EA044665B1 - PHARMACEUTICAL COMPOSITION CONTAINING ANTI-LAG-3 ANTIBODY AND ANTI-PD-1 ANTIBODY - Google Patents

Description

State of the art

Therapeutic antibodies are particularly one of the fastest growing segments of the pharmaceutical industry. To maintain efficacy (i.e., potency) and minimize immunogenicity, antibodies and other protein drugs must be protected from physical and chemical degradation during manufacture and storage. Indeed, one of the major challenges in antibody drug development is the potential immunogenic response when administered to a subject, which may result in rapid clearance or even cause life-threatening side effects, including anaphylactic shock. The immunogenicity of an antibody is influenced by various factors, such as its physiochemical properties (eg, purity, stability, or solubility), clinical factors (eg, dose, route of administration, disease heterogeneity, or patient characteristics), and concomitant treatment with other agents (Swann et al. ( 2008) Curr Opinion Immunol 20:493-499).

Antibody immunogenicity and/or loss of antibody activity is often associated with deamidation. Deamidation is a chemical degradation process that occurs spontaneously in proteins (such as antibodies). Deamidation removes the amide functional group from an amino acid residue such as asparagine and glutamine, thereby damaging its amide-containing side chains. This in turn causes structural and biological changes throughout the protein, thereby creating heterogeneous forms of the antibody. Deamidation is one of the most common post-translational modifications that occur in recombinantly produced therapeutic antibodies. For example, heterogeneity in the heavy chain of the monoclonal antibody h1B4 (humanized anti-CD18 antibody) due to deamidation during cell culture was noted by Tsai et al. (Pharm Res 10(11): 1580 (1993)). In addition, the reduction/loss of biological activity due to deamidation has been a known problem in the past. For example, Kroon et al. characterized various deamidation sites in the therapeutic antibody OKT3 and reported that samples from a batch of OKT3 (14 months to 3 years old) had less than 75% activity (Pharm Res 9(11): 1386 (1992), p. 1389, second column) . Additionally, OKT3 samples with large amounts of oxidized peptides in their maps had significantly reduced activity in the antigen binding efficiency assay (p. 1390, first column).

The authors concluded that specific sites of chemical modification that occur during OKT3 storage were identified by peptide mapping and correlated with observed changes in chemical and antibody bioassays (p. 1392, first column). Loss of biological activity has also been noted for a number of other deamidated therapeutic proteins, including recombinant human DNase (Cacia et al. (1993) J. Chromatogr. 634:229-239) and recombinant soluble CD4 (Teshima et al. (1991) Biochemistry 30 :3916-3922).

Overall, deamidation poses a significant and unpredictable challenge to the pharmaceutical industry. Efforts related to monitoring variability caused by deamidation in antibody drugs in particular, as well as FDA-related concerns about this variability, increase costs and delay clinical trials. Moreover, related modifications, including changing conditions (e.g., temperature, pH, and cell type) associated with recombinant production and/or alteration of amino acids that are susceptible to deamidation (e.g., site-directed mutagenesis), can negatively affect stability and activity, especially when making changes to the complementarity determining regions (CDRs) of the antibody. Accordingly, there is a need for more stable versions of therapeutic antibodies.

The essence of the invention

The present invention provides isolated monoclonal antibodies (eg, human monoclonal antibodies) that bind LAG-3 (eg, human LAG-3) and have optimized physical stability compared to previously described anti-LAG-3 antibodies. In particular, the invention relates to a modified form of antibody 25F7 (US 2011/0150892 Al), which has significantly increased thermal and chemical stability compared to the unmodified antibody. Specifically, by modifying the critical binding region of the heavy chain CDR2 domain of antibody 25F7, it was shown that the modified antibody exhibited significantly higher physical and thermal stability, reduced levels of deamidation, higher thermal reversibility, and lower aggregation. At the same time, it was unexpectedly found that the modified antibody retains the same high binding affinity to human LAG-3 and the functional activity of the unmodified antibody, including the ability to inhibit LAG-3 binding to MHC class II molecules and stimulate antigen-specific T-cell responses. The combined significant increase in stability and retention of binding efficiency/biological activity of the modified antibody was unexpected, especially in light of the criticality of the CDR regions for antibody function.

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The antibodies of the invention can be used for a variety of purposes, including detecting LAG-3 protein and stimulating antigen-specific T-cell responses in subjects with tumors or viruses.

Accordingly, in one aspect, the invention provides an isolated monoclonal antibody (e.g., a human antibody) or an antigen-binding portion thereof having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In another embodiment, the antibody also includes a light chain variable region including the amino acid sequence SEQ ID NO:14. In another embodiment, the antibody or antigen-binding portion thereof comprises the CDR1, CDR2 and CDR3 regions of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:12 (eg, SEQ ID NO: 15, 16 and 17, respectively). In another embodiment, the antibody also includes the CDR1, CDR2 and CDR3 regions of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (eg, SEQ ID NO: 18, 19 and 20, respectively). In a preferred embodiment, the antibody has improved physical properties (ie, thermal and chemical stability) compared to the 25F7 antibody, while maintaining at least the same human LAG-3 binding affinity as 25F7. For example, the antibody has reduced sequence variability in the heavy chain CDR2 region due to deamidation compared to antibody 25F7, such as approximately 2.5% or less amino acid sequence modification after 12 weeks at 4°C (i.e., in real-time stability assays as described here) and/or approximately 12.0% or less amino acid sequence modification after 12 weeks at 40°C (i.e., under accelerated stress conditions as described herein), while maintaining human LAG-3 binding affinity at levels at least a KD of about 1 x 10 ^-7 M or less (more preferably a KD of 1 x 10 ^-8 M or less, a KD of 5 x 10 ^-9 M or less, or a _KD of 1 x 10 ^-9 M or less). In another embodiment, the antibody has a thermal reversibility of about 40% in PBS at pH 8.0.

In another embodiment, the antibody has a higher melting point (indicating greater overall stability in vivo) compared to the unmodified antibody (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 2:361-71). In one embodiment, the antibody has a T _M1 (initial folding temperature) value greater than 60°C, such as greater than 65°C or greater than 70°C. The melting point of an antibody can be determined using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) or the circular dichroism method (Murray et al. ( 2002) J Chromatogr Sci 40:343-9).

In another embodiment, the antibody is characterized by its resistance to rapid degradation. Antibody decay can be determined by capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

In another embodiment, the antibody has minimal aggregation properties, such as 25% or less aggregation, such as 20% or less, 15% or less, 10% or less, 5% or less, or 4% or less. Aggregation may result in the triggering of an undesirable immune response and/or altered or undesirable pharmacokinetic properties. Aggregation can be measured using a variety of techniques, including size exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

In another embodiment, the antibody also has at least one of the following properties:

(a) binding to monkey LAG-3;

(b) lack of binding to mouse LAG-3;

(c) inhibiting the binding of LAG-3 to class II major histocompatibility complex molecules; and (d) stimulating immune responses, in particular antigen-specific T-cell responses.

Preferably, the antibody has at least two of the properties of (a), (b), (c) and (d). More preferably, the antibody has at least three of properties (a), (b), (c) and (d). Even more preferably, the antibody has all four of properties (a), (b), (c) and (d).

In another embodiment, the antibody stimulates an antigen-specific T cell response, such as the production of interleukin-2 (IL-2) in an antigen-specific T cell response. In other embodiments, the antibody stimulates an immune response, such as an antitumor response (eg, inhibition of tumor growth in an in vivo xenograft tumor model) or an autoimmune response (eg, development of diabetes in NOD mice).

In another embodiment, the antibody binds to a human LAG-3 epitope comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 21). In another embodiment, the antibody binds a human LAG-3 epitope comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 22) or PAAPSSWG (SEQ ID NO: 23).

In other embodiments, the antibody stains pituitary tissue using immunostaining

- 2 044665 stochemistry or does not stain pituitary tissue using immunohistochemistry. Antibodies of the invention may be full-length antibodies, for example of the IgG1, IgG2 or IgG4 isotype, optionally with a serine-to-proline mutation at the heavy chain constant region hinge region (at a position corresponding to position 241, as described by Angal et al. (1993) Mol. Immunol. 30:105-108), so that the heterogeneity of the disulfide bridge between the heavy chains is reduced or eliminated. In one aspect, the constant region isotype is IgG4 with a mutation at amino acid residues 228, such as S228P. Alternatively, antibodies may be antibody fragments such as Fab, Fab' or Fab'2 fragments or single chain antibodies.

In another aspect of the invention, the antibody (or an antigen-binding portion thereof) is part of an immunoconjugate that includes a therapeutic agent, such as a cytotoxin or radioactive isotope, bound to the antibody. In another aspect, the antibody is part of a bispecific molecule that includes a second functional group (eg, a second antibody) having a different binding specificity than the antibody or antigen-binding portion thereof. Also included within the scope of the invention are compositions comprising antibodies or antigen-binding portions thereof, immunoconjugates or bispecific molecules of the invention, optionally associated with a pharmaceutically acceptable carrier. Also included within the scope of the invention are nucleic acid molecules of the invention encoding antibodies or antigen binding portions thereof (eg, variable regions and/or CDRs), as well as expression vectors comprising these nucleic acids and host cells comprising these expression vectors. Also included within the scope of the invention are methods for producing anti-LAG-3 antibodies using host cells comprising these expression vectors, which may include the steps of (i) expressing the antibody in the host cell and (ii) isolating the antibody from the host cell.

In another aspect, the invention relates to methods of stimulating immune responses using anti-LAG-3 antibodies of the invention. In one embodiment, the method includes stimulating an antigen-specific T cell response by contacting the T cells with an antibody of the invention such that an antigen-specific T cell response is stimulated. In a preferred embodiment, interleukin-2 production is stimulated by an antigen-specific T cell. In another embodiment, the subject is a subject with a tumor, and an immune response against the tumor is stimulated. In another embodiment, the subject is a subject with a virus, and an immune response against the virus is stimulated.

In another embodiment, the invention provides a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof of the invention such that the growth of a tumor in the subject is inhibited. In another embodiment, the invention provides a method of treating a viral infection in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof of the invention such that the viral infection is treated in the subject. In another embodiment, the methods include administering a composition, bispecific antibody, or immunoconjugate of the invention. In another embodiment, the invention provides a method of stimulating an immune response in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof of the invention and at least one additional immunostimulatory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody such that an immune response is stimulated in the subject, for example to inhibit tumor growth or promote an antiviral response. In one embodiment, the additional immunostimulatory antibody is an anti-PD-1 antibody. In another embodiment, the additional immunostimulatory agent is an anti-PD-LI antibody. In another embodiment, the additional immunostimulating agent is an anti-CTLA-4 antibody. In another embodiment, an antibody or antigen-binding portion thereof of the invention is administered with a cytokine (eg, IL-2 and/or IL-21) or a co-stimulatory antibody (eg, anti-CD137 and/or anti-GITR antibody). Antibodies can be, for example, human antibodies, chimeric or humanized antibodies.

In another aspect, the invention relates to anti-LAG-3 antibodies and compositions of the invention for use in the above methods or for the manufacture of a medicament for use in the above methods (eg, for treatment).

Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which are not to be construed as limiting. The contents of all references, Genbank databases, patents and published patent applications cited in this application are incorporated herein by reference in their entirety.

