. The variants maintain the binding specificity of the (bispecific) antibody. The functional parts, derivatives and / or analogs maintain the binding specificity of the (bispecific) antibody. As described herein, the binding specificity is defined by the ability to bind to the extracellular parts of PD-1 and TIM-3.

[0067] The functional part of the antibody as described herein or preferably the functional part of the bispecific antibody is a part comprising a variable domain that binds to the extracellular part of PD-1 and a variable domain that binds to the extracellular part of TIM-3. Suitable parts are, for example, F(ab')2 fragments produced by digesting the bispecific antibody with pepsin. Other parts comprising the variable domains are also included in the present invention.

[0068] Functional derivatives of antibodies as described herein or preferably functional derivatives of bispecific antibodies are proteins comprising variable domains of the extracellular part of the binding PD-1 and the variable domains of the extracellular part of the binding TIM-3 connected by a joint. The variable domain can be a variable domain as is, or a Fab fragment or a variable domain-like molecule, such as a single-chain Fv fragment comprising VH and VL connected together via a joint. Other examples of variable domain-like molecules are so-called single-domain antibody fragments. Single-domain antibody fragments (single-domain antibody fragment, sdAb) are antibody fragments with a single monomer variable antibody region. Like a complete antibody, it can selectively bind to a specific antigen. Due to the molecular weight of only 12 to 15 kDa, single-domain antibody fragments are much smaller than ordinary antibodies (150 to 160 kDa) composed of two heavy protein chains and two light chains, and are even smaller than Fab fragments (about 50 kDa, one light chain and half heavy chain) and single-chain variable fragments (about 25 kDa, two variable regions, one from a light chain and one from a heavy chain). Single domain antibodies themselves are not much smaller than normal antibodies (usually 90 to 100 kDa). Single domain antibody fragments are primarily engineered from heavy chain antibodies found in camels; these are called VHH fragments Some fish also have heavy chain-only antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single domain antibody fragments called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains of the common immunoglobulin G (IgG) from humans or mice into monomers. Although most studies on single domain antibodies are currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Other non-limiting examples of variable domain-like molecules are VHH, human domain antibodies (dAb) and single antibodies (Unibody). Preferred functional parts are parts that contain variable domains containing heavy chain variable regions and light chain variable regions. Some non-limiting examples of such variable domains are F(ab) fragments and single-chain Fv fragments. The bispecific format of variable domain (like) connection is, for example, human serum albumin (HSA) bound to two different scFvs; bispecific minibodies comprising two different scFvs, which are bound together by a dimerization motif or self-associating secondary structure (e.g., helical bundle or coiled coil) to dimerize the scFv fragments (Morrison (2007) Nat. Biotechnol 25: 1233-34). Some examples of suitable HSA linkers and methods for coupling scFvs to linkers are described in WO2009 / 126920.

[0069] The antibodies or functional parts, derivatives and / or analogs thereof or preferably bispecific antibodies or functional parts, derivatives and / or analogs thereof of the present invention are preferably used in humans. For this reason, the antibodies or functional parts, derivatives and / or analogs thereof of the present invention are preferably human antibodies or humanized antibodies. Human tolerance to polypeptides is determined by many different aspects. Immunity, whether it is T cell-mediated, B cell-mediated or other, is one of the variables covered in human tolerance to polypeptides. The constant region of the bispecific antibody of the present invention preferably comprises a human heavy chain constant region, which preferably comprises such as Figure 2 and a human light chain constant region, which preferably comprises Figure 1The sequence shown in C. Constant region may include one or more, preferably no more than 10, preferably no more than 5 amino acid differences with the constant region of naturally occurring human antibodies. Preferably, the constant part is completely derived from naturally occurring human antibodies. Such source sequences can be transformed for specific purposes, for example to promote heterodimerization or reduce ADCC and / or CDC activity, and other reasons. The various antibodies produced herein are derived from the common light chain mice immunized with the corresponding targets, as described in WO2009 / 157771. The various antibodies produced herein are derived from human antibody variable domain libraries. Therefore, these variable domains are human. Unique CDR regions can be derived from humans, are synthetic, or derived from another organism. When the variable region has (except the CDR region) the same amino acid sequence as the amino acid sequence of the variable region of naturally occurring human antibodies, it is at least human variable region. In some such embodiments, the variable domain VH of the antibody of the present invention that binds to the extracellular portion of PD-1 or TIM-3, or the light chain in the antibody may contain one or more, preferably no more than 10, preferably no more than 5 amino acid differences from the variable region of a naturally occurring human antibody, excluding possible differences in the amino acid sequence of the CDR region. Such mutations also occur naturally in the context of somatic hypermutation.

[0070] At least for the heavy chain variable region, the antibodies can be derived from a variety of animal species. Common practice is humanization, such as humanizing the murine heavy chain variable region. There are a variety of ways in which humanization can be achieved, including grafting the CDRs into a human heavy chain variable region having a 3D structure that matches the 3D structure of the murine heavy chain variable region; deimmunization of the murine heavy chain variable region, preferably by removing known or suspected T cell or B cell epitopes from the murine heavy chain variable region. The removal is usually performed by replacing one or more amino acids in the epitope with another (usually conservative) amino acid, so that the sequence of the epitope is modified so that it is no longer a T cell or B cell epitope.

[0071] The immunogenicity of the deimmunized murine heavy chain variable region in humans is lower than that of the original murine heavy chain variable region. Preferably, the variable region or domain of the present invention is further humanized, for example, as veneer. By using veneer technology, external residues that are easily encountered by the immune system are selectively replaced by human residues to provide a hybrid molecule comprising a veneered surface that is weakly immunogenic or substantially non-immunogenic. The animal used in the present invention is preferably a mammal, more preferably a primate, and most preferably a human.

[0072] The antibody or bispecific antibody according to the present invention or its functional part, derivative and / or analogue preferably comprises the constant region of human antibody. According to the difference of its heavy chain constant domain, antibody is divided into five categories or isotypes: IgG, IgA, IgM, IgD and IgE. These categories or isotypes comprise at least one of the heavy chains named with corresponding Greek letters. In a preferred embodiment, the invention provides an antibody according to the present invention, wherein the constant region is selected from IgG constant region, i.e. selected from IgG1, IgG2, IgG3 and IgG4. Preferably, the constant region is IgG4 or IgG1 constant region ( Figure 2 ), more preferably a mutated IgG1 constant region. Some changes in the IgG1 constant region occur naturally and / or are allowed without changing the immunological properties of the resulting antibody. Generally, about 1 to 10 amino acid insertions, deletions, substitutions, or combinations thereof are allowed in the constant region. The constant region may be mutated as described herein to enable efficient heterodimerization, to reduce effector function, or for other reasons, including half-life, stability, etc.

[0073] Rational approaches have been developed towards minimizing the content of non-human residues in the human environment. A variety of methods can be used to successfully graft the antigen binding properties of an antibody onto another antibody. The binding properties of an antibody may be primarily present in the exact sequence of the CDR3 region, which is generally supported by the sequence of the CDR1 and CDR2 regions in the variable domains in combination with the appropriate structure of the variable domain as a whole. Currently, a variety of methods are available to graft the CDR region onto the appropriate variable domain of another antibody. Some of these methods are summarized in JC Almagrol and J. Fransson (2008) Frontiers in Bioscience 13, 1619-1633, which is included herein by reference.

[0074] Contains Figure 3 The light chain variable region of the variable domain of the variable heavy chain sequence shown in is preferably an O12 or O12-based germline light chain, preferably a rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01 or a fragment or functional derivative thereof (according to the nomenclature of the IMGT database World Wide Web at imgt.org). The terms rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01, IGKV1-39 / IGKJ1, huVκ1-39 light chain or simply huVκ1-39 are used. The light chain may have 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof. The 1, 2, 3, 4 or 5 amino acid substitutions mentioned are preferably conservative amino acid substitutions, and the insertions, deletions, substitutions or a combination thereof are preferably not in the CDR3 region of the VL chain, preferably not in the CDR1, CDR2 or CDR3 region or FR4 region of the VL chain. The preferred sequence of the common light chain is shown in Figure 1 middle.

[0075] Bispecific antibodies can be produced in a variety of ways. One method involves expressing two different heavy chains and two different light chains in cells and collecting antibodies produced by cells. Antibodies produced in this way will generally include a collection of antibodies with different combinations of heavy chains and light chains, some of which are desired bispecific antibodies. The bispecific antibodies can then be purified from the collection. The ratio of bispecific antibodies produced by cells to other antibodies can be increased in a variety of ways. In a preferred embodiment of the present invention, the ratio is increased by expressing two substantially identical light chains instead of two different light chains in the cell. The two substantially identical light chains can be light chains with substantially identical light chain variable regions and different light chain constant regions, or preferably two substantially identical light chain constant regions. This concept is also referred to as the "common light chain" method in the art. When substantially identical light chains act together with two different heavy chains to allow the formation of variable domains with different antigen binding sites and with different binding properties, the ratio of bispecific antibodies produced by cells to other antibodies is significantly increased relative to the expression of two substantially different light chains. The ratio of bispecific antibodies produced by cells can be further increased by stimulating the pairing of two different heavy chains relative to the pairing of two identical heavy chains. The art describes a variety of ways in which such heterodimerization of heavy chains can be achieved. A preferred method is described in U.S. Provisional Application 61 / 635,935, followed by U.S. Formal Application No. 13 / 866,747 and PCT Application No. PCT / NL2013 / 050294 (WO 2013 / 157954 A1), which are incorporated herein by reference. Methods and means for producing bispecific antibodies (from a single cell) are disclosed, thereby providing means for the formation of bispecific antibodies that are more conducive to the formation of monospecific antibodies. These methods may also be advantageously used in the present invention. Therefore, the present invention provides a method for producing a bispecific antibody according to the present invention (from a single cell), wherein the bispecific antibody comprises two CH3 domains capable of forming an interface, the method comprising providing in the cell: a) a first nucleic acid molecule encoding a heavy chain containing a first CH3 domain, b) a second nucleic acid molecule encoding a heavy chain containing a second CH3 domain, wherein the nucleic acid molecule provides a preference pairing for the heavy chain containing the first CH3 domain with the heavy chain containing the second CH3 domain, the method further comprising culturing the host cell and allowing the expression of the two nucleic acid molecules and harvesting the bispecific antibody from the culture. The first and second nucleic acid molecules can be part of the same nucleic acid molecule, vector or gene delivery vector, and can be integrated at the same site of the host cell genome. Alternatively, the first and second nucleic acid molecules are provided to the cell separately. The host cell comprises at least one light chain, and preferably a common light chain.

[0076] A preferred embodiment provides a method for producing a bispecific antibody according to the present invention from a single cell, wherein the bispecific antibody comprises two CH3 domains capable of forming an interface, the method comprising providing:

[0077] - a cell having: a) a first nucleic acid molecule encoding a heavy chain comprising an antigen binding site that can bind to the extracellular portion of PD-1 and containing a first CH3 domain; and b) a second nucleic acid molecule encoding a heavy chain comprising an antigen binding site that can bind to the extracellular portion of TIM-3 and containing a second CH3 domain, wherein the nucleic acid molecules provide for preferential pairing of the first CH3 domain with the second CH3 domain,

[0078] The method further comprises the steps of: culturing the cell and allowing expression of the protein encoded by the two nucleic acid molecules, and harvesting the bispecific IgG antibody from the culture. In a particularly preferred embodiment, the cell also has a third nucleic acid molecule encoding a common light chain. The first, second and third nucleic acid molecules can be part of the same nucleic acid molecule, vector or gene delivery vector, and can be integrated at the same site of the host cell genome. Alternatively, the first, second and third nucleic acid molecules are provided to the cell, respectively. As described above, the preferred common light chain is based on O12, which is preferably rearranged germline human κ light chain IgVκ1 39*01 / IGJκ1*01. The means for the preferred pairing of the first CH3 domain and the second CH3 domain are preferably corresponding mutations in the CH3 domain of the heavy chain coding region. The preferred mutations that substantially only produce bispecific antibodies are amino acid replacements L351K and T366K in the first CH3 domain (numbered according to EU numbering) and amino acid replacements L351D and L368E in the second CH3 domain, or vice versa ( Figure 2). Therefore, a method for producing a bispecific antibody according to the present invention is also provided, wherein the first CH3 domain comprises the amino acid substitutions L351K and T366K (numbered according to EU numbering), and wherein the second CH3 domain comprises the amino acid substitutions L351D and L368E, and the method further comprises the following steps: culturing the cell and allowing expression of the protein encoded by the nucleic acid molecule, and harvesting the bispecific antibody from the culture. A method for producing a bispecific antibody according to the present invention is also provided, wherein the first CH3 domain comprises the amino acid substitutions L351D and L368E (numbered according to EU numbering), and wherein the second CH3 domain comprises the amino acid substitutions L351K and T366K, and the method further comprises the following steps: culturing the cell and allowing expression of the nucleic acid molecule, and harvesting the bispecific antibody from the culture. Antibodies that can be produced by these methods are also part of the present invention. The CH3 heterodimerization domain is preferably an IgG1 heterodimerization domain. The heavy chain constant region comprising the CH3 heterodimerization domain is preferably an IgG1 constant region.