Brief description of the figures

In fig. 1A shows the nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of the heavy chain variable region of human monoclonal antibody 25F7. CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6) and CDR3 (SEQ ID NO:7) regions are shown with a broken line and germline derivations V, D and J are indicated. CDR regions are shown with a broken line with

- 3 044665 using the Kabat system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

In fig. 1B shows the nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of the kappa light chain variable region of the human monoclonal antibody 25F7. The CDR1 (SEQ ID NO:8), CDR2 (SEQ ID NO:9) and CDR3 (SEQ ID NO:10) regions are indicated by a broken line and the germline derivations V and J are indicated. The full-length amino acid sequences of the heavy and light chain of the 25F7 antibody are shown in SEQ ID NO: 32 and 34, respectively. In fig. 2A shows the nucleotide sequence (SEQ ID NO: 12) of the heavy chain variable region of the monoclonal antibody LAG-3.5. CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO: 16) and CDR3 (SEQ ID NO: 17) regions are outlined. The full-length amino acid sequences of the LAG3.5 antibody heavy and light chain are shown in SEQ ID NO: 35 and 37, respectively.

In fig. 2B shows the nucleotide sequence (SEQ ID NO: 13) and amino acid sequence (SEQ ID NO: 14) of the kappa light chain variable region of the monoclonal antibody LAG3.5. CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19) and CDR3 (SEQ ID NO: 20) regions are outlined.

In fig. 3 shows the CDR2 amino acid sequences of the heavy chain variable region of LAG-3 variants LAG3.5 (SEQ ID NO: 42), LAG3.6 (SEQ ID NO: 43), LAG3.7 (SEQ ID NO: 44) and LAG3.8 ( SEQ ID NO: 45) compared to the CDR2 amino acid sequence of the heavy chain variable region of antibody 25F7 (LAG3.1) (SEQ ID NO: 41) and the corresponding human germline sequence (SEQ ID NO: 27). The LAG3.5 heavy chain variable region CDR2 differs from the 25F7 heavy chain variable region CDR2 by arginine (R) at position 54 (versus asparagine (N)) and serine (S) at position 56 (versus asparagine (N)). The remaining CDR LAG3.5 25F7 are identical. In fig. 3 also shows SEQ ID NO: 40.

Fig. 4A and 4B are graphs showing the binding activity (EC ₅₀ and affinity, respectively) of antibodies LAG3.1 (25F7), LAG3.2, LAG3.5, LAG3.6, LAG3.7 and LAG3.8 to activated CD4+ T- human cells. In fig. 4B shows SEQ ID NOS 41, 42, 45, 44 and 43, respectively, in order.

Fig. 5A, B, C, D and E are graphs showing thermal melting curves (ie, thermal stability) of antibodies LAG3.1 (25F7), LAG3.5, LAG3.6, LAG3.7 and LAG3.8, respectively.

Fig. 6A, B, C, D and E are graphs showing thermal reversibility (ie thermal stability) curves of antibodies LAG3.1 (25F7), LAG3.5, LAG3.6, LAG3.7 and LAG3.8, respectively.

Fig. 7 is a graph showing the binding activity of antibodies LAG3.1 (25F7) and LAG3.5 to activated human CD4+ T cells and antigen binding (Biacore).

In fig. Figure 8 shows peptide mapping results using mass spectrometry (chemical modifications/molecular stability) for antibodies LAG3.1 (25F7) and LAG3.5, reflecting deamidation and isomerization after culture for 5 days under accelerated stress conditions as described here.

In fig. 8 shows SEQ ID NO: 46-52, respectively, in order.

Fig. 9 is a graph comparing the hydrophobicity profiles of LAG3.1 (25F7) and LAG3.5 antibodies.

Fig. 1OA-D are graphs comparing the affinity and physical stability (i.e. thermal and chemical stability) of antibodies LAG3.1 and LAG3.5 at 4°C and 40°C, i.e. both accelerated stress conditions and real-time stability assays as described here.

Fig. 11A and B are graphs comparing the percentage of modification of the amino acid sequences of antibodies LAG3.1 and LAG3.5 at 4°C and 40°C.

Detailed Description of the Invention

One aspect of the invention includes an isolated monoclonal antibody or antigen binding portion thereof that binds human LAG-3 comprising heavy and light chain variable regions, wherein the heavy chain variable region includes the CDR1, CDR2 and CDR3 regions of the heavy chain variable region SEQ ID NO: 12.

In one aspect, the antibody or antigen binding portion thereof is characterized in that the heavy chain CDR1, CDR2 and CDR3 regions comprise the amino acid sequences of SEQ ID NO: 15, 16 and 17, respectively.

In one aspect, the antibody or antigen binding portion thereof is characterized in that the light chain variable region includes the CDR1, CDR2, and CDR3 light chain variable region regions of SEQ ID NO: 14.

In one aspect, the antibody or antigen binding portion thereof is characterized in that the light chain CDR1, CDR2 and CDR3 regions comprise the amino acid sequences of SEQ ID NO: 18, 19 and 20, respectively.

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In one aspect, the antibody or antigen binding portion thereof is characterized in that the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12.

In one aspect, the antibody or antigen binding portion thereof is characterized in that the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14.

One aspect of the invention includes an isolated monoclonal antibody or antigen-binding portion thereof that binds human LAG-3, comprising CDR1, CDR2 and CDR3 heavy and light chain regions comprising the amino acid sequences of SEQ ID NO: 15, 16, 17 and SEQ ID NO: 18 , 19 and 20 respectively.

One aspect of the invention includes an isolated monoclonal antibody or antigen binding portion thereof that binds human LAG-3 comprising heavy and light chain variable regions comprising the amino acid sequences of SEQ ID NO: 12 and 14, respectively.

One aspect of the invention includes an antibody or antigen-binding portion thereof according to any of the preceding claims, having one or a combination of the following properties:

(a) monkey LAG-3 binding;

(b) lack of mouse LAG-3 binding;

(c) binding of LAG-3 to class II major histocompatibility complex molecules;

(d) inhibiting the binding of LAG-3 to class II major histocompatibility complex molecules; or (e) stimulating an immune response.

In one aspect, the antibody or antigen-binding portion thereof stimulates the production of interleukin-2 (IL-2) in an antigen-specific T cell response.

In one aspect, the antibody or antigen-binding portion thereof stimulates an antitumor immune response.

In one aspect, the antibody or antigen-binding portion thereof binds a human LAG-3 epitope comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 21).

In one aspect, the antibody or antigen-binding portion thereof binds a human LAG-3 epitope comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 22) or PAAPSSWG (SEQ ID NO: 23).

In one aspect, the antibody or antigen-binding portion thereof binds human LAG-3 with a KD value of 0.27 x 10 ^-9 M or less.

In one aspect, the antibody or antigen-binding portion thereof is a human, humanized, or chimeric antibody.

In one aspect, the antibody or antigen-binding portion thereof is of the IgG1, IgG2, or IgG4 isotype.

In one aspect, the antibody or antigen-binding portion thereof is an antibody fragment or single chain antibody.

One aspect of the invention includes a bispecific molecule comprising an antibody or antigen-binding portion thereof and a second antibody or antigen-binding portion thereof.

One aspect of the invention includes an immunoconjugate comprising an antibody or antigen-binding portion thereof associated with a therapeutic agent.

One aspect of the invention includes an immunoconjugate, characterized in that the therapeutic agent is a cytotoxin or radioactive isotope.

One aspect of the invention includes a composition comprising an antibody or antigen-binding portion thereof, a bispecific molecule or immunoconjugate, and a pharmaceutically acceptable carrier.

In one aspect, the composition further comprises an anticancer agent. In one aspect, the composition is characterized in that the agent is an antibody or a chemotherapeutic agent.

One aspect of the invention includes an isolated nucleic acid encoding a variable region of the heavy and/or light chain of an antibody or antigen-binding portion thereof, as defined above.

One aspect of the invention includes an expression vector comprising a nucleic acid as defined above.

One aspect of the invention includes a host cell including an expression vector as defined above.

One aspect of the invention includes a method for producing an anti-LAG-3 antibody, comprising expressing the antibody in a host cell as defined above and isolating the antibody from the host cell.

One aspect of the invention includes a method of stimulating an immune response in a subject, comprising administering an antibody or an antigen-binding portion thereof, as defined above, a bispecific molecule, as defined above, or an immunoconjugate, as defined above, to a subject such that an immune response is stimulated in the subject.

In one aspect, the method is characterized in that the subject is a subject with a tumor,

- 5 044665 this stimulates the immune response against the tumor.

In one aspect, the method is characterized in that the subject is a subject with a virus and an immune response against the virus is stimulated.

In one aspect, the method is characterized in that the immune response is an antigen-specific T-cell response such that the antigen-specific T-cell response is stimulated.

In one aspect, the method is characterized in that it stimulates the production of interleukin-2 by an antigen-specific T cell.

One aspect of the invention includes a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject an antibody or an antigen binding portion thereof, as defined above, a bispecific, as defined above, or an immunoconjugate, as defined above, such that the growth of a tumor in the subject is inhibited.

One aspect of the invention includes a method of treating a viral infection in a subject, comprising administering to the subject an antibody or an antigen-binding portion thereof, as defined above, a bispecific, as defined above, or an immunoconjugate, as defined above, such that the viral infection in the subject is treated.

In one aspect, the method also includes administering at least one additional immunostimulatory antibody.

In one aspect, the method is characterized in that the at least one immunostimulatory additional antibody is an anti-PD-1 antibody.

In one aspect, the method is characterized in that the at least one additional immunostimulatory antibody is an anti-PD-L1 antibody.

In one aspect, the method is characterized in that the at least one additional immunostimulatory antibody is an anti-CTLA-4 antibody.

One aspect of the invention includes the use of an antibody or antigen-binding portion thereof, as defined above, bispecific, as defined above, or an immunoconjugate as defined above, to stimulate an immune response, optionally an antigen-specific T cell response, or inhibit the growth of tumor cells or treat a viral infection in a subject.

One aspect of the invention includes the use of an antibody or an antigen-binding portion thereof as defined above, a bispecific as defined above, or an immunoconjugate as defined above in the manufacture of a medicament for stimulating an immune response, optionally an antigen-specific T cell response, or inhibiting the growth of tumor cells or treating a viral infection in subject.

For a better understanding of this specification, certain terms are defined below. Additional definitions are provided in the text of the detailed description. The terms 25F7, 25F7 antibody, LAG3.1 antibody and LAG3.1 refer to the human LAG-3 specific antibody described in US2011/0150892 A1. The nucleotide sequence (SEQ ID NO: 1) encoding the 25F7 heavy chain variable region (LAG3.1) and the corresponding amino acid sequence (SEQ ID NO: 2) is shown in FIG. 1A (with CDR sequences designated SEQ ID NO: 4, 5 and 7, respectively). The nucleotide sequence (SEQ ID NO: 3) encoding the 25F7 light chain variable region (LAG3.1) and the corresponding amino acid sequence (SEQ ID NO: 4) are shown in FIG. 1B (with CDR sequences designated SEQ ID NO: 8, 9 and 10, respectively).

The term LAG-3 stands for lymphocyte activation gene 3. The term LAG-3 includes variants, isoforms, homologs, orthologs and paralogs. For example, antibodies specific to the human LAG-3 protein may, in some cases, cross-react with the LAG-3 protein of non-human species. In other embodiments, antibodies specific for human LAG-3 protein may be completely specific for human LAG-3 protein and may not exhibit species or other types of cross-reactivity or may cross-react with LAG-3 of some other species but not all other species (eg, cross-reacts with monkey LAG-3 but not mouse LAG-3). The term human LAG-3 means a human LAG-3 sequence, such as the complete amino acid sequence of human LAG-3 having Genbank accession number NP_002277 (SEQ ID NO: 29). The term mouse LAG-3 means a mouse LAG-3 sequence, such as the complete amino acid sequence of mouse LAG-3 having Genbank accession number NP_032505. LAG-3 is also known in the art as, for example, CD223. The sequence of human LAG-3 may differ from the human LAG-3 number NP_002277 in the Genbank database due to the presence of, for example, conserved mutations or mutations in non-conserved regions, with LAG-3 having substantially the same biological function as and LAG-3, numbered NP_002277 in the Genbank database. For example, the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3 that is specifically bound by an antibody as described herein, or the biological function of human LAG-3 is to bind to MHC class II molecules.

The term monkey LAG-3 is intended to describe LAG-3 proteins expressed by monkeys

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Old and New World, including, without limitation, LAG-3 cynomolgus monkey and LAG-3 rhesus macaque. The corresponding monkey LAG-3 amino acid sequence is the rhesus monkey LAG-3 amino acid sequence, which is also deposited in Genbank under accession number XM_001108923. Another corresponding amino acid sequence of monkey LAG-3 is the alternative sequence of the rhesus macaque clone pa23-5, as described in US 2011/0150892 A1. The alternative Rh sequence has one amino acid difference at position 419 compared to the sequence deposited in Genbank.

A particular human LAG-3 sequence will be at least 90% identical to the human LAG-3 amino acid sequence Genbank NP_002277 and will contain amino acid residues that allow the amino acid sequence to be identified as human when compared with the LAG-3 amino acid sequences of other species (eg mice). In certain cases, human LAG-3 may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to LAG-3 Genbank NP_002277. In certain embodiments, the human LAG-3 sequence will exhibit a difference of no more than 10 amino acids from the Genbank LAG-3 sequence NP_002277. In certain embodiments, human LAG-3 may exhibit no more than 5, or even no more than 4, 3, 2, and 1 amino acid differences from the Genbank LAG-3 sequence NP_002277. The identity share can be determined as described here. The term immune response means the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines and complement), which leads to the selective attack, destruction or removal from the human body of invasive pathogens, cells or tissues infected by pathogens, cancer cells or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. Antigen-specific T cell response refers to T cell mediated responses that arise due to stimulation of a T cell by an antigen for which the T cell has specificity. Non-limiting examples of T cell responses upon antigen-specific stimulation include proliferation and cytokine production (eg, IL-2 production).

As used herein, the term antibody means whole antibodies and any antigen-binding fragment (ie, antigen-binding portion) or individual chains thereof. Whole antibodies are glycoproteins containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (herein referred to as _VH ) and a heavy chain constant region. The heavy chain constant region consists of three domains, _CH1 , CH2 and CH3. Each light chain consists of a light chain variable region (herein referred to as VL) and a light chain constant region. The light chain constant region consists of a single domain, CL. The VH and _VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed between regions that are more conserved, called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The light and heavy chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (Clq) of the classical complement system.

The term antigen-binding antibody moiety (or simply antibody moiety) as used herein means one or more antibody fragments that retain the ability to specifically bind an antigen (eg, LAG-3 protein). It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody. Examples of binding fragments encompassed by the term antigen-binding moiety of an antibody include (i) a Fab fragment, a monovalent fragment consisting of V _L , V _H , C _L and _CH 1 domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and CH1 domains; (v) an Fv fragment consisting of the VL and VH domains of one arm of the antibody; (vi) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a _VH domain; (vii) a dedicated complementarity determining region (CDR); and (viii) a nanobody, a heavy chain variable region containing one variable domain and two constant domains. Moreover, although the two domains of the Fv fragment, VL and _VH , are encoded by different genes, they can be joined recombinantly using a synthetic linker, which makes it possible to obtain them in the form of a single protein chain in which VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). These single chain antibodies are also encompassed by the term antigen binding portion of an antibody. These anti fragments

- 7 044665 bodies are prepared using conventional methods known to those skilled in the art, with these fragments being screened for utility in the same manner as intact antibodies.

As used herein, an isolated antibody means an antibody that is substantially free from other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds the LAG-3 protein is substantially free from antibodies that specifically bind antigens other than proteins LAG-3). However, an isolated antibody that specifically binds human LAG-3 protein may be cross-reactive with other antigens, such as LAG-3 proteins of other species. Moreover, the isolated antibody may be substantially free of other cellular material and/or chemical compounds.

As used herein, the terms monoclonal antibody or monoclonal antibody composition mean a preparation of antibody molecules having the same molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

As used herein, the term human antibody includes antibodies having variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a constant region, then the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro, or somatic mutation in vivo). However, the term human antibody, as used herein, does not include antibodies in which CDR sequences derived from the germ line of other mammalian species, such as mouse, have been transplanted into human framework sequences.

The term human monoclonal antibody means antibodies exhibiting a single binding specificity that have variable regions in which framework regions and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced using a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell. The term recombinant human antibody, as used herein, includes all human antibodies that are produced, expressed, created or isolated by recombinant methods, such as (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transchromosomal in against human immunoglobulin genes, or from a hybridoma derived therefrom (described below), (b) antibodies isolated from a host cell transformed to express a human antibody, for example from a transfectome, (c) antibodies isolated from a recombinant combinatorial antibody library human, and (d) antibodies produced, expressed, generated or isolated by any other means that involve splicing human immunoglobulin gene sequences with other DNA sequences. These recombinant human antibodies include variable regions in which the framework and CDR regions are derived from human immunoglobulin germline sequences. However, in certain embodiments, these recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using an animal transgenic for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and V _L regions of the recombinant antibodies represent are sequences that, although derived from or related to human germline VH and VL sequences, may not naturally exist in the human germline antibody repertoire in vivo.

The term isotype refers to a class of antibodies (eg, IgM or IgG1) that is encoded by heavy chain constant region genes.

The expressions antigen-recognizing antibody and antigen-specific antibody are used interchangeably herein with the expression antibody that specifically binds an antigen.

The term human antibody derivatives means any modified form of a human antibody, such as a conjugate of an antibody and another agent or antibody.

The term humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been transplanted into human framework sequences. In human framework sequences, further modifications to the framework region may be made.

The term chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are

- 8 044665 sequences are from a mouse antibody, and the constant region sequences are derived from a human antibody.

As used herein, an antibody that specifically binds human LAG3 is an antibody that binds the human LAG-3 protein (and possibly the LAG-3 protein of one or more non-human species) but does not substantially bind to non-human species. LAG-3 proteins. Preferably, the antibody binds to human LAG-3 protein with high affinity, more particularly with a K _D value of 1 x 10 ^-7 M or less, more preferably 1 x 10 ^-8 M or less, more preferably 5 x 10 ^-9 M or less, more preferably 1 x 10 ^-9 M or less.

The term does not bind substantially to protein or cells, as used herein means does not bind or does not bind with high affinity to protein or cells, that is, binds to protein or cells with a K _D of 1x10 ^-6 M or higher, more preferably 1 x 10 ^-5 M or higher, more preferably 1 x 10 ^-4 M or higher, more preferably 1 x 10 ^-3 M or higher, and even more preferably 1 x 10 ^-2 M or higher.

The term K _associate or K _a , as used herein, is intended to denote the rate of association of a particular antibody-antigen interaction, whereas the term K _guc or K _d , as used herein, is intended to denote the rate of dissociation of a particular antibody-antigen interaction. antigen. The term K _D , as used herein, is intended to denote the dissociation constant, which is calculated from the ratio of Kd to K _a (ie, Kd/K _a ) and expressed as molar concentration (M). K _D values for antibodies can be determined using methods well known in the art. The preferred method for determining the K _D of an antibody is surface plasma resonance, preferably using a biosensor system such as the Biacore® system.

The term high affinity for an IgG antibody means an antibody having a K _D for a target antigen of 1 x 10 ^-7 M or less, more preferably 5 x 10 ^-8 M or less, even more preferably 1 x 10 ^-8 M or less, even more preferably 5 x 10 ^-9 M or less and even more preferably 1 x 10 ^-9 M or less. However, high binding affinity may differ for antibodies of other isotypes. For example, high binding affinity of the IgM isotype means an antibody with a K _D of 10 ^-6 M or less, more preferably 10 ^-7 M or less, more preferably 10 ^-8 M or less. The term deamidation refers to a chemical degradation process that occurs spontaneously in proteins (such as antibodies). Deamidation removes the amide functional group from an amino acid residue such as asparagine and glutamine, thereby damaging its amide-containing side chains. In particular, the asparagine side chain attacks a nearby peptide group, forming a symmetric succinimide derivative. The symmetry of the derivative results in two hydrolysis products, aspartate or isoaspartate. A similar reaction can occur in aspartate side chains, leading to partial conversion to isoaspartate. In the case of glutamine, the rate of deamidation is generally ten times less than in the case of asparagine, but the mechanism is essentially similar and requires only water molecules.

The term subject includes any animal, whether human or non-human. The term non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles, although mammals are preferred, e.g. non-human primates, sheep , dogs, cats, cows and horses. Various aspects of the invention are described in more detail in the following subsections.

Ahtu-LAG-Z antibodies with increased stability and useful functional properties

The antibodies of the invention specifically bind to human LAG-3 and have optimized stability compared to previously described anti-LAG-3 antibodies, in particular compared to antibody 25F7 (LAG3.1). This optimization includes reduced deamidation (eg, increased chemical stability) and increased thermal refolding (eg, increased physical stability), while maintaining high human LAG-3 binding affinity.

Methods for identifying deamidation sites are known in the art (see, for example, ion exchange, reverse phase, and hydrophobic interaction chromatography and peptide proteolytic cleavage mapping (HPLC-MS)). Suitable assays for measuring physical stability include, for example, analysis of melting points and/or refolding of the antibody structure after denaturation (eg, fractional reversibility, as described, for example, in Example 3, Section 3). Binding to human LAG-3 can be analyzed using one or more methods well known in the art. For example, the antibody can be tested using a flow cytometry-based assay in which the antibody reacts with a cell line that expresses human LAG-3, such as CHO cells that have been transfected to express LAG-3 (e.g., human LAG-3 or monkey LAG-3 (eg rhesus or cynomolgus) or mouse LAG-3) on their cell surface. Other suitable cages for use within

- 9044665 flow cytometry-based assays include anti-CD3-stimulated CD4+ activated T cells that express native LAG-3. Additionally or alternatively, antibody binding, including binding kinetics (eg, KD value), can be tested using BIAcore assays. Other suitable binding assays include ELISA assays, for example using recombinant LAG-3 protein.

Antibodies of the invention preferably bind to human LAG-3 protein with a KD value of 1 x 10 ^-7 M or less, more preferably 1 x 10 ^-8 M or less, 5 x 10 ^-9 M or less, or 1 x 10 ^-9 M or less.

Typically, the antibody binds to LAG-3 in lymphoid tissues such as the tonsil, spleen or thymus, which can be detected by immunohistochemical analysis. In one embodiment, the antibody stains pituitary tissue (eg, accumulates in the pituitary gland), which is detected by immunohistochemical analysis. In another embodiment, the antibody does not stain pituitary tissue (ie, does not accumulate in the pituitary gland), as detected by immunohistochemical analysis.