[0079] A variable domain that "blocks" the binding of PD-1 to PD-L1 and / or PD-L2 interferes with the binding of PD-1 to PD-L1 and / or PD-L2. Such a variable domain may bind to PD-1. Such a blocking variable domain may bind to an epitope on PD-1 and compete with PD-L1 and / or PD-L2 for binding to the epitope. Such a blocking variable domain and PD-L1 and / or PD-L2 may also bind to different epitopes on PD-1. In such a case, the blocking activity may, for example, be due to a reduction in the binding of PD-L1 and / or PD-L2, displacement of PD-L1 and / or PD-L2 (when it is already bound to PD-1), or may be prevented from binding to PD-1 by steric hindrance. All of these and other mechanisms may at least partially prevent the binding partner from binding to the first membrane protein.

[0080] The variable domain that "blocks" the binding of TIM-3 to galectin-9 interferes with the binding of TIM-3 to galectin-9. Such variable domains bind to TIM-3. Such blocking variable domains bind to an epitope on TIM-3 and compete with galectin-9 for binding to the epitope. Such blocking variable domains and galectin-9 may also bind to different epitopes on TIM-3. In such cases, the blocking activity may be due to reduced binding of PD-L1 and / or PD-L2, displacement of galectin-9 (when it has bound to TIM-3), or blocking binding to TIM-3 by steric hindrance. All of these and other mechanisms may at least partially prevent the binding partner from binding to the first membrane protein.

[0081] When compared to the binding in the absence of variable domains, the variable domains that block the binding of a specific binding pair (i.e., PD-1 / PD-L1; PD-1 / PD-L2 or TIM-3 / galectin-9) as described herein generally reduce the binding of the pair. This is preferably measured in an in vitro assay. Generally speaking, this is performed by incubating the variable domain with a membrane protein that can bind to it and then incubating the mixture with the other member of the pair. The binding of the pair is then compared with the binding of the pair in the absence of variable domains. The variable domain can completely prevent the binding of the first membrane protein to its binding partner. It can also partially prevent the binding of the binding pair. When compared to the binding in the absence of variable domains, the variable domains that block the binding of the specific binding pair of membrane proteins preferably reduce the binding of the pair by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, and more preferably at least 90%. Binding blocking by variable domains is defined herein as blocking obtained using a bivalent monoclonal antibody comprising the two identical variable domains. Said variable domain will of course also block binding when present in an antibody comprising said variable domain and the variable domain that binds a second membrane protein.

[0082] The specific variable domains that can bind to the extracellular portion of PD-1 and at least partially block the binding of PD-1 to PD-L1 and / or PD-L2 are variable domains comprising the amino acid sequence of the following VH: MF6076, MF6226, MF6236, MF6256, MF6930, MF6932, MF6935, MF6936, MF6972, MF6974 and MF6982. The specific variable domains that can bind to the extracellular domain of TIM-3 and block the binding of TIM-3 to galectin-9 are variable domains comprising the amino acid sequence of the following VH: MF7676, MF7677, MF7678, MF7679. The amino acid sequence is shown in Figure 3 middle.

[0083] The present invention also provides a method for engaging and / or activating T cells, comprising: providing a system comprising T cells and cells (second cells) to be engaged or activated with the T cells; and providing the system with at least one antibody, preferably at least one bispecific antibody, comprising a variable domain that can bind to PD-1 and a variable domain that can bind to TIM-3; and incubating the system under conditions that allow T cell engagement and / or activation. In some embodiments, the method is an in vitro method. The cells to be engaged or activated with the T cells are preferably immune cells, such as antigen presenting cells, macrophages, neoplastic cells, virally infected cells, or intracellular parasitic infected cells. Engaging and / or activating T cells causes T cells to target specific targets. Activated T cells are T cell receptors that activate the T cells. Engaging T cells is usually activating T cells. Engagement can also cause already activated T cells to target targets specified by antibodies. The conditions that allow the T cells to engage and / or activate are usually culture conditions, but can also be incubated in non-human animals. The conditions are such that T cells do not engage in the absence of antibodies. If a pool of T cells is measured, some of them may have been engaged or activated, provided that the pool contains sufficient unengaged or activated T cells.

[0084] The antibodies of the present invention can bring two cells close together, thereby allowing interactions between cells mediated by proteins other than PD-1 and TIM-3 bound by the antibodies of the present invention. One such interaction is the interaction of a T cell receptor of a cell with the MHC on another cell.

[0085] In one aspect, the present invention provides a method for interfering with PD-1 and / or TIM-3-mediated inhibition in PD-1 and / or TIM-3 positive cells, the method comprising contacting the cell with an antibody or a functional part, derivative and / or analog thereof comprising the following, thereby inhibiting PD-1 and / or TIM-3-mediated activity in the cell:

[0086] - a variable domain that can bind to the extracellular portion of PD-1, and

[0087] - Its variable domain that can bind to the extracellular part of TIM-3.

[0088] TIM-3 or PD-1 positive cells express membrane proteins on the cell membrane in a detectable amount, which is usually detected by immunofluorescence of a monoclonal antibody specific for the membrane protein. PD-1 positive cells are T cells. TIM-3 cells are preferably T cells, more preferably so-called exhausted T cells. T cell exhaustion is a state of T cell dysfunction that occurs during many chronic infections and cancers. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state different from that of functional effector or memory T cells. Exhaustion prevents optimal prevention and treatment of infection and tumors. The antibodies of the present invention interfere with PD-1 and TIM-3 mediated inhibition by binding to the corresponding membrane protein and preventing the protein from being stimulated by the corresponding binding partner of the protein. The known binding partners of PD-1 are PD-L1 and PD-L2. The known binding partner of TIM-3 is galectin-9. The antibody blocks the interaction between PD-1 and PD-L1 and / or PD-L2; and / or TIM-3 and galectin-9, thereby at least partially preventing the inhibitory activity of PD-1 in PD-1-positive cells and / or the inhibitory activity of TIM-3 in TIM-3-positive cells. In a preferred embodiment of the present invention, the binding of the PD-1 binding variable domain to PD-1 blocks the binding of PD-1 to PD-L1 and / or PD-L2, preferably PD-L1. In a preferred embodiment of the present invention, the binding of the TIM-3 binding variable domain to TIM-3 blocks the binding of TIM-3 to galectin-9. The inhibition of PD-1 and / or TIM-3-mediated activity in the cell can be measured in a variety of ways. Usually, but not necessarily, by measuring CD4 + or CD8 +Activity is measured by the activation of T cells. This can be done using healthy CD4+ or CD8+ T cells, but the effect of exhaustion is usually best measured on exhausted T cells. Such T cells are, for example, positive for TIM-3. Activity is preferably measured in HIV-specific T cells, which are preferably collected from subjects with progressive diseases. Proliferation is a suitable parameter. The proliferation rate can be determined in the presence and absence of antibodies. The difference in proliferation rate is a measure of the level of inhibition of the activity of TIM-3 and / or PD-1 in these cells. Other examples of suitable T cells are TILs collected from subjects with non-small-cell lung cancer (NSCLC). The production of interferon gamma is a suitable parameter. The production of interferon gamma can be determined in the presence and absence of antibodies. The difference in interferon gamma production is a measure of the level of inhibition of the activity of TIM-3 and / or PD-1 in these cells. Proliferation and / or interferon gamma production increase the inhibition of the activity of TIM-3 and / or PD-1 in these cells. In a preferred embodiment, the increase is at least 10%, preferably at least 20%, more preferably at least 40%, more preferably at least 80%, relative to the level or rate detected in the absence of the antibody.

[0089] The present invention also provides a method for stimulating the formation, stability and / or activity of an immune synapse, comprising providing a system comprising at least two cells capable of associating with each other via an immune synapse, and providing to the system an antibody or a functional part, derivative and / or analog thereof comprising:

[0090] - a variable domain that can bind to the extracellular portion of PD-1, and

[0091] - a variable domain that can bind to the extracellular part of TIM-3.

[0092] The antibody promotes the formation, stability and / or activity of immune synapses by binding to PD-1 and / or TIM-3 on cells containing PD-1 or TIM-3 on the cell membrane. The binding inhibits the activity of PD-1 and / or TIM-3. This has the effect of stimulating the formation, stability and / or activity of immune synapses. The variable domains that can bind to PD-1 preferably block the binding of PD-1 to PD-L1 and / or PD-L2, preferably PD-L1. The variable domains that can bind to TIM-3 preferably block the binding of TIM-3 to galectin-9. The two cells are cells that can form immune synapses. At least one of the cells is a T cell receptor positive cell. Another cell is usually but not necessarily an antigen presenting cell. The immune synapse is formed due to the close engagement of T cells with antigen presenting cells (antigen-presenting cell, APC), and it is a site where the T cell receptor (TCR) is triggered by its antigen ligand (peptide-MHC complex) present in the APC membrane. The immune synapse in the T cell membrane typically has three concentric rings of membrane receptors and their underlying cytoskeleton and signaling proteins. The inner ring or central supramolecular activation cluster (cSMAC) concentrates most of the TCRs and CD28, and it is surrounded by the peripheral SMAC formed by integrins. Finally, the outermost ring or distal SMAC (dSMAC) is where proteins with large extracellular domains are located, such as CD43 and CD45, away from the cSMAC.

[0093] The present invention also provides an antibody or a variant thereof, which comprises a variable domain that can bind to the extracellular portion of PD-1 and a variable domain that can bind to the extracellular portion of TIM-3.

[0094] In a preferred embodiment of the method, antibody (or variant thereof) or use of the present invention, the variable domain that binds to PD-1 preferably blocks the binding of PD-1 to PD-L1 and / or PD-L2. The variable domain that binds to TIM-3 preferably blocks the binding of TIM-3 to galectin-9. Preferably, the two variable domains block the binding of the corresponding binding partners.

[0095] The variable domain that binds the extracellular part of PD-1 is preferably defined as a variable domain that, when in the form of a bivalent monospecific antibody comprising two of said variable domains that bind PD-1, inhibits PD-1 / PD-L1 mediated inhibition of T cell receptor-mediated activation of Jurkat cells in the range of 20% to 150% compared to the inhibition obtained with the antibody nivolumab on Jurkat cells.

[0096] The inhibition of PD-1 inhibition of TCR-mediated activation of said Jurkat cells is preferably in the range of 50% to 150%, preferably 80% to 150%, more preferably 100% to 150%, when compared to the inhibition obtained with the antibody nivolumab on Jurkat cells. In a preferred embodiment, the inhibition is at least 100% when compared to the inhibition obtained with the antibody nivolumab on said Jurkat cells. TCR-mediated PD-1 inhibition of activation of Jurkat cells is preferably measured by measuring the immunoreduction effect of PD-1 / PD-L1 binding in Jurkat cells, which are incubated under conditions that can be activated by the T cell receptor due to the presence of said antibody or its functional part, derivative and / or analog.

[0097] The present invention also provides a composition or kit comprising two or more antibodies containing a variable domain that can bind to the extracellular portion of PD-1 and a variable domain that can bind to the extracellular portion of TIM-3, or functional parts, derivatives and / or analogs thereof;

[0098] wherein the first and second of said antibodies or functional parts, derivatives and / or analogs thereof bind to:

[0099] -Different epitopes on PD-1;

[0100] - a different epitope on TIM-3; or

[0101] -different epitopes on PD-1 and TIM-3. Embodiments comprising methods, uses, compositions or kits involving two or more antibodies or functional parts, derivatives and / or analogs thereof having variable domains that bind to PD-1 and TIM-3 as specified in this paragraph are also referred to as "Oligoclonics" embodiments. Some examples of such Oligoclonics embodiments are embodiments having the first and second antibodies. "Oligoclonics" is a registered trademark. Methods for making such The general method of the product is described in detail in the accompanying drawings and is incorporated herein by reference.

[0102] In the Oligoclonics embodiment, the first and second antibodies comprise variable domains that bind to PD-1 and TIM-3. Antibodies with variable domains that bind to the same PD-1 or TIM-3 can bind to the same individual protein, but this is not necessary. The antibodies of the present invention that bind to PD-1 or TIM-3 bind to epitopes on the protein. An epitope is an antigen, in this case the part of a membrane protein recognized by an antibody. The first and second antibodies that bind to different epitopes on a membrane protein can bind to the same individual protein on the membrane. To this end, different epitopes are preferably non-overlapping epitopes. In other words, different epitopes are sufficiently separated on the membrane protein that two antibodies can bind to the same individual protein at the same time. It was unexpectedly found that Oligoclonics (a combination of the first and second antibodies or more antibodies) can be more effective than the same amount of each individual antibody.

[0103] Preferably, at least one of the two or more antibodies or functional parts, derivatives and / or analogs comprises a PD-1 binding variable domain that blocks the binding of PD-1 to PD-L1 and / or PD-L2, preferably PD-L1. In a preferred embodiment, at least two of the two or more antibodies or functional parts, derivatives and / or analogs comprise a PD-1 binding variable domain that blocks the binding of PD-1 to PD-L1 and / or PD-L2, preferably PD-L1.

[0104] Preferably, at least one of the two or more antibodies or functional parts, derivatives and / or analogs comprises a TIM-3 binding variable domain that blocks the binding of TIM-3 to galectin-9. In a preferred embodiment, at least two of the two or more antibodies or functional parts, derivatives and / or analogs comprise a TIM-3 binding variable domain that blocks the binding of TIM-3 to galectin-9.

[0105] Preferably, at least one of the two or more antibodies or functional parts, derivatives and / or analogs comprises: a PD-1 binding variable domain that blocks the binding of PD-1 to PD-L1 and / or PD-L2, preferably PD-L1; and a TIM-3 binding variable domain that blocks the binding of TIM-3 to galectin-9. Preferably, at least two of the two or more antibodies or functional parts, derivatives and / or analogs comprise: a PD-1 binding variable domain that blocks the binding of PD-1 to PD-L1 and / or PD-L2, preferably PD-L1; and a TIM-3 binding variable domain that blocks the binding of TIM-3 to galectin-9.

[0106] In one aspect, the present invention provides methods and uses as described herein, wherein two or more antibodies or functional parts, derivatives and / or analogs thereof are used, and wherein these two or more antibodies or functional parts, derivatives and / or analogs thereof comprise a variable domain that can bind to the extracellular part of PD-1 and a variable domain that can bind to the extracellular part of TIM-3;

[0107] wherein the first and second of said antibodies or functional parts, derivatives and / or analogs thereof bind to:

[0108] -Different epitopes on PD-1;

[0109] - a different epitope on TIM-3; or

[0110] - Different epitopes on PD-1 and TIM-3. The priority of blocking the binding of PD-L1 and / or PD-L1 by binding to the variable domain of PD-1 and blocking the binding of TIM-3 to galectin-9 by binding to the variable domain of TIM-3 is the same as described in the Oligoclonics embodiment.

[0111] The antibody or its part, derivative or analogue preferably comprises two variable domains as described. Such an antibody is preferably a bispecific antibody or its functional part, derivative or analogue. Two or more antibodies or its functional part, derivative and / or analogue can be linked together. Various methods are known in the art. A suitable method is conjugation. In addition, the technology of making multispecific antibodies has progressed to also include bispecific antibodies with the same overall structure as normal monospecific antibodies, but wherein the two arms of the antibody each bind to different targets. The bispecific antibody or its functional part, derivative and / or analogue preferably has two heavy chains with compatible heterodimerization domains. The light chain is preferably a common light chain. The antibody is preferably a full-length bispecific antibody, which consists of two heavy chains with compatible heterodimerization domains. The light chain is preferably a common light chain.

[0112] The term "conjugate" as used herein refers to two or more molecules that have been covalently linked, optionally covalently linked by a connecting region. For example, in some embodiments, the conjugate is a first protein or non-protein part connected to a second protein or non-protein part by a connecting region. For example, in some embodiments of the binding molecules of the present invention, it comprises or consists of two or more antibodies that have been covalently linked. The conjugate is not limited to the first and second parts, and in some embodiments, it may also have a third, fourth or more parts connected by another connecting region. Some examples of protein parts include, but are not limited to: polypeptides, peptide mimetics or antibodies (or antibody parts, derivatives or analogs as described elsewhere in this application). Some examples of non-protein parts include, but are not limited to aptamers. Various types of joints can be used, and suitable joints will be selected according to the type of molecules in the conjugate and the desired properties of the joint (length, flexibility, resistance to protease activity and other similar features). Such joints may include nucleotides, polypeptides or suitable synthetic materials. For example, the joint may be a flexible peptide joint. In certain embodiments, the joint may be a cleavable joint, thereby allowing the parts of the conjugate to be separated from each other. In other embodiments, the peptide joint may be a spiral joint. Various examples and kits for connecting proteins and other molecules are well known in the art. The term "fusion protein" as used herein refers to a protein comprising two or more polypeptides or proteins that have been connected at the DNA level by recombination and expressed together as a single polypeptide. The fusion protein may also include a peptide linker region that is also encoded by the DNA and expressed together with the fusion protein. The peptide linker that is part of the fusion protein can be designed to have specific properties, such as flexibility, hydrophilicity, protease resistance, cleavage, etc. All of these properties can be designed in the DNA sequence, and methods for designing linkers are well known in the art. For example, antibodies can be linked together by methods well known in the art, and as described herein, to form bispecific or multi-targeted antibodies. In addition, bispecific antibodies can be prepared by a variety of methods known in the art, such as by using, for example (See, for example, WO2013 / 157954). Bispecific monoclonal antibodies (BsMAbs, BsAbs) generally contain the binding domains of two different monoclonal antibodies and thus bind to two different epitopes. The molecules and other full-length IgG bispecific antibodies have two different antigen-binding specificities encoded by two different variable regions of the full-length IgG molecule of the Fab of the scFv. Can be produced by co-transfecting separate cells with genetic constructs encoding two different common light chain (cLC) antibodies, as described elsewhere herein. CH3 engineering ensures efficient heterodimerization and the formation of essentially pure bispecific antibodies.

[0113] The antibodies of the present invention are preferably bispecific antibodies. Antibodies typically bind to their targets through a so-called antigen binding site. Unmodified antigen binding sites are typically formed by and present in the variable domains of antibodies. The variable domains contain antigen binding sites. Variable domains that can bind to antigens are variable domains that contain antigen binding sites that can bind to antigens.

[0114] The antibody variable domain generally comprises a heavy chain variable region (VH) and a light chain variable region (VL). The antigen binding site may be present in the combined VH / VL variable domain, or only in the VH region or only in the VL region. When the antigen binding site is present in one of the two regions of the variable domain, the corresponding variable region may promote the folding and / or stability of the bound variable region, but does not significantly promote the binding of the antigen itself.

[0115] Antigen binding as used herein refers to the typical binding capacity of an antibody to its antigen. The binding of an antibody to an antigen can be assessed in a variety of ways. One way is to incubate the antibody with an antigen (preferably a cell expressing the antigen), remove unbound antibody (preferably by a washing step), and detect the bound antibody by a labeled antibody bound to the bound antibody.

[0116] Antigen binding of antibodies is usually mediated by the complementary determining regions (CDRs) of the antibodies and the specific three-dimensional structure of both the antigen and the variable domains, thereby allowing the two structures to be precisely bound together (similar to the interaction of a lock and a key), which is relative to random, non-specific protein adhesion. Since antibodies usually recognize a portion of an antigen called an antigenic epitope, and since such an epitope may also be present in other compounds, antibodies according to the present invention may also recognize other proteins if such other compounds contain the same epitope. Therefore, the term "binding" does not exclude the binding of an antibody to another protein or more proteins containing the same epitope. Such other proteins are preferably not human proteins.

[0117] The antibodies generally do not bind to other proteins than the specific target protein on the cell membrane in postnatal humans, preferably adults.

[0118] The variable domains of the antibodies of the present invention or their functional parts, derivatives and / or analogs that can bind to the extracellular part of PD-1 bind to PD-1, and under otherwise identical conditions, bind to the extracellular part of another member of the CD28 family of the same species at a level at least 100 times lower. The variable domains of the antibodies or their functional parts, derivatives and / or analogs that bind to PD-1 bind to PD-1, and under otherwise identical conditions, bind to CD28, CTLA4, ICOS, BTLA, NKp30 and TMIGD2 of the same species at a level at least 100 times lower. Considering that PD-1 is a cell surface protein, the binding is usually evaluated on cells expressing members on the cell surface.

[0119] The variable domains of the antibodies of the present invention or their functional parts, derivatives and / or analogs that can bind to the extracellular portion of TIM-3 bind to TIM-3, and under otherwise identical conditions, bind to the extracellular portion of another member of the TIM family of the same species at a level at least 100-fold lower. The variable domains of the antibodies or their functional parts, derivatives and / or analogs that bind to TIM-3 bind to TIM-3, and under otherwise identical conditions, bind to TIM-1 or TIM-4 of the same species at a level at least 100-fold lower. Considering that TIM-3 is a cell surface protein, the binding is usually assessed on cells expressing the member on the cell surface.

[0120] The present invention also provides a method for treating an individual with cancer, the method comprising administering an antibody or functional portion, derivative and / or analog of the present invention or a bispecific antibody of the present invention to an individual in need thereof. The individual is preferably an individual with cancer. In some embodiments, the cancer is a cancer comprising cancer cells expressing membrane proteins. In a preferred embodiment, the cancer is a cancer comprising cancer cells expressing PD-L1, PD-L2 and / or galectin-9.

[0121] The cancer is preferably adenocarcinoma. Some preferred cancers are colorectal cancer; pancreatic cancer; lung cancer; breast cancer; liver cancer; prostate cancer; ovarian cancer; cervical cancer; endometrial cancer; head and neck cancer; melanoma; testicular cancer; urothelial cancer; kidney cancer; gastric cancer; or carcinoid. In a preferred embodiment, the cancer is colorectal cancer; pancreatic cancer; lung cancer; breast cancer; liver cancer; prostate cancer; ovarian cancer; cervical cancer; endometrial cancer; head and neck cancer; or melanoma. In a particularly preferred embodiment, the cancer is colorectal cancer; pancreatic cancer; lung cancer; breast cancer; or liver cancer. In a particularly preferred embodiment, the cancer is gastrointestinal cancer. In a preferred embodiment, the cancer is colorectal cancer. In this embodiment, the antibody or its functional part, derivative and / or analog is preferably an antibody having a variable domain that can bind to PD-1 and a variable domain that can bind to TIM-3. The PD-1 binding variable domain preferably blocks the binding of PD-1 to PD-L1 and / or PD-L2, preferably PD-L1. The TIM-3 binding variable domain preferably blocks the binding of TIM-3 to galectin-9. Preferably, the method comprises two or more of said antibodies or functional parts thereof, as described for the Oligoclonics embodiment.