Additional functional properties include cross-reactivity with LAG-3 of other species. For example, the antibody may bind to monkey LAG-3 (eg, cynomolgus monkeys, rhesus monkeys) but has little or no binding to mouse LAG-3. Preferably, the antibody of the invention binds to human LAG-3 with high affinity.

Other functional properties include the ability of the antibody to stimulate an immune response, such as an antigen-specific T-cell response. This can be determined, for example, by analyzing the ability of the antibody to stimulate the production of interleukin-2 (IL-2) in an antigen-specific T-cell response. In some embodiments, the antibody binds to human LAG-3 and stimulates an antigen-specific T cell response. In other embodiments, the antibody binds to human LAG-3 but does not stimulate an antigen-specific T cell response. Other means for assessing the ability of an antibody to stimulate an immune response include testing its ability to inhibit tumor growth, such as an in vivo xenograft tumor model (see, for example, Example 6) or its ability to stimulate an autoimmune response, such as the ability to promote the development of an autoimmune disease in an autoimmune model , such as the ability to promote the development of diabetes in a NOD mouse model.

Preferred antibodies of the invention are human monoclonal antibodies. Additionally or alternatively, the antibodies may be, for example, chimeric or humanized monoclonal antibodies.

Monoclonal antibody LAG3.5

A preferred antibody of the invention is a human monoclonal antibody, LAG3.5, characterized structurally and chemically as described below in the following examples. The amino acid sequence of VH LAG3.5 is shown in SEQ ID NO:12 (Fig. 2A). The amino acid sequence of V _L LAG3.5 is shown in SEQ ID NO:14 (Fig. 2B).

The VH and V _L sequences (or CDR sequences) of other anti-LAG antibodies that bind human LAG-3 can be mixed and paired with the VH and V _L sequences (or CDR sequences) of the LAG3.5 antibody. Preferably, when mixing and pairing VH and VL chains (or CDRs in these chains), the VH sequence from a particular VH/VL pairing is replaced by a structurally similar _VH sequence. Likewise, the V _L sequence from a particular VH/VL pairing is replaced by a structurally similar VL sequence. Accordingly, in one embodiment, the antibodies of the invention or antigen-binding portions thereof include:

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (ie, VH LAG3.5); and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (ie V _L LAG3.5) or VL of another anti-LAG3 antibody (ie other than LAG3.5);

the antibody specifically binds human LAG-3.

In another embodiment, the antibodies of the invention or antigen-binding portions thereof include:

(a) CDR1, CDR2 and CDR3 regions of the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (ie, CDR sequence LAG3.5, SEQ ID NO: 15, 16 and 17, respectively); and (b) the CDR1, CDR2, and CDR3 regions of a light chain variable region comprising the amino acid sequences of SEQ ID NO: 14 (i.e., CDR sequences LAG3.5, SEQ ID NOs: 18, 19, and 20, respectively) or the CDR of another anti-LAG3 antibodies (ie, other than LAG3.5);

the antibody specifically binds human LAG-3.

In another embodiment, the antibody or antigen-binding portion thereof comprises the heavy chain variable region CDR2 of LAG3.5 combined with the CDRs of other antibodies that bind human LAG-3, such as CDR1 and/or CDR3 from the heavy chain variable region and/or CDR1, CDR2 and /or CDR3 from the light chain variable region of another anti-LAG-3 antibody.

- 10 044665

In addition, it is well known in the art that the CDR3 domain, independently of the CDR1 and/or CDR2 domain(s), can independently determine the binding specificity of an antibody for its antigen and that multiple antibodies with the same binding specificity can be predictably produced based on common CDR3 sequence. See, for example, Klimka et al, British J. of Cancer 83(2):252-260 (2000); Beiboer et al, J. Mol Biol. 296:833-849 (2000); Rader et al, Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533 (1995); Ditzel et al, J. Immunol. 157:739-749 (1996); Berezov et al, BIAjournal 8: Scientific Review 8 (2001); Igarashi et al, J. Biochem (Tokyo) 117:452-7 (1995); Bourgeois et al, J. Virol 72:807-10 (1998); Levi et al, Proc. Natl. Acad. Sci. U.S.A. 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). See also US patents 6951646; 6914128; 6090382; 6818216; 6156313; 6827925; 5833943; 5762905 and 5760185. Each of these publications is incorporated herein by reference in its entirety.

Accordingly, in another embodiment, antibodies of the invention comprise LAG3.5 heavy chain variable region CDR2 and at least LAG3.5 heavy and/or light chain variable region CDR3 (SEQ ID NO: 17 and/or 20) or variable region CDR3 a region of the heavy and/or light chain of another LAG-3 antibody, wherein the antibody is capable of specifically binding to human LAG-3. These antibodies preferably (a) compete for binding; (b) maintain functional characteristics; (c) bind to a single epitope; and/or (d) have the same binding affinity as LAG3.5. In another embodiment, the antibodies may also comprise the light chain variable region CDR2 of LAG3.5 (SEQ ID NO: 17 and/or 20) or the light chain variable region CDR2 of another LAG-3 antibody, wherein the antibody is capable of specifically binding to human LAG-3 . In another embodiment, antibodies of the invention may also include a heavy and/or light chain variable region CDR1 of LAG3.5 (SEQ ID NO: 17 and/or 20) or a heavy/or light chain variable region CDR1 of another LAG-3 antibody, wherein the antibody is able to specifically bind to human LAG-3.

Conservative modifications

In another embodiment, antibodies of the invention include heavy chain variable region sequences and/or light chain CDR1, CDR2 and CDR3 sequences that differ from these LAG3.5 sequences by one or more conservative modifications. However, in a preferred embodiment, CDR2 VH residues 54 and 56 are retained as arginine and serine, respectively (ie, not mutated). It is apparent that conservative sequence modifications can be made such that they do not result in loss of antigen binding ability. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al.. (1997)J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43. Accordingly, in one embodiment, the antibody includes a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and/or a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein:

(a) the heavy chain variable region CDR1 sequence includes SEQ ID NO: 15 and/or conservative modifications thereof, excluding positions 54 and 56; and/or (b) the heavy chain variable region CDR3 sequence includes SEQ ID NO: 17 and conservative modifications thereof; and/or (c) CDR1 and/or CDR2 and/or CDR3 light chain variable region sequences include SEQ ID NO: 18 and/or SEQ ID NO: 19 and/or SEQ ID NO: 20 and/or conservative modifications thereof and ( d) the antibody specifically binds human LAG-3.

Additionally or alternatively, the antibody may have one or more of the following functional properties described above, such as high affinity human LAG-3 binding, monkey LAG-3 binding, lack of mouse LAG-3 binding, the ability to inhibit LAG-3 binding to MHC class II molecules and/or the ability to stimulate antigen-specific T-cell responses. In various embodiments, the antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody. As used herein, the term conservative sequence modifications means amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions or deletions. Modifications can be introduced into the antibody of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by another amino acid residue with a similar side chain. The prior art has identified families of amino acid residues having similar side chains. Such families include amino acids with

- 11 044665 novel side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine ). Thus, one or more amino acid residues in the CDR regions of an antibody of the invention may be replaced by other amino acid residues of the family having the same side chains, and the altered antibody may be tested for retention of function (namely, the functions specified above) using the described here are functional analyses.

Engineered and modified antibodies

Antibodies of the invention can be produced using an antibody having one or more VH and/or _VL sequences of LAG3.5 as starting material for constructing the modified antibody. An antibody can be constructed by modifying one or more residues in one or both variable regions (ie, V _H and/or V _L ), such as one or more CDR regions and/or one or more framework regions. Additionally or alternatively, an antibody can be constructed by modifying residues in the constant region(s), for example, to change the effector function(s) of the antibody. In certain embodiments, CDR grafting may be used to construct antibody variable regions. Antibodies interact with target antigens primarily through amino acid residues that are located in the six complementarity determining regions (CDRs) of the heavy and light chains. For this reason, amino acid sequences within the CDR are more variable between individual antibodies than sequences outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from a specific naturally occurring antibody grafted into framework sequences from another antibody with different properties (see, for example, Riechmann et al. (1998) Nature 332:323-327; Jones et al. (1986) Nature 321:522-525; Queen et al. (1989) Proc. Natl. Acad. See. USA 86:10029-10033; US patents 5225539; 5530101; 5585089; 5693762 and 6180370).

Accordingly, another embodiment of the invention relates to an isolated monoclonal antibody, an antigen binding portion thereof comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO: 15, 16, 17, respectively, and/or a light chain variable region , comprising CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO: 18, 19, 20, respectively (ie CDR LAG3.5). While these antibodies contain the VH and VL CDR sequences of the monoclonal antibody LAG3.5, they may contain other framework sequences.

These framework sequences can be obtained from publicly available DNA databases or published sources that include germline antibody gene sequences. For example, germline DNA sequences for the human heavy and light chain variable region genes can be found in the human germline sequence database VBase (at www.mrc-cpe.cam.ac.uk/vbase) and in publications by Kabat et al. (1991), cited above; Tomlinson et al. (1992) The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of V _H Segments with Different Hypervariable Loops J. Mol. Biol. 227:776-798; and Cox et al. (1994) A Directory of Human Germ-line V _H Segments Reveals a Strong Bias in their Usage Eur. J. Immunol. 24:827-836; the contents of each are included here in full for reference. As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database. For example, the following germline heavy chain sequences included in mouse HCo7 HuMAb are available in Genbank under numbers: 1-69 (NG_0010109, NT_024637 & BC070333), 3-33 (NG_0010109 & NT_024637) and 3-7 (NG_0010109 & NT_024637 ). As another example, the following heavy chain germline sequences comprising the mouse HCo12 HuMAb are available in GenBank under accession numbers: 1-69 (NG_0010109, NT_024637 & BC070333), 5-51 (NG_0010109 & NT_024637), 4-34 (NG_0010109 & NT_024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

The protein sequences of the antibody are compared to the protein sequences available in the compiled database using one of the sequence similarity search methods called Gapped BLAST (Altschul et al. (1997), supra), which are well known in the art.

Preferred framework sequences for use in antibodies of the invention are antibodies that are structurally similar to the framework sequences used in selected antibodies of the invention, for example, similar to the V _H 4-34 framework sequences and/or V _K L6 framework sequences used in the preferred mono

- 12 044665 clonal antibodies according to the invention. The CDR1, CDR2 and CDR3 VH sequences and the CDR1, CDR2 and CDR3 _VK sequences may be transplanted into framework regions that have a sequence identical to the sequence found in the germline immunoglobulin gene from which the framework sequence is derived, or such CDR sequences may be transplanted into scaffolds that contain one or more mutations relative to germline sequences. For example, it has been found that in some cases it is preferable to mutate residues in the framework regions to maintain or increase the antigen-binding ability of the antibody (see, for example, US patents 5530101; 5585089; 5693762 and 6180370).

Another type of variable region modification is the mutation of amino acid residues in the CDR1, CDR2 and/or CDR3 VH and/or _VL regions to thereby improve one or more binding properties (eg, affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutation(s), and the effect on antibody binding or other functional property of interest can be assessed in the in vitro or in vivo assays described herein and listed in the Examples section. Preferably, conservative modifications (discussed above) are introduced. Mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. Moreover, typically no more than one, two, three, four or five residues in the CDR region are modified.