[0122] Also provided is a method for stimulating an immune response against abnormal cells in an individual, the method comprising providing the individual with an antibody of the present invention or a functional portion thereof, a derivative and / or an analog thereof. Abnormal cells are preferably cancer cells, virus-infected cells, parasites or parasite-infected cells. In a preferred embodiment, the cell is a cancer cell or a neoplastic cell. In this embodiment, the antibody or its functional portion, derivative and / or analog is preferably an antibody having a variable domain that can bind to PD-1 and a variable domain that can bind to TIM-3. In this embodiment, the PD-1 binding variable domain preferably blocks the binding of PD-1 to PD-L1 and / or PD-L2, preferably PD-L1. The TIM-3 binding variable domain preferably blocks the binding of TIM-3 to galectin-9. Preferably, the method comprises two or more of the antibodies or their functional portions, as described for the Oligoclonics embodiment.

[0123] A neoplasm is an abnormal growth of tissue and is often referred to as a tumor when it also forms a mass. Neoplasms in the present invention often form a mass. Neoplastic cells are cells from a neoplasm that form a mass on their own. The World Health Organization (WHO) classifies neoplasms into four main groups: benign neoplasms, in situ neoplasms, malignant neoplasms, and neoplasms of uncertain or unknown behavior. Malignant neoplasms (neoplasm) are also referred to as cancer.

[0124] Stimulating an immune response encompasses both inducing an immune response and enhancing an already existing immune response.The immune response in an individual can be measured by measuring the tumor load in the individual, the viral load in the individual, the parasite load in the individual, where applicable.

[0125] The virus-infected cells are preferably cells infected with: immunodeficiency virus; herpes virus, preferably herpes simplex virus, varicella-zoster virus; cytomegalovirus or Epstein-Barr virus; papillomavirus; hepatitis virus, preferably hepatitis A, B or C virus; measles virus or adenovirus. The virus is preferably a virus known to persist in an individual. Persistent infection is characterized by an infection in which the virus is not cleared and remains in specific cells of the infected individual. Persistent infection can involve two stages of silent infection and productive infection without rapidly killing the host cell, or even causing excessive damage to it. Persistent virus-host interaction can be a latent, chronic and / or slow infection.

[0126] The cells infected by parasites are cells infected by intracellular parasites. Such parasites are parasitic microorganisms that can grow and reproduce in the cells of the host. Some intracellular parasites can also live outside the cell. Such parasites are so-called facultative intracellular parasites. Some non-limiting examples are Listeria monocytogenes, Legionella, some species of mycobacterium, and Cryptococcus neoforman. Some preferred intracellular parasites are parasites that are unable to grow outside of a host cell, some preferred examples being Chlamydia and closely related species; certain species of Mycobacterium, such as Mycobacterium leprae; certain protozoa, including: Apicomplexans (Plasmodium spp., Toxoplasma gondii, and Cryptosporidium parvum), and trypanosomatids.

[0127] The present invention also provides a nucleic acid molecule encoding the variable region of the heavy chain of an antibody according to the present invention. The nucleic acid molecule (usually an in vitro, isolated or recombinant nucleic acid molecule) preferably encodes Figure 3 or a heavy chain variable region having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions, or a combination thereof. Figure 3 In a preferred embodiment, the nucleic acid molecule comprises Figure 3 The nucleic acid molecule preferably uses codons optimized for expression in the antibody-producing cells to be used. Preferably, the coding sequence is Figure 3 or a heavy chain variable region having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions, or a combination thereof. Figure 3 The nucleic acid encoding the heavy chain variable region shown in is codon optimized for expression in human cells, preferably Per.C6TM, or Chinese hamsters, preferably CHO. The present invention also provides a nucleic acid encoding the heavy chain variable region and Figure 2 The nucleic acid sequence of the heavy chain constant region.

[0128] The nucleic acid molecules used in the present invention are usually, but not absolutely, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Alternative nucleic acids can be used by those skilled in the art. The nucleic acid molecules according to the present invention are, for example, contained in cells. When the nucleic acid molecules are expressed in the cells, the cells can produce antibodies according to the present invention. Therefore, in one embodiment, the present invention provides cells comprising antibodies according to the present invention and / or nucleic acid molecules according to the present invention. When the cells produce heavy chains and light chains, antibodies are produced. Cells that can produce antibodies according to the present invention are provided. The cells preferably contain nucleic acid molecules encoding antibody heavy chains comprising antibody heavy chain variable regions, which can bind to the first membrane protein when combined with a common light chain. The cells preferably also contain nucleic acid molecules encoding antibody heavy chains comprising antibody heavy chain variable regions, which can bind to the second membrane protein when combined with a common light chain. The cells preferably also contain nucleic acid molecules encoding common light chains. The cells are preferably animal cells, more preferably mammalian cells, more preferably primate cells, and most preferably human cells. For the purposes of the present invention, suitable cells are any cells that can contain and preferably produce antibodies according to the present invention and / or nucleic acids according to the present invention.

[0129] The present invention also provides cells comprising antibodies according to the present invention. Cells comprising one or more nucleic acid molecules encoding antibodies of the present invention alone or together are also provided. One or more nucleic acid molecules are expressible nucleic acid molecules, meaning that they contain cis signals required for RNA transcription and protein coding domain translation. Preferably, the cell (typically an in vitro, isolated or recombinant cell) produces the antibody. In a preferred embodiment, the cell is a hybridoma cell, a Chinese hamster ovary (CHO) cell, a NS0 cell or a PER-C6TM cell. In a particularly preferred embodiment, the cell is a CHO cell. A cell culture comprising cells according to the present invention is also provided. Many institutions and companies have developed cell lines for large-scale production of antibodies, such as for clinical applications. Some non-limiting examples of such cell lines are CHO cells, NS0 cells or PER.C6TM cells. These cells are also used for other purposes, such as the production of proteins. Cell lines developed for industrial-scale production of proteins and antibodies are also referred to herein as industrial cell lines. Therefore, in a preferred embodiment, the present invention provides cell lines developed for large-scale production of antibodies for the production of antibodies of the present invention. The present invention also provides a cell for producing antibodies, which comprises a gene encoding Figure 3 , 1 and 2. Preferably, the nucleic acid molecule comprises a nucleic acid molecule of the VH, VL and / or heavy chain shown in Figure 1 and 2 The sequence shown in .

[0130] The present invention also provides a method for producing an antibody, comprising culturing the cells of the present invention and harvesting the antibody from the culture. Preferably, the cells are cultured in a serum-free medium. Preferably, the cells are suitable for suspension growth. Also provided are antibodies obtainable by the method for producing an antibody according to the present invention. Preferably, the antibody is purified from the culture medium of the culture. Preferably, the antibody is affinity purified.

[0131] The cells of the present invention are, for example, hybridoma cell lines, CHO cells, 293F cells, NS0 cells or any other cell type known in the art to be suitable for antibody production for clinical purposes, in particular suitable for producing antibodies for administration in humans. In a particularly preferred embodiment, the cells are human cells, preferably cells transformed by the E1 region of adenovirus or its functional equivalent. A preferred example of such a cell line is the PER.C6TM cell line or its equivalent. In a particularly preferred embodiment, the cells are CHO cells or variants thereof, preferably variants of antibodies expressed using a glutamine synthetase (GS) vector system.

[0132] The present invention also provides a pharmaceutical composition comprising one or more antibodies or variants thereof according to the present invention.The pharmaceutical composition preferably comprises an excipient or carrier, which is preferably pharmaceutically acceptable.

[0133] The antibodies of the present invention or their variants may also include a label, preferably a label for in vivo imaging. Such labels are generally optional for therapeutic applications. For example, in a diagnostic context, labels may be helpful. For example, they can be used to visualize target cells in vivo. A variety of labels are suitable, and many labels are well known in the art. In a preferred embodiment, the label is a radioactive label for detection. In another preferred embodiment, the label is an infrared label. Preferably, the infrared label is suitable for in vivo imaging. A variety of infrared labels are available to those skilled in the art. Some preferred infrared labels are, for example, IRDye 800; IRDye 680RD; IRDye 680LT; IRDye 750; IRDye 700DX; IRDye 800RS IRDye 650; IRDye 700 phosporamidite; IRDye 800 phosporamidite (LI-COR USA; 4647 Superior Street: Lincoln, Nebraska).

[0134] The amount of the antibody according to the invention to be administered to the patient is generally within the therapeutic window, meaning that a sufficient amount is used to obtain a therapeutic effect without exceeding the threshold that causes an unacceptable degree of side effects. The lower the amount of antibody required to obtain the desired therapeutic effect, the larger the therapeutic window is generally. Therefore, antibodies according to the invention that exert a sufficient therapeutic effect at low doses are preferred. The dose may be within the range of the dosing regimen of nivolumab. The dose may also be lower.

[0135] Compared with the combination of bivalent monospecific antibodies with variable domains, antibodies according to the present invention or their variants and particularly bispecific antibodies or their variants may have less side effects.Compared with the combination of bivalent monospecific antibodies with variable domains, antibodies according to the present invention or bispecific antibodies or their functional parts, derivatives and / or analogs have lower immune-related toxicity.The antibody or bispecific antibody reduces the safety responsibility in the patient.The combination of antibodies that block inhibitory and / or co-stimulatory molecules is beneficial to patients who are unresponsive to existing immunotherapy.However, it has been shown that the dual blocking of immunomodulatory receptors (immuno-modulatory receptor, iMOD) improves immune-related toxicity.Antibodies according to the present invention or their variants and particularly bispecific antibodies or their variants are suitable for solving the dual blocking of iMOD, because they can play the functional activity that monoclonal antibody combinations cannot reproduce, and can more selectively target specific cell populations, which reduces the safety responsibility in the patient.

[0136] Antibodies are produced as bispecific antibodies by cloning the antibodies into complementary expression vectors comprising mutations in the CH3 region that drive the heavy chain to heterodimerize. Many bispecific antibodies are produced on a small scale and tested on cancer cell lines in binding and functional assays. Antibodies of the present invention, particularly bispecific antibodies of the present invention, can combine low toxicity properties with high efficacy. Antibodies of the present invention can be used for immune targeted therapy of various types and lines. When compared with antibodies that bind the same antigen with two arms, antibodies of the present invention can have an expanded therapeutic window.

[0137] Also provided is the use of a bispecific antibody according to the present invention or a functional part, derivative and / or analog thereof for the preparation of a medicament for treating or preventing abnormal cells, tumors and / or metastasis formation. The tumor from which the metastasis originates is preferably a tumor that is positive for PD-L1, PD-L2 and / or galectin-9.

[0138] After transient transfection in suspension 293F cells, the antibodies of the invention can be produced at levels >50 mg / L. The bispecific antibodies can be purified to a purity greater than 98% with a yield >70%. Analytical characterization studies have shown that the bispecific IgG1 antibody spectrum is comparable to that of a bivalent monospecific IgG1.

[0139] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it is to be understood that the scope of the present invention may include embodiments having a combination of all or some of the described features.

[0140] Also provided is a method for treating an individual suffering from cancer, the method comprising administering a protein of the invention or a bispecific antibody of the invention to an individual in need thereof.

[0141] The present invention also provides a protein of the present invention or a bispecific antibody of the present invention for use in treating an individual suffering from cancer.

[0142] The antibody or variant thereof of the present invention preferably comprises a variable domain that can bind to the extracellular portion of PD-1 and comprises a heavy chain variable region having a CDR3 region, wherein the CDR3 region comprises Figure 3 The amino acid sequence of the CDR3 of the heavy chain variable region of one of the following VHs in the embodiment of the present invention: MF6076, MF6236, MF6256, MF6226, MF6930, MF6932, MF6935, MF6936, MF6972, MF6974 or MF6982, preferably MF6226, MF6256 or MF6930. The variable domain that binds to PD-1 preferably comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, wherein the CDR1, CDR2 and CDR3 regions comprise Figure 3 The amino acid sequence of CDR1, CDR2 and CDR3 of the heavy chain variable region of one of the following VHs shown in: MF6076, MF6236, MF6256, MF6226, MF6930, MF6932, MF6935, MF6936, MF6972, MF6974 or MF6982, preferably MF6226, MF6256 or MF6930.

[0143] The antibody or variant thereof preferably comprises a variable domain that can bind to the extracellular portion of PD-1 and comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of the heavy chain variable region as shown below: MF6076, MF6256, MF6226, MF6930, MF6932, MF6935, MF6936, MF6972, MF6974 or MF6982, preferably MF6226, MF6256 or MF6930 (such as Figure 3 ), said amino acid sequence having at most 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of VH as shown in the designated MF.