Accordingly, in another embodiment, the invention provides isolated anti-LAG-3 monoclonal antibodies or antigen binding portions thereof comprising a heavy chain variable region comprising: (a) a VH CDR1 region comprising SEQ ID NO: 15 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 15; (b) a V _H CDR2 region comprising SEQ ID NO: 16 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 16 (preferably where positions 54 and 56 same as SEQ ID NO: 16); (c) a V _H CDR3 region comprising SEQ ID NO: 17 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 17; (d) a V _L CDR1 region comprising SEQ ID NO: 18 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 18; (f) a V _L CDR2 region comprising SEQ ID NO: 19 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 19; and (f) a V _L CDR3 region comprising SEQ ID NO: 20 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 20. Engineered antibodies of the invention include antibodies in which modifications have been made to the framework residues in VH and/or _VL , for example to improve the properties of the antibody. Typically, such modifications in the framework region are made to reduce the immunogenicity of the antibody. For example, one approach is to mutate one or more framework residues back to the corresponding germline sequence residues. More specifically, an antibody that has been somatically mutated may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequences of the antibody with the germline sequences from which the antibody is derived.

Another type of framework modification involves mutation of one or more residues in the framework region or even in one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as deimmunization and is described in more detail in US Patent Publication 20030153043.

As a complement or alternative to modifications made in the framework or CDR regions, antibodies of the invention can be designed to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation , Fc receptor binding and/or antigen-dependent cellular cytotoxicity. Moreover, the antibody of the invention may also be chemically modified (eg, one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these options is described in more detail below. The numbering of residues in the Fc region corresponds to the numbering according to the Kabat EU index.

In a preferred embodiment, the antibody is an IgG4 isotype antibody comprising a serine to proline mutation at a position corresponding to position 228 (S228P; EU index) in the heavy chain constant region hinge region. It was noted that this mutation eliminates the heterogeneity of disulfide bridges between heavy chains in the hinge

- 13 044665 site (Angal et al. supra; position 241 based on Kabat numbering system).

In one embodiment, the CH1 hinge region is modified such that the number of cysteine residues in the hinge region is changed, such as increased or decreased. This approach is also described in US Pat. No. 5,677,425. The number of cysteine residues in the CH1 hinge region is varied, for example, to facilitate the assembly of light and heavy chains or to increase or decrease the stability of the antibody. In another embodiment, a mutation in the Fc hinge region of an antibody reduces the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 interface of the Fc hinge domain such that binding of the antibody to Staphylococcyl protein A (SpA) is reduced compared to binding of the native SpA Fc hinge domain. This approach is also described in US patent 6165745.

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in US Pat. No. 6,277,375. Alternatively, to increase the biological half-life of the antibody, the antibody may be altered at the CH1 or CH2 region such that it contains a salvage receptor binding epitope , formed from two loops of the CH2 domain of the IgG Fc region, as described in US Patents 5,869,046 and 6,121,022. In other embodiments, the Fc region is altered by replacing at least one amino acid residue with another amino acid residue to change the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 may be replaced by another amino acid residue such that the antibody has altered affinity for the effector ligand but retains antigen-binding ability original antibody. The effector ligand for which the affinity is changed may be, for example, the Fc receptor or the C1 complement component. This approach is also described in US patents 5624821 and 5648260.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 may be replaced with another amino acid residue such that the antibody has altered C1q binding and/or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is also described in US patent 6194551.

In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered, thereby altering the ability of the antibody to fix complement. This approach is also described in PCT publication WO 94/29351. In another example, the Fc region is modified to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the Fcy receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252 , 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301 , 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382 , 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is also described in PCT publication WO 00/42072. Moreover, the binding sites on human IgG1 for FcyR1, FcyRII, FcyRIII and FcRn have been mapped, and variants with improved binding have been described (see Shields et al. (2001) J. Biol. Chem. 276:6591-6604). Certain mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcyRIII. In addition, the following combination mutations have been shown to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A, and S298A/E333A/K334A.

In another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (ie, an antibody without glycosylation) can be produced. Glycosylation can be altered to, for example, increase the affinity of an antibody for an antigen. Such modifications of carbohydrates can be accomplished, for example, by changing one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions may be made that result in the elimination of one or more glycosylation sites of the variable region framework, thereby eliminating glycosylation at that site. This aglycosylation may increase the affinity of the antibody for the antigen. See, for example, US Patents 5,714,350 and 6,350,861.

Additionally or alternatively, the antibody may be prepared to have an altered glycosylation pattern, such as a hypofucosylated antibody containing a reduced amount of fucose residues, or an antibody containing an increased amount of biantennary GlcNac structures. These altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. These carbohydrate modifications can be accomplished, for example, by expressing an antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies of the invention therein, thereby obtaining an altered glycosylation antibody. For example, in the Ms704, Ms705 and Ms709 cell lines there is no

- 14044665 contains the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), such that the antibodies expressed in the Ms704, Ms705 and Ms709 cell lines lack fucose in the carbohydrates. FUT8 ^-/- cell lines Ms704, Ms705 and Ms709 were generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see US Patent Publication 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614- 22). As another example, EP 1176195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation due to the reduction or elimination of the α-1,6-related enzyme. communications. EP 1176195 also describes cell lines that have low enzymatic activity for the addition of fucose to N-acetylglucosamine that binds to the Fc region of the antibody or no enzymatic activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT publication WO 03/035835 describes a variant of the CHO cell line, Lec13 cells, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also leads to hypofucosylation of antibodies expressed in that host cell (see also Shields et al (2002) J Biol Chem 277:26733–26740). Antibodies with a modified glycosylation profile can be produced in chicken eggs as described in PCT publication WO 06/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells such as Lemna. Methods for producing antibodies in a plant system are described in US Application Serial No. Alston & Bird LLP 040989/314911, filed August 11, 2006. PCT Publication WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases ( e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in such engineered cell lines exhibit increased bipartitioning of GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana et al (1999) Nat Biotech 17:176-180). Alternatively, the fucosyl residues of an antibody can be cleaved by the enzyme fucosidase, for example fucosidase aL-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516-23). Another modification of the antibodies of the invention is pegylation. The antibody may be pegylated, for example, to increase the biological half-life of the antibody (eg, in serum). To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is carried out through an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term polyethylene glycol covers any of the forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10)alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the pegylated antibody is an aglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the invention. See, for example, EP 0154316 and EP 0401384.

Physical properties of antibodies

Antibodies of the invention can be characterized by their various physical properties to detect and/or distinguish between their classes.

For example, antibodies may contain one or more glycosylation sites in the variable region of the heavy or light chain. These glycosylation sites may provide increased antibody immunogenicity or a change in antibody pK due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702: Gala and Morrison (2004) J Immunol 172:5489-94: Wallick et al ( 1988) J ExpMed 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al (2000) Mol Immunol 37:697-706). Glycosylation has been shown to occur in motifs containing the N-X-S/T sequence. In some cases, it is preferable to have an anti-LAG-3 antibody without variable region glycosylation. This can be achieved either by selecting antibodies that do not contain a glycosylation motif in the variable region, or by introducing mutations to residues in the glycosylation site.

In a preferred embodiment, the antibodies do not contain asparagine isomerism sites. Deamidation of asparagine can occur in N-G or D-G sequences and result in the formation of an isoaspartic acid residue, which causes looping of the polypeptide chain or a decrease in its stability (isaspartic acid effect).

Each antibody has a unique isoelectric point (pI), which typically lies in the pH range from 6 to 9.5. The pI of an IgG1 antibody is typically in the pH range of 7-9.5, and the pI of an IgG4 antibody is typically in the pH range of 6-8. It has been suggested that antibodies whose pI is outside the normal range may exhibit some misfolding and instability under in vivo conditions. Thus, it is preferable to obtain anti-LAG-3 antibodies whose pI value lies within

- 15 044665 normal range. This can be achieved either by selecting an antibody with a pI in the normal range or by introducing mutations to charged surface residues.

Nucleic acid molecules encoding antibodies of the invention

In another aspect, the invention relates to nucleic acid molecules that encode heavy and/or light chain variable regions, or CDRs, of antibodies of the invention. Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. A nucleic acid is isolated or substantially pure if it is purified from other cellular components or other impurities, such as other cellular nucleic acids or proteins, by standard methods including alkali/SDS treatment, CsC1 density gradient centrifugation, column chromatography, agarose electrophoresis gel and other methods well known from the prior art. See, Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid of the invention may be, for example, DNA or RNA, and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acids of the invention can be obtained using standard molecular biology techniques. In the case of antibodies expressed in hybridomas (for example, hybridomas obtained from transgenic mice having human immunoglobulin genes, as described in more detail below), cDNAs encoding the light and heavy chains of the antibody produced using the hybridoma can be obtained by conventional PCR methods. amplification or cDNA cloning. In the case of antibodies obtained from an immunoglobulin gene library (eg, using phage display methods), the nucleic acid encoding such antibodies can be obtained from the gene library.

Preferred nucleic acids of the invention include nucleic acids that encode the VH and _VL sequences of the monoclonal antibody LAG3.5 (SEQ ID NOs: 12 and 14, respectively) or CDRs. Once DNA fragments encoding the VH and _VL segments are obtained, these DNA fragments can be further processed by standard recombinant DNA techniques, for example to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a DNA fragment encoding V _L or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term operatively linked, as used in this context, is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in reading frame.

The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by functionally linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3). Human heavy chain constant region gene sequences are known in the art (see Kabat et al. (1991), supra), and DNA fragments comprising such regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For a heavy chain Fab gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the CL light chain constant region. Human light chain constant region gene sequences are known in the art (see, eg, Kabat et al., supra), and DNA fragments comprising such regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda constant region.

To create the scFv gene, the V _H and V L encoding DNA fragments are operably linked to another flexible linker encoding fragment, for example, encoding the amino acid sequence (Gly4-Ser) ₃ (SEQ ID NO: 28), so that the VH and V _L sequences can be expressed as a continuous single-chain protein with V _H and V _L regions connected by a flexible linker (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Preparation of monoclonal antibodies according to the invention

The monoclonal antibodies (mAbs) of the present invention can be produced using the well-known somatic cell hybridization (hybridoma) method according to Kohler and Milstein (1975) Nature 256: 495. Other options for producing monoclonal antibodies include methods based on viral or oncogenic transformation B -lymphocytes and phage display. Chimeric or humanized antibodies are also well known in the art. See, for example, US patents 4816567; 5225539; 5530101; 5585089; 5693762 and 6180370, the contents of which are given in full

- 16 044665 here for reference.

In a preferred embodiment, the antibodies of the invention are human monoclonal antibodies. Such human monoclonal antibodies directed against human LAG-3 can be produced using transgenic or transchromosomal mice having parts of the human immune system instead of the mouse system. These transgenic or transchromosomal mice include mice referred to herein as HuMAb Mouse® and KM Mouse®, respectively, and are collectively referred to herein as human Ig mice.

HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin gene miniloci that encode rearranged sequences of the human immunoglobulin heavy (μ and γ) and light chains, together with targeted mutations that inactivate the endogenous μ and k chain loci (see, e.g. Lonberg, (1994) Nature 368(6474): 856-859).

Thus, these mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human monoclonal IgGK (Lonberg et al. (1994), supra described in Lonberg (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern Rev. Immunol. 13: 65-93, and Harding and Lonberg (1995) Ann. NY Acad Sci 764:536-546). The preparation and use of HuMAb Mouse®, as well as the genomic modifications present in such mice, are also described by Taylor et al. (1992) Nucleic Acids Research 20:62876295; Chen et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117–123; Chen et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor et al. (1994) International Immunology 6: 579-591; and Fishwild et al. (1996) Nature Biotechnology 14: 845-851, the contents of which are incorporated herein by reference in their entirety. See also US patents 5545806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299; 5770429; and 5545807; PCT publication WO 92/03918; WO 93/12227; WO 94/25585; WO 97/13852; WO 98/24884; WO 99/45962 and WO 01/14424, the contents of which are incorporated herein by reference in their entirety.

In another embodiment, human antibodies of the invention can be produced using mice having human immunoglobulin sequences in transgenes and transchromosomes, for example mice having a human heavy chain transgene and a human light chain transchromosome. These mice are referred to here as KM mouse® and are described in detail in PCT publication WO 02/43478. A modified form of such a mouse, which is also characterized by a homozygous disruption of the endogenous FcyRIIB receptor gene, is also described in PCT publication WO 02/43478 and is referred to herein as the KM/FCGR2D mouse®. In addition, mice with HCo7 or HCo12 heavy chain transgenes, or both, can be used. Additional transgenic animal options include Xenomouse (Abgenix, Inc., US patents 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963). Other variants include mouse TC (Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727) and cows having human heavy and light chain transchromosomes (Kuroiwa et al. (2002) Nature Biotechnology 20: 889-894; PCT publication WO 02/092812). The contents of these patents and publications are incorporated herein in their entirety by reference.

In one embodiment, human monoclonal antibodies of the invention are produced using phage display methods to screen human immunoglobulin gene libraries. See, for example, US patents 5223409; 5403484; 5571698; 5427908; 5580717; 5969108; 6172197; 5885793; 6521404; 6544731; 6555313; 6582915 and 6593081, the contents of which are incorporated herein by reference in their entirety. The human monoclonal antibodies of the invention can also be produced using SCID mice in which human immune cells have been engineered such that a human antibody response can be generated following immunization. See, for example, US Pat. Nos. 5,476,996 and 5,698,767, the contents of which are incorporated herein by reference in their entirety.

In another embodiment, human anti-LAG-3 antibodies are produced using phage display, in which the phages include nucleic acids encoding antibodies produced in transgenic animals previously immunized with LAG-3. In a preferred embodiment, the transgenic animal is a HuMab, KM or Kirin mouse. See, for example, US Pat. No. 6,794,132, the contents of which are incorporated herein by reference in their entirety.

Immunization of mice with human Ig

In one embodiment, mice with human Ig are immunized with a purified or enriched preparation of LAG-3 antigen, recombinant LAG-3 protein, or cells expressing LAG-3 protein. See, for example, Lonberg et al. (1994), supra; Fishwild et al. (1996), supra; PCT publication WO 98/24884 or WO 01/14424, the contents of which are incorporated herein by reference in their entirety. In a preferred embodiment, mice 6-16 weeks old are immunized with 5-50 μg of LAG-3 protein. Alternatively, a fusion polypeptide of a LAG-3 portion and a non-LAG-3 portion is used.

In one embodiment, transgenic mice are immunized intraperitoneally (I/P)

- 17 044665 or intravenous (I/V) LAG-3 antigen in complete Freund's adjuvant followed by I/P or I/V immunization with antigen in incomplete Freund's adjuvant. In other embodiments, adjuvants other than Freund's adjuvant or whole cells without adjuvant are used. Plasma can be screened using ELISA, and cells from mice with sufficient levels of human anti-LAG-3 immunoglobulin titer can be used to generate fusion constructs.

Preparation of hybridomas producing human monoclonal antibodies according to the invention

To produce hybridomas producing human monoclonal antibodies of the invention, splenocytes and/or lymph node cells from immunized mice can be isolated and fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

Preparation of transfectomes producing monoclonal antibodies according to the invention

Antibodies of the invention can also be produced in a host cell transfectome, for example using a combination of recombinant DNA techniques and gene transfection techniques, as is well known in the art (eg, Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding portions or full-length light and heavy chains obtained by standard molecular biological techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. As used herein, the term operably linked means that the antibody gene is ligated into the vector such that the transcriptional and translational control sequences in the vector perform their function of regulating the transcription and translation of the antibody gene.

The term regulatory sequence includes promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control transcription or translation of antibody chain genes. These regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Preferred regulatory sequences for expression in a mammalian host cell include viral elements that result in high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (eg , major late promoter of adenovirus (AdMLP)) and polyoma virus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter may be used. In addition, regulatory elements composed of sequences from different sources can be used, such as the SRa promoter system, which contains sequences from the SV40 early promoter, and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al. (1988) Mol. Cell Biol 8:466–472). The expression vector and expression control sequences are selected to be compatible with the host cell being used.

The antibody light chain gene and the antibody heavy chain gene may be inserted into the same or different expression vectors. In preferred embodiments, variable regions are used to create full-length antibody genes of any isotype by inserting them into expression vectors that already encode a heavy chain constant region and a light chain constant region of the desired isotype, such that the V _H segment is operably linked to the C _H segment(s) in the vector, and the V _L segment is functionally linked to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that causes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene allows selection of host cells into which the vector has been introduced (see, for example, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, in the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with selection/amplification in the presence of methotrexate) and the peo gene (for selection in the presence of G418).

For expression of light and heavy chains, expression vector(s) encoding the heavy and light chains are transfected into a host cell using standard methods. Various forms of termi

- 18 044665 for transfection cover a wide range of methods widely used for introducing exogenous DNA into a prokaryotic or eukaryotic host cell, for example electroporation, calcium phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, it is most preferred to express the antibodies in eukaryotic cells and, most preferably, in mammalian host cells, since such eukaryotic cells, and in particular mammalian cells, are more suitable for assembly than prokaryotic cells. and secreting an appropriately folded and immunologically active antibody.

Preferred mammalian host cells for expression of recombinant antibodies of the invention include Chinese hamster ovary cells (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, for example as described by R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene-based expression system described in WO 87/04462, WO 89/01036 and EP 338,841. If recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to permit expression of the antibody in the host cells, or more preferably secreting the antibody into a culture medium in which host cells are grown. Antibodies can be isolated from the culture medium using standard protein purification methods.

Immunoconjugates

Antibodies of the invention can be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylated agents, DNA minor groove binding agents, DNA intercalates, DNA cross-linking agents, histone acetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibodies and therapeutic agent are preferably conjugated through a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 39), Ala-Asn- Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. ADCs can be prepared as described in US Pat. No. 7,087,600; 6989452 and 7129261; PCT publications WO 02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312 and WO 08/103693; US Patent Publications 20060024317; 20060004081 and 20060247295, which are described here for reference.

Bispecific molecules

In another aspect, the present disclosure discloses bispecific molecules comprising one or more antibodies of the invention linked to at least one other functional molecule, such as another peptide or protein (eg, another antibody or ligand for a receptor) to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, the term bispecific molecule includes molecules having three or more specificities. In a preferred embodiment, the bispecific molecule includes a first binding specificity for LAG-3 and a second binding specificity for initiating a molecule that engages cytotoxic effector cells that can kill a LAG-3-expressing target cell. Examples of suitable initiation molecules are CD64, CD89, CD16 and CD3. See, for example, Kufer et al, TRENDS in Biotechnology, 22 (5), 238-244 (2004).

In one embodiment, the bispecific molecule has a third specificity in addition to the anti-Fc binding specificity and the anti-LAG-3 binding specificity. A third specificity may be directed toward an enhancing factor (EF), for example, a molecule that binds to a surface protein involved in cytotoxic activity and thereby enhances the immune response against the target cell. For example, an anti-enhancement factor may bind a cytotoxic T cell (eg, via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) or other immune cell, resulting in an increased immune response against the target cell.

Bispecific molecules can have different formats and sizes. At one end of the size spectrum, a bispecific molecule retains the traditional antibody format except that instead of having two binding sites with identical specificity, it has two binding sites, each with a different specificity.

At the other border are bispecific molecules consisting of two single-chain antibody fragments (scFv's) linked by a peptide chain - the so-called Bs(scFv) ₂ construct. Bispecific molecules with an intermediate size include two different F(ab) fragments linked by a peptide linker. Bispecific molecules of this and other formats can be obtained from

- 19 044665 by the power of genetic modification, somatic hybridization or chemical methods. See, for example, Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8), 391-397 (2000), and references therein.

Pharmaceutical compositions

In another aspect, the present description includes a pharmaceutical composition comprising one or more antibodies of the present invention, combined with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the invention may also be administered as part of a combination therapy, for example, with another immunostimulating agent, an anticancer agent, an antiviral agent or a vaccine, such that the anti-LAG-3 antibody stimulates an immune response against the vaccine. The pharmaceutical composition may include any number of excipients. Excipients that may be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending agents, solvents, coloring agents, flavoring agents, coating agents, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. . The selection and use of suitable excipients is described in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), described here for reference.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound may be coated with a material to protect it from acids and other natural conditions that may cause it to inactivate. The expression parenteral administration as used herein means methods of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal , subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intracapsular, epidural and epigastric injection and infusion. Alternatively, the antibody of the invention may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually or topically. The pharmaceutical compositions of the invention may include pharmaceutically acceptable salts. The term pharmaceutically acceptable salt means a salt that retains the desired biological activity of the parent compound and does not introduce any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as from non-toxic organic amines such as N,N-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

Pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They may also be prepared in the form of a microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

The amount of active ingredient that can be combined with the carrier material to formulate a single dosage form will vary depending on the subject being treated and the particular route of administration and will generally correspond to the amount of the composition that produces a therapeutic effect. Typically, on a one hundred percent basis, this amount will range from about 0.01% to about ninety-nine percent of the active ingredient, preferably from about 0.1 to about 70%, most preferably from about 1 to about 30% of the active ingredient in the combination with a pharmaceutically acceptable carrier. Dosage regimens are adjusted to provide the optimal desired response (eg, response to therapy). For example, a single bolus may be administered, or multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased depending on the therapeutic situation. Particularly preferred is the formulation of parenteral compositions in dosage form for ease of administration and uniformity of dosage.

The term dosage form as used herein means physically discrete units suitable as unit dosage forms for subjects being treated; each unit contains a predetermined amount of active compound, calculated to ensure the desired therapeutic effect

- 20 044665 tic effect together with the required pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained release formulation, which requires less frequent administration.

For administration of the antibody, the dose ranges from about 0.0001 to 100 mg/kg and more commonly from 0.01 to 5 mg/kg of host body weight. For example, doses may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, or 10 mg/kg body weight, or in the range of 1-10 mg/kg body weight. kg. An exemplary treatment regimen includes administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months. Preferred dosing schedules for the anti-LAG-3 antibody of the invention include 1 mg/kg body weight or 3 mg/kg body weight when administered intravenously, the antibody being administered using one of the following dosing schedules: (i) every four weeks for the first six doses, then every three months; (ii) every three weeks; (w) 3 mg/kg body weight once and then 1 mg/kg body weight every three weeks. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg/ml, and in some methods, about 25-300 μg/ml.