[0144] The antibody or variant thereof preferably comprises a variable domain that can bind to the extracellular portion of TIM-3 and comprises a heavy chain variable region having a CDR3 region, wherein the CDR3 region comprises Figure 3The amino acid sequence of the CDR3 region of the heavy chain variable region shown below: MF7676, MF7677, MF7678 or MF7679. The variable domain that binds to TIM-3 preferably comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, wherein the CDR1, CDR2 and CDR3 regions comprise Figure 3 The amino acid sequence of CDR1, CDR2 and CDR3 of the heavy chain variable region of one of the following VHs shown in: MF7676, MF7677, MF7678 or MF7679.

[0145] The antibody or variant thereof preferably comprises a variable domain that can bind to the extracellular portion of TIM-3 and comprises a heavy chain variable region comprising the amino acid sequence of a heavy chain variable region as shown below: MF7676, MF7677, MF7678 or MF7679 ( Figure 3 The amino acid sequence has at most 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably has 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the specified MF.

[0146] The present invention is further explained in the following examples. These examples do not limit the scope of the present invention, but are only used to illustrate the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 : Common light chain used in monospecific and bispecific IgG. Figure 1 A: Common light chain amino acid sequence. Figure 1 B: Common light chain variable domain DNA sequence and translation (IGKV1-39 / jk1). Figure 1 C: Common light chain constant region DNA sequence and translation. Figure 1 D: IGKV1-39 / jk5 common light chain variable domain translation. Figure 1 E: V region IGKV1-39A.

[0149] Figure 2 : IgG heavy chain used to generate bispecific molecules.

[0150] Figure 2 A: VH gene. Figure 2 B: CH1 area. Figure 2 C: Hinge region. Figure 2 D: CH2 region. Figure 2 E: CH2 region containing L235G and G236R silent substitutions. Figure 2 F: CH3 domain containing L351K and T366K substitutions (KK). Figure 2G: CH3 domain containing L351D and L368E substitutions (DE).

[0151] Figure 3 : Amino acid sequence of the heavy chain variable region:

[0152] Figure 3 A: Heavy chain variable region of PD-1 specific clone

[0153] Figure 3 B: Heavy chain variable region of TIM-3 specific clone

[0154] The label MF refers to the fab containing the heavy chain variable region and the common light chain as shown. The amino acid sequence of the light chain is shown in Figure 1 In A, the underlined sequences indicate the CDR1, CDR2, and CDR3 regions of each amino acid sequence, respectively.

[0155] Figure 4 :Vector map and characteristics of pIRES-Neo3(MV1363).

[0156] Figure 5 : Vector map and characteristics of pVAX1.

[0157] Figure 6 : Vector map and characteristics of the phagemid vector MV1473 used to generate an “immune” phage display library.

[0158] Figure 7 : Vector map and features of the IgG expression vector MV1452, which is used to express PD-1 and PD-L1 specific Fab arms in the KK variant heavy chain for bispecific IgG production.

[0159] Figure 8 : The amino acid sequence of the VH gene, which has tetanus toxin specificity when combined with the common light chain as MF1337, and is present in the DE variant heavy chain used to generate PD-L1xTT and PD-1xTT bispecific IgG molecules. The underlined sequences indicate the CDR1, CDR2 and CDR3 regions of each amino acid sequence, respectively.

[0160] Fig. 9 : Vector map and features of the IgG expression vector MV1377, which is used to express the TT-specific Fab arm MF1337 in the DE variant heavy chain for bispecific IgG production.

[0161] Fig.10 : PD-1 / PD-L1 Blockade Assay

[0162] The anti-PD-1 antibody panel was evaluated for its ability to block the interaction of PD-L1 with coated PD-1 at a concentration of 10 μg / ml bispecific IgG. Data were normalized to data obtained using the bivalent benchmark PD-L1 antibody MPDL3280A at a concentration of 10 μg / ml (100% blocking). A representative example of the PD-1 panel is shown. Maximum binding was established by incubation with non-PD-1 / PD-L1 specific human isotype antibodies (normalized to 0% blocking). Figure 3 All PD-1 variable domains of the MF sequences shown in and not represented here blocked PD-1 / PD-L1 interaction by >70%.

[0163] Fig.11 : Effects of the tested antibodies on IFN-γ production in allogeneic mMLR.

[0164] Mo-DCs were prepared from CD14+ monocytes cultured for 7 days. Immature DCs were used on day 7 and mature DCs were generated by culturing in maturation medium for an additional 3 days and then cultured for 4 days with T cells isolated by negative selection and the test antibody (mMLR). IFN-γ was measured in the culture supernatant by ELISA. Data were normalized to vehicle control. Six separate MLRs were performed. Example

[0165] As used herein, "MFXXXX" wherein X is independently a number from 0 to 9 refers to a Fab comprising a variable domain wherein VH has an amino acid sequence identified by 4 numbers. Unless otherwise specified, the light chain variable region of the variable domain generally has Figure 1 A. Usually Figure 1 B. "MFXXXX VH" refers to the amino acid sequence of VH identified by 4 numbers. MF also contains the constant region of the light chain and the constant region of the heavy chain that usually interacts with the constant region of the light chain. PG refers to a monospecific antibody comprising the same heavy chain and light chain. PB refers to a bispecific antibody with two different heavy chains. The variable region (VH) of the heavy chain is different, and usually the CH3 region is also different, wherein one heavy chain has a KK mutation in its CH3 domain and the other heavy chain has a complementary DE mutation in its CH3 domain (reference PCT / NL2013 / 050294 (published as WO2013 / 157954)).

[0166] Example: 1

[0167] Generation of materials for selection and screening

[0168] Cell line culture

[0169] Freestyle 293F cells (cat. no. p / n 51-0029) were obtained from Invitrogen and routinely maintained in 293 FreeStyle medium. HEK293T (cat. no. ATCC-CRL-11268) cells were purchased from ATCC and routinely maintained in DMEM / F12 (Gibco) supplemented with L-glutamine (Gibco) and FBS (Lonza), and CHO-S (cat. no. 11619-012) cell lines were purchased from Gibco and routinely maintained in Freestyle CHO expression medium (Invitrogen) supplemented with L-glutamine.

[0170] Generation of PD-1 and TIM-3 expression vectors for immunization and for generation of stable cell lines and transient transfection

[0171] The full-length cDNA of each target, containing unique restriction sites for cloning and a kozak consensus sequence for efficient translation, was synthesized or obtained by PCR amplification of commercially available expression constructs containing the target cDNA using specific primers that introduced unique restriction sites for cloning and a kozak consensus sequence for efficient translation. The cDNA of each target was cloned into a eukaryotic expression construct such as pIRES-Neo3 (Clontech; Figure 4 ) or pVAX1 (Thermo Fisher Scientific; Figure 5 ), thereby generating pIRES-Neo3_[target name] and pVAX1_[target name]. Insert sequences were verified by comparison with the NCBI reference amino acid sequence. The pIRES-Neo3 construct was used to generate stable cell lines and transient transfections. The pVAX1 construct was used for immunization purposes. For an overview of the names of the resulting constructs, see Table 1.

[0172] Amino acid sequence for expression on the cell surface Full-length huPD-1 insert sequence (in both pIRES-Neo3 and pVAX1) (same as GenBank: NP_005009.2):

[0173]

[0174] in:

[0175] MQIPQAPWPVVWAVLQLGWR: signal peptide.

[0176] ECD of huPD-1.

[0177] V GVVGGLLGSLVLLVWVLAVI: predicted TM region.

[0178] Intracellular tail region.

[0179] Amino acid sequence for expression on the cell surface Full-length macaque ( macaca fascicularis ) PD-1 insert sequence (in both pIRES-Neo3 and pVAX1) (same as GenBank: ABR15751.1):

[0180]

[0181] in:

[0182] MQIPQAPWPVVWAVLQLGWR: signal peptide.

[0183] ECD of maPD-1.

[0184] VGVVGGLLGSLVLLVWVLAVI: predicted TM region.

[0185] Intracellular tail region.

[0186] Amino acid sequence for expression on the cell surface Full-length human TIM-3 insert sequence (in both pIRES-Neo3 and pVAX1) (same as GenBank: AAH63431.1):

[0187]

[0188] in:

[0189] MFSHLPFDCVLLLLLLLLTRS: signal peptide.

[0190]

[0191] IYIGAGICAGLALALIFGALI: Predicted TM region.

[0192] Intracellular tail region.

[0193] Amino acid sequence for expression on the cell surface Full-length macaque TIM-3 (cynomolgus monkey) insert sequence (in both pIRES-Neo3 and pVAX1) (identical to GenBank: EHH54703.1):

[0194]

[0195] in:

[0196] MFSHLPFDCVLLLLLLLLTRS: signal peptide.

[0197]

[0198]

[0199] IYIAAGISAGLALALIFGALI: Predicted TM region.

[0200] Intracellular tail region.

[0201] Generation of stable cell lines expressing PD-1 and TIM-3

[0202] The pIRES-Neo3_[target name] expression construct (Table 1) is used to generate CHO-S or Freestyle 293F clones that stably express the corresponding protein. The construct was transiently transfected into CHO-S and Freestyle293F cells using lipofectamine transfection and screened by FACS using antibodies that react with the corresponding protein. After confirming the expression, the transiently transfected cells were inoculated with limiting dilutions and cultured under the selection pressure associated with the expression construct used to obtain stable cell clones. After 2 to 3 weeks of selection, the clones were screened by FACS. The selected clones were amplified by serial passages, retested in FACS and frozen to -150°C. The names of clones that stably express heterologous proteins are CHO-S[target name] cells or Freestyle 293F_[target name] cells. For an overview of the constructs used to generate stable cell lines and their resulting names, see Table 1.

[0203] Example 2

[0204] Immunization, selection and screening

[0205] Mice used for immunization

[0206] To generate human antibodies that bind to huPD-1 and huTIM-3, transgenic mice were immunized against the human VK1-39 light chain (common light chain mice, see WO2009 / 157771) and against the human heavy chain (HC) mini-locus (containing a selection of human V gene segments, all human Ds, and all human Js). These mice are called Mice. Mice are immunized with the recombinant protein antigen or DNA encoding the protein antigen as briefly described below.

[0207] Protein immunization

[0208] The recombinant protein and Gerbu adjuvant MM (Gerbu Biotechnik; cat. no. 3001) were injected subcutaneously. Mice were immunized. Recombinant huPD-1-Fc (R&D; cat.no.1086-PD), huTIM-3-Fc (R&D; cat.no.2365-TM) and maTIM-3-Fc (R&D; cat.no.7914-TM) were used for immunization. Mice were immunized with 40 μg of recombinant protein in PBS mixed with 40 μl of adjuvant in a total volume of 100 μl. Mice were subsequently boosted on days 14 and 28 with 20 μg of recombinant protein in PBS and 20 μl of adjuvant in a total volume of 50 μl. Mouse sera were collected on day 35 to determine serum titers. Mice with low serum reactivity against human and / or macaque targets received additional cycles of booster immunizations and serum analysis with recombinant human or macaque protein antigens. Each cycle consisted of two weekly immunizations using 20 μg of recombinant protein in 50 μl PBS, followed by serum collection one week later for titer analysis. Mice that showed high serum titers against human and macaque targets received a final booster immunization consisting of 20 μg of recombinant protein injected daily in 50 μl PBS for three consecutive days. One day after the final injection, mouse lymphoid tissue was collected.

[0209] DNA immunization

[0210] DNA tattooing using microtexture devices Mice were immunized. DNA tattoo immunization was performed with 20 μg plasmid DNA (pVAX1_[target name], Table 1) encoding the target antigen. Mice were immunized with DNA encoding the human target. Mice were immunized on days 0, 3, 6, 14, 17, 28 and 31. Mouse serum was collected on day 35 to determine serum titer. Mice with low serum reactivity to human and / or macaque targets received additional cycles of booster immunization with human DNA antigens and serum analysis. Each cycle consisted of DNA immunization twice a week, followed by serum collection for titer analysis one week later. Mice that showed strong serum reactivity to cells expressing human and macaque targets received the final booster immunization, followed by lymphoid tissue collection 3 days later.