A therapeutically effective dose of the anti-LAG-3 antibody of the invention preferably results in a reduction in the severity of symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or the prevention of deterioration or disability due to the disease. For example, for treating subjects with a tumor, a therapeutically effective dosage preferably inhibits tumor growth by at least 20%, more preferably by at least 40%, even more preferably by at least 60%, and more preferably by at least , by 80% relative to the subject in the absence of treatment. A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise alleviate symptoms in a subject, which is typically a human or other mammal.

The pharmaceutical composition may be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Therapeutic compositions can be administered using medical devices, such as needleless hypodermic devices (for example, US Pat. 4596556); (2) microinfusion pumps (US Pat. No. 4,487,603); (3) transdermal devices (US Patent 4,486,194); (4) infusion devices (US patents 4447233 and 4447224); and (5) osmotic devices (US patents 4439196 and 4475196); which are described here for reference. In some embodiments, human monoclonal antibodies of the invention may be formulated to ensure proper distribution in vivo. For example, to enable therapeutic compounds of the invention to cross the blood-brain barrier, they may be formulated in liposomes, which may further include targeting moieties to provide selective transport to specific cells or organs. See, for example, US patents 4522811; 5374548; 5416016 and 5399331; V.V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al. (1995) Am. J. Physiol. 1233:134; Schreiere et al. (1994) J Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

Applications and methods of the invention

The antibodies (compositions, bispecific antibodies and immunoconjugates) of the present invention have a variety of in vitro and in vivo applications, including, for example, detecting LAG-3 or promoting immune responses by blocking LAG-3. In a preferred embodiment, the antibodies are human antibodies. Such antibodies can be administered to cells in culture, in vitro or ex vivo, or to human subjects, for example, in vivo, to stimulate immunity in various situations. Accordingly, in one aspect, the invention provides a method for modifying an immune response in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof of the invention to modify the immune response in the subject. Preferably, the response is enhanced, stimulated or up-regulated.

Preferred subjects include human patients in need of an enhanced immune response. These methods are preferably suitable for treating human patients having a disorder that can be treated by enhancing an immune response (eg, a T cell-mediated immune response). In a particular embodiment, these methods are particularly suitable for treating cancer in vivo. To achieve antigen-specific immune stimulation, anti-LAG-3 antibodies may be coadministered with the antigen of interest, or the antigen may already be present in the subject being treated (eg, a subject with a tumor or virus). When administering antibodies to LAG-3 together with another agent, the two agents can be administered in any order or one

- 21 044665 temporarily.

The invention also relates to methods for detecting the presence of an antigen in a sample or measuring the amount of human LAG-3 antigen, comprising contacting the sample and a control sample with a human monoclonal antibody or an antigen-binding portion thereof that specifically binds to human LAG-3 under conditions that allow formation of a complex between an antibody or part thereof and human LAG-3. Complex formation is further detected, with the difference between complex formation between the sample and the control sample indicating the presence of human LAG-3 antigen in the sample. Moreover, the anti-LAG-3 antibodies of the invention can be used to purify human LAG-3 by immunoaffinity purification.

In view of the ability of the anti-LAG-3 antibodies of the invention to inhibit the binding of LAG-3 to MHC class II molecules and stimulate antigen-specific T cell responses, the invention also provides in vitro and in vivo methods of using antibodies to stimulate, enhance or upregulate antigen-specific T-cell responses. cellular responses. For example, the invention relates to a method of stimulating an antigen-specific T-cell response, comprising contacting said T-cell with an antibody of the invention such that the antigen-specific T-cell response is stimulated. Any suitable indicator of antigen-specific T-cell response can be used to measure the antigen-specific T-cell response. Non-limiting examples of these suitable indicators include increased T cell proliferation in the presence of an antibody and/or increased cytokine production in the presence of an antibody. In a preferred embodiment, interleukin-2 production is stimulated by an antigen-specific T cell.

The invention also provides a method of stimulating an immune response (eg, an antigen-specific T-cell response) in a subject, comprising administering an antibody of the invention to the subject such that the immune response (eg, an antigen-specific T-cell response) of the subject is stimulated. In a preferred embodiment, the subject is a subject with a tumor, and an immune response against the tumor is stimulated. In another preferred embodiment, the subject is a subject with a virus, and an immune response against the virus is stimulated.

In another embodiment, the invention provides a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject an antibody of the invention such that the growth of a tumor in the subject is inhibited. In another embodiment, the invention provides methods for treating a viral infection in a subject, comprising administering to the subject an antibody of the invention such that the viral infection in the subject is treated. These and other methods of the invention are described in more detail below.

Cancer

Blockade of LAG-3 by antibodies may enhance a patient's immune response to cancer cells. In one aspect, the present invention relates to treating a subject in vivo with an anti-LAG-3 antibody such that the growth of cancerous tumors is inhibited. Ahtu-LAG-3 antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, the anti-LAG-3 antibody may be used in conjunction with other immunogenic agents, conventional cancer treatments, or other antibodies, as described below.

Accordingly, in one embodiment, the invention provides a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of an anti-LAG-3 antibody or antigen-binding portion thereof. Preferably, the antibody is a human anti-LAG-3 antibody (such as any of the anti-human LAG-3 antibodies described herein). Additionally or alternatively, the antibody may be a chimeric or humanized anti-LAG-3 antibody.

Preferred types of cancers whose growth can be inhibited using the antibodies of the invention include those typically responsive to immunotherapy. Non-limiting examples of preferred types of cancer for treatment include melanoma (eg, metastatic malignant melanoma), kidney cancer (eg, hypernephroma), prostate cancer (eg, hormone-unresponsive prostate adenocarcinoma), breast cancer, colon cancer, and lung cancer (eg, non-small cell lung cancer). In addition, the invention includes intractable or recurrent malignant tumors, the growth of which can be inhibited using the antibodies of the invention.

Examples of other types of cancer that can be treated using the methods of the invention include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer anal cancer, stomach cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestinal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer , adrenal cortex cancer, soft tissue sarcoma, urethral cancer, penile cancer, chromosomal

- 22 044665 nic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvic carcinoma, tumor of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced malignancies, including asbestos-induced malignancies , and combinations of these types of cancer. The present invention can also be used to treat metastatic cancers, in particular metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17:133-144). Antibodies to LAG-3 may optionally be combined with an immunogenic agent such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells and cells transfected with genes encoding immunostimulatory cytokines (He et al (2004) J Immunol 173:4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides, such as gp100 peptides, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (described further below).

Some human tumors, such as melanomas, have been found to be immunogenic. Anti-tumor responses in the host can be activated by increasing the threshold of T cell activation through LAG-3 blockade.

LAG-3 blockade appears to be more effective when combined with a vaccination protocol. Many experimental tumor vaccination strategies have been developed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023- 3043 in DeVita et al (eds.), 1997, Cancer: Principles and Practice of Oncology, 5th ed.). In one such strategy, a vaccine is prepared using autologous or allogeneic tumor cells. Such cell vaccines have been shown to be most effective at transducing tumor cells to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43). The study of gene expression and large-scale gene expression patterns in various tumors has allowed the identification of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, tumor-specific antigens are differentiation antigens expressed in tumors and in the cell from which the tumor arose, such as melanocyte antigens gp100, MAGE and Trp-2 antigens. Most importantly, many of these antigens have been shown to be targets of tumor-specific T cells found in the host. LAG-3 blockade can be used in conjunction with a set of recombinant proteins and/or peptides expressed in the tumor to produce an immune response to these proteins. These proteins are normally considered by the immune system as autoantigens and, therefore, are tolerant to it. The tumor antigen may also include the protein telomerase, which is required for the synthesis of chromosomal telomeres and which is expressed in more than 85% of human cancer types and only in a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013). (These somatic tissues can be protected from immune attack in various ways.) Tumor antigen can also be neoantigens expressed in cancer cells due to somatic mutations that change the protein sequence or create fusion proteins between two unrelated sequences (eg, bcr-abl in the Philadelphia chromosome) or idiotype from B-cell tumors.

Other tumor vaccines may include proteins from viruses implicated in human cancers, such as human papillomaviruses (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used in conjunction with LAG-3 blockade is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. Heat shock proteins contain protein fragments from tumor cells, and these HSPs are highly effective in delivering antigen presenting cells to induce antitumor immunity (Suot & Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117- 120).

Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime antigen-specific responses. DCs can be produced ex vivo and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. DCs have also been fused directly to tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination modality, DC immunization can be effectively combined with LAG-3 blockade to promote stronger antitumor responses.

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LAG-3 blockade could be combined with standard cancer treatments. LAG-3 blockade can be effectively combined with chemotherapy treatments. In these cases, it is possible to reduce the dose of the chemotherapy reagent administered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is an anti-LAG-3 antibody in combination with decarbazine for the treatment of melanoma. Another example of this combination is an anti-LAG-3 antibody in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale for the combined use of LAG-3 blockade and chemotherapy is that cell death, which is a consequence of the cytotoxic effects of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway. Other combination treatments that may synergize with LAG-3 blockade via cell death are radiation, surgery, and endocrine shutdown. Each of these protocols allows the creation of a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with LAG-3 blockade. Inhibition of angiogenesis results in the death of tumor cells, which can supply tumor antigen to the host antigen presentation pathway.

LAG-3 blocking antibodies can also be used in combination with bispecific antibodies that target Fca or Fcy receptor expressing effector cells to tumor cells (see, for example, US Pat. Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor/tumor antigen bispecific antibodies (eg, Her-2/neu) have been used to target macrophages to tumor sites. Such targeting may more effectively activate tumor-specific responses. The T cell lineage of these responses may be enhanced by LAG-3 blockade. Alternatively, the antigen can be delivered directly to DCs using bispecific antibodies that bind to the tumor antigen and a dendritic cell-specific cell surface marker.

Tumors evade immune control through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins that are expressed by tumors and that are immunosuppressive. These include, among others, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200) , and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each of these agents can be used in combination with an anti-LAG-3 antibody to counteract the effects of the immunosuppressive agent and promote host immune responses against the tumor.

Other antibodies that activate the host immune response may be used in combination with an anti-LAG-3 antibody. They include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively replace T-helper cell activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in conjunction with anti-LAG-3 antibodies (Ito et al. (2000) Immunobiology 201 (5 ) 527-40). Activation of antibodies to T cell co-stimulatory molecules such as CTLA-4 (eg, US Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero et al. ( 1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) may also provide increased levels of T cell activation.

Currently, bone marrow transplantation is used to treat various tumors of hematopoietic origin. While graft rejection is a consequence of such treatment, beneficial therapeutic effects may be achieved through graft-versus-tumor response. LAG-3 blockade can be used to enhance the effectiveness of transplanted donor tumor-specific T cells.

There are also several experimental treatment protocols that involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to recipients to stimulate antigen-specific T cells against the tumor (Greenberg & Riddell (1999) Science 285: 546-51). Such methods may also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-LAG-3 antibodies can increase the occurrence and activity of adoptively transferred T cells.

Infectious diseases

Other methods of the invention are used to treat patients infected with specific toxins or pathogens. Thus, another aspect of the invention relates to a method of treating an infectious disease in a subject, comprising administering to the subject an anti-LAG-3 antibody or an antigen-binding portion thereof such that the subject is treated for said infectious disease. Preferably, the antibody is a human anti-LAG-3 antibody (eg, any of the human anti-LAG-3 antibodies described herein). Additionally or alternatively, the antibody may be a chimeric or humanized antibody.