[0211] Determination of serum titer

[0212] Serum titers were determined by FACS analysis using cell lines expressing human and macaque target antigens.

[0213] Generation of synthetic phage Fab libraries

[0214] The synthetic library is based on the library construction of germline human VH genes, and the germline human VH genes are selected for frequent use in natural libraries and canonical sequence diversity. PCR is used to add synthetic HCDR3 regions to these VH genes. This is carried out using a forward primer that anneals to the framework 1 of the VH gene and comprises a SfI restriction site for cloning. The reverse primer comprises a sequence annealed to the framework 3 of the VH gene, followed by a random sequence for encoding HCDR3 diversity and a framework 4 encoding sequence that also comprises BstEII and XhoI restriction sites for cloning. Based on the frequency of use of amino acid residues at certain positions in HCDR3, the synthetic CDR3 region is completely random or encodes a more restrictive diversity. Using the above-mentioned restriction enzymes, the PCR product encoding the VH gene is cloned into a phage display vector that is fused to the phage M13 gene 3 protein and also comprises a common light chain encoding gene. Large-scale ligation and E. coli TG1 transformation generate large libraries of synthetic Fab fragments displayed on phage, which are used for panning with antigen or cells to identify antigen-specific Fab fragments.

[0215] Generation of an “immune” phage Fab library from tissues of immunized mice by RT-PCR

[0216] Spleens and draining lymph nodes were removed from mice in which significant humoral responses against the corresponding target protein were observed.

[0217] Single cell suspensions were generated from both spleen and inguinal lymph nodes, and these tissues were subsequently lysed in Trizol LS reagent (Thermo Scientific c#10296028) and stored at -80°C until use.

[0218] Inguinal lymph nodes from successfully immunized mice were used to construct an "immune" phage antibody library. RNA was extracted from single cell suspensions of lymphoid tissue. 1 μg of total RNA was used for RT reaction using IgG-CH1 specific primers. The resulting cDNA was then used to amplify a polyclonal library of cDNA encoding VH using internally adapted VH specific primers essentially as described in Marks et al. (J Mol Biol. 1991 Dec 5; 222(3): 581-97). The resulting PCR product was then cloned into a phagemid vector ( Figure 6 ) for displaying Fab fragments on phage as described in de Haard et al. (J Biol Chem. 1999 Jun 25; 274(26): 18218-30), except that the light chain ( Figure 1A and 1B) are identical for each antibody and are encoded by the vector. After ligation, the phagemids were used to transform E. coli TG1 bacteria, and the transformed bacteria were plated onto LB agar plates containing ampicillin and glucose. All phage libraries contained >4×10 5 Bacteria were harvested after overnight growth and used to prepare phage according to established protocols (deHaard et al., J Biol Chem. 1999 Jun 25; 274(26): 18218-30).

[0219] Selection of phage carrying Fab fragments that specifically bind to human target proteins from synthetic and “immune” phage Fab libraries using recombinant proteins

[0220] The generated phage Fab library was used to select target-specific Fab using phage display on directly coated recombinant proteins. For PD-1, huPD-1-Fc (R&D; cat. no. 1086-PD) and huPD-1 biotin (BPSbioscience; cat. no. 71109) were used. For TIM-3, huTIM-3-His (CreativeBiomart; cat. no. HAVCR2-3166H), huTIM-3-Fc (R&D; cat. no. 2365-TM) and maTIM-3-Fc (R&D; 7914-TM) were used.

[0221] For selection using non-biotinylated recombinant proteins ("panning"), proteins were coated onto MAXISORP TM Place MAXISORP TM ELISA plates were blocked with 4% dried skim milk powder (Marvel) in PBS. The phage Fab library was also blocked with 4% Marvel and when Fc-tagged recombinant proteins were used, excess human IgG was also used to deplete Fc region binders before adding the phage library to the coated antigen.

[0222] The phage library was incubated with the coated protein at room temperature for 1.5 hours under shaking conditions. The plate or tube was then washed 15 times with 0.05% Tween-20 in PBS, followed by 5 washes with PBS. The bound phage was eluted using trypsin for 20 minutes, after which trypsin was neutralized with AEBSF trypsin inhibitor (Sigma).

[0223] For selection using biotinylated proteins ("in-solution selection"), neutravidin was coated on the MAXISORP TMPlace MAXISORP TM ELISA plates were blocked with 1% casein in PBS. In parallel, in separate Eppendorf tubes, in 0.5% casein in PBS containing excess human IgG, biotinylated protein and phage Fab library were blocked for 30 minutes. After that, the blocked phage and biotinylated protein were mixed and incubated at room temperature for 2 hours. After that, the mixture was added to the neutravidin coated wells for 20 minutes to capture the phage Fab particles bound to the biotinylated protein. The plate was then washed 15 times with 0.05% Tween-20 in PBS, followed by 5 times with PBS. The bound phage was eluted with trypsin for 20 minutes, after which trypsin was neutralized with AEBSF trypsin inhibitor (Sigma).

[0224] The eluates from both selection strategies ("panning and in solution") were added to E. coli TG-1 and incubated at 37°C for phage infection. The infected bacteria were then plated on agar plates containing ampicillin and glucose and incubated overnight at 37°C. Single clones from the selection output were screened for target binding in ELISA or FACS depending on the target.

[0225] For selections using the synthetic phage Fab library, after rescue of the first round of selection output, a second round of selection was performed using the same protocol as described above for the first round of selection. All three selection antigens used for the TIM-3 round 1 selection were also used for the second round of selection.

[0226] Select phage carrying Fab fragments that specifically bind to human targets from an “immune” phage Fab library using cells stably expressing the target protein

[0227] Phage display was used to select phage Fab libraries produced from mice immunized with target on cells expressing the corresponding targets. Stable cell lines expressing PD-1 or TIM-3 (Table 1) were used for the first round of selection. Cells were blocked with 10% FBS in PBS. After blocking, the rescued phages were incubated with the blocked cells. Cells were incubated with phages at 4 ° C for 1 hour. 1 ml of 10% FBS in PBS was used to wash cells (5 times). The bound phages were eluted with trypsin for 20 minutes, after which trypsin was neutralized with AEBSF trypsin inhibitor (Sigma). The eluate was added to Escherichia coli TG-1 and incubated at 37 ° C for phage infection. Subsequently, the phage-infected bacteria were plated on agar plates containing ampicillin and glucose, and incubated overnight at 37 ° C.

[0228] Screening of target-specific Fab clones in ELISA

[0229] For monoclonal, soluble Fab or phage were prepared (J Mol Biol. 1991 Dec 5; 222 (3): 581-97; J Biol Chem. 1999 Jun 25; 274 (26): 18218-30). The obtained soluble Fab or phage samples were diluted in 4% dry skim milk powder (Marvel) in PBS (blocking buffer) (1: 5 or 1: 10, respectively) and tested in ELISA for binding to wells coated with the same antigen used for selection.

[0230] The bound Fab was detected by staining with anti-myc antibody (Roche; cat. no. 11667203001) diluted 1:1000 in blocking buffer, followed by HRP-conjugated anti-mouse IgG antibody (Jackson Immunoresearch; cat. no. 715-035-150) diluted 1:5000 in blocking buffer. The bound phage was detected by staining with HRP-conjugated monoclonal anti-M13 antibody (GE healthcare; cat. no. 27-9421-01) diluted 1:5000 in blocking buffer.

[0231] After each antibody staining, the wells were washed with PBS-T (PBS-0.05% v / v Tween 20). Bound secondary antibodies were visualized by TMB / H2O2 staining and OD 450nm The staining was quantified by measurement. A clone was considered to be bound to the target when the OD450nm was at least three times higher than the background signal obtained with the negative control Fab.

[0232] The VH encoding cDNAs of all target-specific clones were sequenced. Unique clones were then selected based on sequence identity and cluster analysis based on binding analysis with PD-L1 expressed on cells in FACS, as described below for clones obtained from cell selection output.

[0233] Screening of target-specific Fab clones in FACS

[0234] For monoclones selected on cells expressing the corresponding target, soluble Fab or phage were prepared as described (J Mol Biol. 1991 Dec 5; 222 (3): 581-97; J Biol Chem. 1999 Jun 25; 274 (26): 18218-30). Binding of Fab samples to cells expressing human and macaque targets (Table 1) was tested in FACS by incubation with a mixture of 1:5 diluted Fab samples and 1:1000 diluted anti-myc antibody (Gentaur; cat. no. 04-CMYC-9E10) in FACS buffer (0.5% HI-FBS in PBS). Bound Fab / anti-myc complexes were detected by incubation with APC-conjugated goat anti-mouse IgG antibody (BD Bioscience; cat. no. 550826) diluted 1:500 in FACS buffer.

[0235] The binding of phage samples was tested in FACS by diluting the phage samples 1:3 in blocking buffer and incubating with target expressing cells for 1 hour. The bound phage was detected by staining with biotinylated anti-M13 antibody (Fitzgerald, cat.nr.61R-M101ABTB62-FEZ, 1:125 in FACS buffer, 30 minutes on ice) and PE-labeled streptavidin (Invitrogen, cat.nr.SA1004-4; 1:400 in FACS buffer, 15 minutes on ice). After each antibody incubation, the wells were washed three times with FACS buffer. The stained cells were analyzed using a FACSAccuri C6 instrument (Becton Dickinson). The clone was considered positive when the mean fluorescence intensity was at least three times higher than the background signal obtained with the negative control Fab.

[0236] Example 3

[0237] Characterization of huTIM-3 and huPD-1 specific Fab clones in IgG format

[0238] Human TIM-3 and PD-1 specific Fabs were recloned into IgG format

[0239] Selection of unique clones based on CDR3 sequence and VH germline differences that bind human and macaque target proteins expressed on cells are then recloned into IgG expression plasmids, such as MV1452 ( Figure 7 ), the plasmid comprising a common light chain ( Figure 1 ).

[0240] Expression of bispecific IgG containing human TIM-3 or human PD-1 specific Fab and tetanus toxin specific Fab

[0241] Bispecific antibodies are produced by transiently co-transfecting two plasmids encoding IgG with different VH domains using a patented CH3 modification technique to ensure efficient heterodimerization and bispecific antibody formation. The common light chain present on the two plasmids containing the heavy chain is also co-transfected in the same cell. In our co-pending applications (e.g., WO2013 / 157954 and WO2013 / 157953; incorporated herein by reference), we disclose methods and means for producing bispecific antibodies from a single cell, thereby providing means that are more conducive to the formation of bispecific antibodies relative to the formation of monospecific antibodies. These methods can also be advantageously used in the present invention. In particular, preferred mutations that substantially only generate bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366, e.g., L351K and T366K (numbering according to EU numbering) in the first CH3 domain ("KK variant" heavy chain), and amino acid substitutions at positions 351 and 368, e.g., L351D and L368E ("DE variant" heavy chain), in the second CH3 domain, or vice versa ( Figure 2 ). It has been previously demonstrated in our co-pending applications that negatively charged DE variant heavy chains and positively charged KK variant heavy chains prefer to pair to form heterodimers (so-called "DEKK" bispecific molecules). Due to the strong repulsion between charged residues in the CH3-CH3 interface between the same heavy chains, homodimerization of DE variant heavy chains (DE-DE homodimers) or homodimerization of KK variant heavy chains (KK-KK homodimers) hardly occurs.

[0242] The VH gene encoding the antibody binding to human TIM-3 and PD-1 was cloned into the MV1452 IgG expression vector encoding the positively charged CH3 domain. Figure 8 ) was cloned into the MV1377 IgG expression vector encoding the negatively charged CH3 domain ( Fig. 9 ). To express the TIM-3 and PD-1 antibody panel in IgG format, the entire panel was also cloned into a negatively charged CH3 domain vector to enable the production of bivalent TIM-3 or PD-1 IgG. 293F Freestyle cells suitable for suspension growth were cultured in T125 flasks on a shaking platform until the density reached 3.0 × 10 6 cells / ml. 6 to 0.5×10 6The density of 100 viable cells / ml was inoculated in each well of a 24-deep-well plate. The cells were transiently transfected with a mixture of two plasmids encoding different antibodies cloned into a patented vector system. Seven days after transfection, the cell supernatant was harvested and filtered through a 0.22 μM filter (Sartorius). The sterile supernatant was stored at 4 ° C until the antibody was purified.

[0243] Purification of bispecific IgG

[0244] Purification of IgG was performed on a small scale (<500 μg) using protein A affinity chromatography. Small-scale purification was performed in a 24-well filter plate using filtration under sterile conditions. First, the pH of the culture medium was adjusted to pH 8.0, and then the supernatant containing IgG was incubated with protein A Sepharose CL-4B beads (50% v / v) (Pierce) on a shaking platform at 600 rpm at 25 ° C for 2 hours. Next, the beads were harvested by filtration. The beads were washed twice with PBS at pH 7.4. The bound IgG was then eluted at pH 3.0 with 0.1M citrate buffer, and the eluent was neutralized using Tris pH 8.0 immediately. Buffer exchange was performed by centrifugation using a multi-screen Ultracel 10 multi-plate (Millipore). Finally, the sample was harvested in the PBS at pH 7.4. The IgG concentration was measured using Octet. The protein samples were stored at 4 ° C.

[0245] IgG quantification using Octet

[0246] To determine the amount of purified IgG, total human IgG (Sigma Aldrich, cat. nr. 14506) was used as a standard and the concentration of the antibody was determined by Octet analysis using a protein A biosensor (Forte-Bio, according to the supplier's recommendations).

[0247] Specificity analysis of huTIM-3 and huPD-1 IgG

[0248] The antibodies (bispecific TIM-3xTT and PD-1xTT antibodies) were tested for binding to stable cell lines expressing the relevant human and macaque orthologs (Table 1) and wt cells in FACS. Therefore, cells were harvested and diluted to 10% in FACS buffer (PBS / 0.5% BSA / 0.5 mM EDTA). 6 Add 1×10 cells / ml to each well of a U-bottom 96-well plate. 5 Up to 2×10 5cells. The cells were centrifuged at 300g for 2 minutes at 4°C. The supernatant was discarded by inverting the plate. 50 μl of each IgG sample at a concentration of 10 μg / ml was added and incubated on ice for 1 hour. The cells were centrifuged once, the supernatant was removed, and the cells were washed twice with 150 μl of FACS buffer. 50 μl of goat anti-human IgG PE (Invitrogen) diluted 1:400 was added and incubated on ice for 30 minutes in the dark. After adding FACS buffer, the cells were centrifuged once, the supernatant was removed, and the cells were washed twice with FACS buffer. The cells were analyzed on a FACSCanto flow cytometer (Becton Dickinson) under HTS settings. The binding of the antibody to the cells was evaluated by measuring the mean fluorescence intensity (MFI) of the stained cell population. When the MFI is at least five times the MFI of the same cell population stained with a (negative control) non-binding antibody (against tetanus toxoid), the antibody is considered to be bound to its target.

[0249] Characterization of the huPD-1-specific Fab arm present in the PD-1xTT bispecific IgG based on ligand blocking ability

[0250] The huPD-1 binding clones were tested for their ability to block the interaction of PD-L1 with PD-1. Therefore, PD1-Fc (R&D systems; cat. no. 1086-PD) was coated to maxisorp plates at 1 μg / ml. The coated wells were blocked with 4% BSA in PBS. After this, 0.55 μg / ml of biotinylated PD-L1 (BPS bioscience; cat. no. 71105) was added in the presence or absence of 0.15 to 20 μg / ml of IgG. The bound biotinylated PD-L1 was detected with HRP-conjugated streptavidin (BD bioscience: cat. no. 554066) diluted 1:2000 in blocking buffer. After each incubation step, the ELISA plate was washed three times with PBS-T (PBS-0.05% v / v Tween 20). The bound streptavidin was visualized by TMB / H2O2 staining and the OD 450nm The staining was quantified by measurement. A clone was considered to block the interaction of PD-1 with PD-L1 when the ELISA signal was reduced by more than 70% at a concentration of 10 μg / ml of IgG (PD-1xTT) compared to a control to which a TT-specific competitor antibody was added. For results obtained with a representative selection of PD-1 antibody panels tested as PD-1xTT bispecific molecules, see Fig.10 .

[0251] Identification of huTIM-3-specific Fab arms present in TIM-3xPD-1 IgG based on their ability to block GAL9 interaction with TIM-3

[0252] The wells of a Maxisorp 96-well plate were coated with recombinant human galectin-9 (R&D; cat. no. 2045-GA) at 5 μg / ml in PBS and incubated overnight at 4°C. The wells were washed three times with PBS and then blocked with 3% BSA (blocking buffer) in PBS for one hour at room temperature. After this, the wells were incubated with 0.1875 μg / ml huTIM-3Fc (R&D; cat. no. 2365-TM) diluted in blocking buffer in the presence or absence of 25 μg / ml IgG for one hour at room temperature. The wells were washed three times with PBST (0.05% v / v Tween 20 in PBS) and incubated with biotin-conjugated anti-human Fc antibody (Invitrogen; cat. no. AHI1309) diluted 1:2000 in blocking buffer for one hour. Next, the washing step was repeated and the wells were incubated with HRP-conjugated streptavidin (Becton Dickinson; cat. no. 554066) diluted 1:2000 in blocking buffer at room temperature for 15 minutes. To detect bound streptavidin, the wells were washed three times with PBST and incubated with TMB substrate components A and B (1:1 ratio) (Becton Dickinson; cat. no. 51-2606KC and 51-2607KC, respectively). After 10 minutes, the reaction was stopped with 1M H2SO4 and the OD450 was measured using an ELISA plate reader. ELISA signals below 95% of the control were considered to represent blocking activity (Table 2).

[0253] Affinity ranking of the huTIM-3 and huPD-1 specific Fab arms present in TIM-3xTT and PD-1xTT bispecific IgGs

[0254] Bispecific antibodies that showed binding to the corresponding human and macaque orthologs in FACS were ranked based on the apparent affinity of the two orthologs in FACS. Therefore, stable cell lines expressing the corresponding orthologs (Table 1) were harvested and diluted to 10% in FACS buffer (PBS / 0.5% BSA / 0.5 mM EDTA). 6Cells / ml. The cells were centrifuged at 300g for 2 minutes at 4°C. The supernatant was discarded by inverting the plate. 50 μl of each IgG sample in an 11-step, 2-fold dilution series (10 to 0.01 μg / ml) was added and incubated on ice for 1 hour. The cells were centrifuged once, the supernatant was removed, and the cells were washed twice with 150 μl of FACS buffer. 50 μl of goat anti-human IgG PE (Invitrogen) diluted 1:400 was added and incubated on ice for 30 minutes in the dark. After adding FACS buffer, the cells were centrifuged once, the supernatant was removed, and the cells were washed twice with FACS buffer. Cells were analyzed under HTS settings on a FACSCanto flow cytometer (Becton Dickinson). The binding of antibodies to cells was evaluated by measuring the mean fluorescence intensity (MFI) of the stained cell population. An antibody is considered bound to its target when the MFI is at least five times the MFI of the same cell population stained with a (negative control) non-binding antibody (against tetanus toxoid).

[0255] Reference Antibodies

[0256] Antibodies that inhibit PD-1 function are known in the art. Monoclonal bivalent antibodies were constructed according to published information and expressed in CHO-S cells. The anti-PD-1 antibody nivolumab was produced based on information disclosed in CA 02607147.

[0257] PD-1 / PD-L1 Blockade Reporter Assay

[0258] The PD-1 / PD-L1 blocking reporter assay used was developed by Promega and is based on a dual cell system; CHO cells expressing PD-L1 and T cell activators, and Jurkat / NFAT-RE reporter cell lines overexpressing PD.1. The PD-1 / PD-L1 blocking reporter assay was performed using Promega's thawing and use format. Cells expressing PD-L1 (cat.no.C187103) were thawed in 14.5 ml of cell recovery medium (DMEM / F12 containing 10% FBS). Next, 50 μl of cell suspension was added to the inner well of a 96-well half-area plate (Coming, cat.no.3688). The plate was incubated overnight at 37 ° C and 5% CO in 95% relative humidity. The next day, the culture medium was removed and 20 μl of the test antibody in the assay medium (RPMI 1640 containing 4% FBS) was added to each well in a serial dilution (starting concentration 10 g / ml). Each plate contains a serial dilution of a negative (Ctrl Ab) and a positive control antibody (nivolumab) used as a reference control. PD-1 effector cells (cat no. C187105) were thawed in 5.9 ml of assay medium and 20 μl of cell suspension was added to each well. The plate was incubated at 37 ° C, 5% CO in 95% relative humidity for 6 hours or overnight. 40 μl of luciferase (Bio-Glo Luciferase Assay System, cat. no. G794L) was added on the second day, and the amount of luciferase activity was measured using a BioTek Synergy 2 multi-mode microplate reader. Efficacy was measured as luciferase activity compared to a negative control antibody.

[0259] Screening of PD-1 antibody panel

[0260] VHs from the PD-1 antibody panel were produced in a 24-well format and tested as bivalent antibodies in a PD-1 / PD-L1 blocking reporter assay in a semi-logarithmic continuous titration (starting concentration 10 μg / ml) to rank the antibodies for blocking potency compared to nivolumab. Based on the activity data, antibodies were selected from the PD-1 antibody panel for subsequent PD1xLAG-3 bispecific screening. The activity of the selected candidates in the reporter assay is shown in Table 3. The PD-1 Fab group consists of functionally active variants in two antibody clusters, A and B.

[0261] The galectin-9 blocking Fab arm was combined with the PD-1 Fab arm and tested for its ability to restore functionality of exhausted antigen-specific T cells.

[0262] Example 4

[0263] Bispecific PD-1 x TIM-3 antibody enhances IFNγ production by CD4+ T cells in mixed lymphocyte reactions born.

[0264] Mixed lymphocyte reaction (mixed lymphocyte reaction, MLR) assay is commonly used to understand the effect of antibodies on T cell activation and proliferation. Such an assay helps to understand whether such a compound will affect the potential of T cells to trigger responses in the tumor microenvironment. Here, we use an allogeneic MLR protocol with immature DCs to determine that the bispecific PD-1x TIM-3 antibody enhances the ability of IFNγ produced by CD4+T cells compared to the ability of the benchmark reference antibody. The responsiveness of T cells is quantified by measuring the level of IFNγ in the culture supernatant.

[0265] To this end, human peripheral blood mononuclear cells (PBMC) from healthy donors were prepared from buffy coats. CD14+ cells (EasySep Stemcell, lot 16C69672) were separated using magnetic activated cell sorting (MACS) and these cells were cultured in differentiation medium for seven days to prepare immature monocyte-derived dendritic cells (Mo-DC). Using a T cell isolation kit (EasySep Stemcell, lot 16D70573) for obtaining untouched T cells, responsive T cells derived from donors different from those used for Mo-DC were prepared from cryopreserved PBMCs on the day required. Six separate MLRs were performed to provide biological repeats.

[0266] For the assay, 1×10 4 Immature Mo-DCs and 1×10 5 CD4+ T cells were co-cultured for 4 days. The culture was performed in triplicate. Supernatants were collected at the end of the culture period and evaluated for IFNγ by ELISA (R&D BioTechne, lot number 342687) according to the manufacturer's instructions and the plate was read at 450 nm.

[0267] result

[0268] The MLR study included an experimental group of Tim3 / PD1 bispecific (PB16465=MF7677 x MF6930) and 3 isotype control groups (monospecific antibodies against TIM-3 (surrogate antibody), tetanus and mIgG1). Single cell controls and vehicle controls were also included.

[0269] The cultures were performed in triplicate to provide technical replicates. At the end of the 4-day culture period, supernatants were collected and subjected to ELISA according to the manufacturer's instructions to assess the effect of the antibodies on IFN-γ production, and the plates were read at 450 nm.

[0270] CD14+ cells were sorted at DO and cultured for 7 days, immature DCs were used at D7, and mature DCs were obtained by culturing in maturation medium for another 3 days. CD14 positivity was assessed at DO to confirm the purity of the initial sorting, and CD14 positivity was assessed in immature and mature DCs at D7 and D10, respectively, to confirm the downregulation of CD14 to indicate differentiation to Mo-DCs (data not shown). Viability and activation markers (CD80, CD83, and CD86) were also assessed for both immature and mature DCs to confirm differentiation and maturation. Mo-DCs (immature or mature) were cultured with responsive T cells for 4 days, after which supernatants were collected and ELISA was performed to assess the effect of the tested antibodies on IFN-γ production.

[0271] exist Fig.11 The results of the test are shown in . The bispecific antibody specific for PD-1 / TIM-3 performed better in IFN-γ production by CD14+ T cells in MLR relative to the control and Tim3-surrogate monospecific antibodies.

[0272] Table 1: Expression constructs for each target used for DNA immunization (based on pVAX1 vector) and for the generation of stable Freestyle293F or CHO-S cell lines (based on pIRES-neo3 vector or similar)

[0273]

[0274] hu = human, ma = macaque, NA = not applicable

[0275] Table 2

[0276] PB# First Fab Arm Second Fab Arm Comparison OD PB16464 MF6930 MF7676 1.198 PB16465 MF6930 MF7677 0.764 PB16466 MF6930 MF7678 0.872 PB16467 MF6930 MF7679 1.187 ctrl mAb 1.151

[0277] Galectin-9 blocking activity of TIM-3xPD-1 bispecific antibodies as determined by ELISA compared to a negative control antibody (Ctrl mAb). The composition of the PB and Fab arms is shown.

[0278] Table 3

[0279] Functional activity of the PD-1 Fab arm measured as a bivalent antibody in a PD-1 / PD-L1 blocking reporter assay compared to the positive control nivolumab. Variants of the same cluster (B) showing different PD-1 blocking activities were tested.

[0280] clone CDR3 Clustering %Activity of positive control MF6226 GGYSGYGGDSFDL A 47.77614647 MF6256 GTVEATLLFDF B 57.85260834 MF6930 GTVEATLLFDY B 51.50445453

Claims

1. An in vitro method for interfering with PD-1 and TIM-3-mediated inhibition in programmed cell death protein 1 (PD-1) and / or T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3)-positive cells, the method comprising contacting the cells with a bispecific antibody or a functional portion thereof, thereby inhibiting PD-1 and / or TIM-3-mediated activity in the cells, the bispecific antibody or a functional portion thereof comprising: - a variable domain that binds to the extracellular portion of PD-1, and - a variable domain that binds to the extracellular part of TIM-3; The variable domain that binds to the extracellular part of PD-1 comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are shown in the following sequences: They are FYFWS, YIYYSGSANFNPSLKS and GGYTGYGGDWFDP. SYVIS, MIIPVFDTSSYEKKFQG, and GTVEATLLFDF, NYVIS, MIIPVFETATYEKKFQG, and GTVEATLLFDY, or SYAMS, TISGGGANIYYADSVKG, and PYGSGYFDV, respectively; The variable domain that binds to the extracellular portion of TIM-3 comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are shown in the following sequences: They are TSGVGVG, FIYSNDDKRYSPSLKS and RRGWGPFDY, respectively, and the variable domain that binds to the extracellular part of PD-1 and TIM-3 comprises a light chain variable region, the CDR1 sequence of the light chain variable region is QSISSY, the CDR2 sequence is AAS, and the CDR3 sequence is QQSYSTPPT.

2. The method of claim 1, wherein the binding of the PD-1 binding variable domain to PD-1 blocks the binding of PD-1 to PD-L1 and / or PD-L2.

3. An in vitro method for stimulating the formation, stability and / or activity of an immune synapse, comprising providing a system comprising at least two cells capable of associating with each other via an immune synapse, and providing to the system a bispecific antibody or a functional portion thereof comprising: - a variable domain that binds to the extracellular portion of PD-1, and - a variable domain that binds to the extracellular part of TIM-3; The variable domain that binds to the extracellular part of PD-1 comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are shown in the following sequences: They are FYFWS, YIYYSGSANFNPSLKS and GGYTGYGGDWFDP. SYVIS, MIIPVFDTSSYEKKFQG, and GTVEATLLFDF, NYVIS, MIIPVFETATYEKKFQG, and GTVEATLLFDY, or SYAMS, TISGGGANIYYADSVKG, and PYGSGYFDV, respectively; The variable domain that binds to the extracellular portion of TIM-3 comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are shown in the following sequences: They are TSGVGVG, FIYSNDDKRYSPSLKS and RRGWGPFDY, respectively, and the variable domain that binds to the extracellular part of PD-1 and TIM-3 comprises a light chain variable region, the CDR1 sequence of the light chain variable region is QSISSY, the CDR2 sequence is AAS, and the CDR3 sequence is QQSYSTPPT.

4. A bispecific antibody or a functional portion thereof, comprising a variable domain that binds to the extracellular portion of PD-1 and a variable domain that binds to the extracellular portion of TIM-3; The variable domain that binds to the extracellular part of PD-1 comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are shown in the following sequences: They are FYFWS, YIYYSGSANFNPSLKS and GGYTGYGGDWFDP. SYVIS, MIIPVFDTSSYEKKFQG, and GTVEATLLFDF, NYVIS, MIIPVFETATYEKKFQG, and GTVEATLLFDY, or SYAMS, TISGGGANIYYADSVKG, and PYGSGYFDV, respectively; The variable domain that binds to the extracellular portion of TIM-3 comprises a heavy chain variable region, wherein the heavy chain variable region has CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are shown in the following sequences: They are TSGVGVG, FIYSNDDKRYSPSLKS and RRGWGPFDY, respectively, and the variable domain that binds to the extracellular part of PD-1 and TIM-3 comprises a light chain variable region, the CDR1 sequence of the light chain variable region is QSISSY, the CDR2 sequence is AAS, and the CDR3 sequence is QQSYSTPPT.

5. The antibody or functional portion thereof of claim 4, wherein the variable domain that binds to PD-1 blocks the binding of PD-1 to PD-L1 and / or PD-L2.

6. The antibody or functional portion of claim 4, wherein the variable domain that binds to TIM-3 blocks the binding of TIM-3 to galectin-9.

7. The antibody or functional portion thereof of claim 4, wherein the variable domain that binds to the extracellular portion of PD-1 is defined as a variable region that, when in a bivalent monospecific antibody comprising two of said variable domains that bind to PD-1, inhibits PD-1 / PD-L1 inhibition in Jurkat cells in the range of 20% to 150% compared to the inhibition obtained with the antibody nivolumab on Jurkat cells.

8. The antibody or functional part thereof of claim 4, wherein the variable domain that binds to the extracellular part of PD-1 comprises a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to any one of SEQ ID NO: 43, 46, 47 or 52, and the amino acid sequence has at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the heavy chain variable region.

9. The antibody or functional part thereof of claim 4, wherein the variable domain that binds to the extracellular portion of PD-1 comprises a heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to any one of SEQ ID NO: 43, 46, 47 or 52.

10. The antibody or functional part thereof of claim 8, wherein the variable domain that binds to the extracellular part of TIM-3 comprises a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to any one of SEQ ID NO: 55 or 56, wherein the amino acid sequence has at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region.

11. The antibody or functional part thereof according to claim 9, wherein the variable domain that binds to the extracellular part of TIM-3 comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence of the heavy chain variable region according to any one of SEQ ID NO: 55 or 56.

12. The antibody or functional portion thereof of claim 4, wherein the antibody comprises: A variable domain that binds to the extracellular part of TIM-3, comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to any one of SEQ ID NO: 55 or 56, the amino acid sequence having at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region; and A variable domain that binds to the extracellular portion of PD-1, comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to SEQ ID NO: 47, wherein the amino acid sequence has at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region.

13. The antibody or functional portion thereof of claim 4, wherein the antibody comprises: A variable domain that binds to the extracellular part of TIM-3, comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to any one of SEQ ID NO: 55 or 56, the amino acid sequence having at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region; and A variable domain that binds to the extracellular portion of PD-1, comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to SEQ ID NO: 46, wherein the amino acid sequence has at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region.

14. The antibody or functional portion thereof of claim 4, wherein the antibody comprises: A variable domain that binds to the extracellular part of TIM-3, comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to any one of SEQ ID NO: 55 or 56, the amino acid sequence having at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region; and A variable domain that binds to the extracellular portion of PD-1, comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to SEQ ID NO: 43, wherein the amino acid sequence has at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region.

15. The antibody or functional portion thereof of claim 4, wherein the antibody comprises: A variable domain that binds to the extracellular part of TIM-3, comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to any one of SEQ ID NO: 55 or 56, the amino acid sequence having at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region; and A variable domain that binds to the extracellular portion of PD-1, comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of a heavy chain variable region according to SEQ ID NO: 52, wherein the amino acid sequence has at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof relative to the amino acid sequence of the heavy chain variable region.

16. The antibody or functional part thereof according to claim 10, comprising a light chain variable region having a sequence that is at least 90% identical to the amino acid sequence according to SEQ ID NO:

26.

17. The antibody or functional part thereof according to claim 11, comprising a light chain variable region, the amino acid sequence of which is the amino acid sequence according to SEQ ID NO:

26.

18. The method of any one of claims 1 to 3, wherein the antibody or a functional part thereof is an antibody or a functional part thereof according to any one of claims 4 to 17.

19. The antibody or functional part thereof according to any one of claims 4 to 17 for use as a medicament.

20. The antibody or functional part thereof according to any one of claims 4 to 19, for use in a method for treating cancer or infection by pathogenic viruses or parasites.

21. A composition or kit comprising at least one antibody or a functional part thereof according to any one of claims 4 to 17.

22. A pharmaceutical composition comprising at least one antibody or a functional part thereof according to any one of claims 4 to 17, and a pharmaceutically acceptable carrier.

23. A pharmaceutical composition comprising at least one antibody according to any one of claims 4 to 17 and a pharmaceutically acceptable excipient.

24. A pharmaceutical composition comprising at least one antibody according to any one of claims 4 to 17 and a pharmaceutically acceptable diluent.

25. A nucleic acid molecule or a collection of nucleic acid molecules encoding the antibody or functional part according to any one of claims 4 to 17.

26. A vector comprising the nucleic acid molecule according to claim 25.

27. An isolated or recombinant cell comprising one or more nucleic acid molecules according to claim 25 or a vector according to claim 26.

28. Use of a therapeutically effective amount of an antibody or functional portion according to any one of claims 4 to 17, a composition or kit according to claim 21, a pharmaceutical composition according to any one of claims 22 to 24, a nucleic acid molecule according to claim 25, or a vector according to claim 26 for the preparation of a medicament for treating cancer or pathogen infection, wherein the treatment of cancer or pathogen infection comprises administering to a subject in need thereof.

29. A method for producing the antibody or functional portion thereof according to any one of claims 4 to 17 from a single cell, wherein the antibody or functional portion thereof comprises two CH3 domains capable of forming an interface, the method comprising providing: - A cell having: a) a first nucleic acid molecule encoding an IgG heavy chain that specifically recognizes the extracellular portion of PD-1 and comprises a first CH3 domain; b) a second nucleic acid sequence encoding an IgG heavy chain that specifically recognizes the extracellular portion of TIM-3 and comprises a second CH3 domain, wherein the nucleic acid sequence encodes residues for preferential pairing of the first and second CH3 domains; and (c) a third nucleic acid sequence encoding a common light chain; The method further comprises the following steps: The cells are cultured and the nucleic acid sequence is allowed to be expressed, and the antibody or a functional portion thereof is harvested from the culture.

30. The method of claim 29, wherein the third nucleic acid sequence encoding a common light chain is a rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01.

31. The method of claim 29 or 30, wherein the first nucleic acid encodes the first CH3 domain comprising the amino acid substitutions L351K and T366K numbered according to EU numbering, and wherein the second nucleic acid encodes the second CH3 domain comprising the amino acid substitutions L351D and L368E numbered according to EU numbering, the method further comprising the following steps: The cells are cultured and the nucleic acid sequence is allowed to be expressed, and the antibody or a functional portion thereof is harvested from the culture.

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