Similar to its use against tumors as discussed above, antibody-mediated

- 24 044665 LAG-3 blockade can be used alone or as an adjunct in combination with vaccines to stimulate an immune response to pathogens, toxins and autoantigens. Examples of pathogens against which this therapeutic approach may be particularly effective include pathogens that are not eradicated by currently available vaccines or pathogens for which standard vaccines are not sufficiently effective. These include, but are not limited to, HIV, hepatitis viruses (A, B and C), influenza viruses, herpes viruses, giardiasis, malaria, leishmaniasis, Staphylococcus aureus, Pseudomonas aeruginosa. LAG-3 blockade is particularly useful against common infections with agents such as HIV that present altered antigens during the period of infection. These new epitopes are recognized as foreign when anti-LAG-3 antibodies are administered, thereby provoking a strong T cell response that is not overwhelmed by negative signals through LAG-3.

Some examples of pathogenic viruses that cause infections that can be treated with the methods of the invention include HIV, hepatitis virus (A, B or C), herpes virus (for example, VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, measles rubella virus, parvovirus, vaccinia virus, HTLV virus, virus Dengue, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections that can be treated by the methods of the invention include chlamydia, bacteria of the genus Rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria bacteria, Salmonella, bacilli , the causative agent of cholera, the causative agent of tetanus, the causative agent of botulism, the causative agent of anthrax, the causative agent of plague, the causative agent of leptospirosis and the bacteria that causes Lyme disease.

Some examples of pathogenic fungi causing infections that can be treated with the methods of the invention include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum. Some examples of pathogenic parasites causing infections that can be treated with the methods of the invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi , Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis. In all of the above methods, LAG-3 blockade can be combined with other forms of immunotherapy, such as cytokine treatment (eg, interferons, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides enhanced presentation of tumor antigens ( see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

Autoimmune reactions Anti-LAG-3 antibodies can provoke and enhance autoimmune responses. Indeed, the induction of antitumor responses using tumor cell and peptide vaccines has revealed that many antitumor responses involve reactivity against self-antigens (van Elsas et al. (2001) J. Exp. Med. 194:481-489; Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987; Hurwitz, (2000) above; Rosenberg & White (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4). Thus, the use of anti-LAG-3 blockade in conjunction with various autologous proteins can be considered to create vaccination protocols to effectively generate immune responses against these autologous proteins for the treatment of diseases. For example, Alzheimer's disease involves the unwanted accumulation of Av peptide in amyloid deposits in the brain; Anti-amyloid antibody reactions are capable of clearing these amyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other autologous proteins, such as IgE, may also be used as targets for the treatment of allergies and asthma, and TNFa may be used to treat rheumatoid arthritis. Finally, antibody responses to various hormones can be induced by the use of an anti-LAG-3 antibody. Neutralizing antibody responses against reproductive hormones can be used for contraception. Neutralizing antibody responses against hormones and other soluble factors required for the growth of certain types of tumors can also be considered as possible targets for vaccination.

Methods similar to those described above for the use of anti-LAG-3 antibodies can be used to induce therapeutic autoimmune responses to treat patients who have undesirable accumulation of other autoantigens, such as amyloid deposits, including Alzheimer's disease, cytokines, such as TNFa, and IgE.

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Anti-LAG-3 antibody vaccines can be used to stimulate antigen-specific immune responses by coadministering an anti-LAG-3 antibody with an antigen of interest (eg, a vaccine). Thus, in another aspect, the present invention provides a method of stimulating an immune response to an antigen in a subject, comprising administering to said subject: (i) an antigen and (ii) an anti-LAG-3 antibody or antigen-binding portion thereof such that the immune response to the given the subject's antigen is enhanced. Preferably, the antibody is a human anti-LAG-3 antibody (eg, any of the human anti-LAG-3 antibodies described herein). Additionally or alternatively, the antibody may be a chimeric or humanized antibody. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include those described in the sections above, such as the tumor antigens (or tumor vaccines) described above, or antigens from viruses, bacteria or other pathogens described above.

Suitable routes for administering antibody compositions (eg, human monoclonal antibodies, polyspecific and bispecific molecules, or immunoconjugates) of the invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art. For example, antibody compositions can be administered by injection (eg, intravenous or subcutaneous). Suitable dosages of the molecules used will depend on the age and body weight of the subject and the concentration and/or composition of the antibody composition.

As described above, the human anti-LAG-3 antibodies of the invention may be co-administered with one or more other therapeutic agents, such as a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. The antibody may be associated with the agent (as an immunocomplex) or it may be administered separately from the agent. In the latter case (in the case of separate administration), the antibody may be administered before, after, or in conjunction with the specified agent, or may be administered in conjunction with other known therapies, such as anticancer therapy, such as radiation. Such therapeutic agents include, but are not limited to, antineoplastic agents such as doxorubicin (Adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which themselves are effective only at levels that are toxic or subtoxic to the patient. Cisplatin is administered intravenously as a 100 mg/mL dose once every four weeks, and Adriamycin is administered intravenously as a 60-75 mg/mL dose once every 21 days. Co-administration of human anti-LAG-3 antibodies or antigen-binding fragments thereof of the present invention with chemotherapeutic agents provides two anti-cancer agents that act through different mechanisms that are cytotoxic to human tumor cells. Such co-administration may overcome problems caused by the development of drug resistance or changes in the antigenicity of tumor cells, making them non-reactive to the antibody. Also included within the scope of the present invention are kits comprising antibody compositions of the invention (eg, human antibodies, bispecific or polyspecific molecules, or immunoconjugates) and instructions for use. The kit may also contain at least one additional reagent or one or more additional human antibodies of the invention (eg, a human antibody having complementary activity that binds to an epitope in the LAG-3 antigen that is different from the epitope of the first human antibody). Kits typically include a label indicating the intended use of the kit's contents. The term label includes any text or printed material affixed to or included in the packaging of this kit, or included in any other form with this kit.

Combination therapy

In another aspect, the invention provides methods of combination therapy in which an anti-LAG-3 antibody (or an antigen-binding portion thereof) of the present invention is administered in conjunction with one or more additional antibodies that are effective in stimulating immune responses to further enhance, stimulate, or upregulate thus immune responses in the subject. In one embodiment, the invention provides a method of stimulating an immune response in a subject, comprising administering to the subject an anti-LAG-3 antibody and one or more additional immunostimulatory antibodies, such as an anti-PD1 antibody, an anti-PD-L1 antibody, and/or an anti-CTLA -4 antibody such that an immune response is stimulated in the subject, for example to inhibit tumor growth or stimulate an antiviral response. In another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-PD-1 antibody. In another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-PD-L1 antibody. In another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-CTLA-4 antibody. In one embodiment, the anti-LAG-3 antibody is a human antibody, such as the antibody described. Alternatively, the anti-LAG-3 antibody may be, for example, a chimeric or humanized antibody (eg, derived from a mouse anti-LAG-3 mAb). In another embodiment, it is realized

- 26 044665 at least one additional immunostimulatory antibody (eg, anti-PD1, anti-PD-L1 and/or anti-CTLA-4 antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody may be, for example, a chimeric or humanized antibody (eg, derived from anti-PD-1, anti-PD-L1 and/or anti-CTLA-4 mouse antibody). In another embodiment, the invention provides a method of treating a hyperproliferative disease (eg, cancer) comprising administering an anti-LAG-3 antibody and an anti-CTLA-4 antibody. In other embodiments, the anti-LAG-3 antibody is administered at a subtherapeutic dose, the anti-STEA-4 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. In another embodiment, the present invention provides a method of modifying a side effect associated with treatment of a hyperproliferative disease with an immunostimulating agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose of an anti-CTLA-4 antibody to a subject. In some embodiments, the subject is a human. In other embodiments, the anti-CTLA-4 antibody is a human sequence 10D1 monoclonal antibody (described in PCT publication WO 01/14424), and the anti-LAG-3 antibody is a human sequence monoclonal antibody, such as LAG3.5 described herein. Other anti-CTLA-4 antibodies within the methods of the present invention include, for example, the methods described in: WO 98/42752; WO 00/37504; US patent 6207156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301–5304. In some embodiments, the anti-CTLA-4 antibody binds to human CTLA-4 at a KD value of 5 x ^10'8 M or less, binds to human CTLA-4 at a KD value of 1x10'8 M or less, binds to human CTLA-4 with a KD value of 5 x 10' ⁹ M or less, or binds to human CTLA-4 with a KD value of 1 x 10' ⁸ M to 1 x 10' ¹⁰ M or less. In another embodiment, the present invention provides a method of treating a hyperproliferative disease (eg, cancer) comprising administering a LAG-3 antibody and a PD-1 antibody to a subject. In other embodiments, the anti-LAG-3 antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. In another embodiment, the present invention provides a method of modifying a side effect associated with treatment of a hyperproliferative disease with an immunostimulating agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose of an anti-PD-1 antibody to a subject. In some embodiments, the subject is a human. In some embodiments, the anti-PD-1 antibody is a human sequence monoclonal antibody and the anti-LAG-3 antibody is a human sequence monoclonal antibody, such as LAG3.5 described herein. Examples of anti-PD-1 antibodies with human sequence include 17D8, 2D3, 4H1, 5C4 and 4A11, which are described in PCT publication WO 06/121168. Other anti-PD-1 antibodies include, for example, lambrolizumab (WO 2008/156712) and AMP514 (WO 2010/027423, WO 2010/027827, WO 2010/027828, WO 2010/098788). In some embodiments, the anti-PD-1 antibody binds to human PD-1 at a KD value of 5 x ^10'8 M or less, binds to human PD-1 at a KD value of 1 x ^10'8 M or less, binds to PD-1 1 human with a KD value of 5 x 10' ⁹ M or less, or binds to human PD-1 with a KD value of 1 x 10' ⁸ M to 1 x 10' ¹⁰ M or less.

In another embodiment, the present invention provides a method of treating a hyperproliferative disease (eg, cancer) comprising administering a LAG-3 antibody and a PD-L1 antibody to a subject. In further embodiments, the anti-PD-1 antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. In another embodiment, the present invention provides a method of modifying a side effect associated with treatment of a hyperproliferative disease with an immunostimulating agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose of an anti-PD-L1 antibody to a subject. In some embodiments, the subject is a human. In other embodiments, the anti-PD-L1 antibody is a human sequence monoclonal antibody and the anti-LAG-3 antibody is a human sequence monoclonal antibody, such as LAG3.5 described herein. Examples of anti-PD-L1 antibodies with human sequence include 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT publication WO 07/005874. Other anti-PD-L1 antibodies include, for example, MPDL3280A (RG7446) (WO 2010/077634), MEDI4736 (WO2 011/066389) and MDX1105 (WO 2007/005874). In some embodiments, the anti-PD-L1 antibody binds to human PD-L1 at a KD value of 5 x ^10'8 M or less, binds to human PD-L1 at a KD value of 1 x ^10'8 M or less, binds to PD-L1 Human L1 with a KD value of 5 x 10' ⁹ M or less, or binds to human PD-L1 with a KD value of 1 x 10' ⁸ M to 1 x 10' ¹⁰ M or less.

Blockade of LAG-3 and one or more second target antigens, such as CTLA-4 and/or PD-1 and/or PD-L1, with antibodies can stimulate an immune response against cancer cells in a patient. Types of cancer whose growth can be inhibited by the antibodies of the present invention include those

- 27 044665 cancers that are usually sensitive to immunotherapy. Relevant examples of cancers to be treated using the combination therapy of the present invention include the cancers listed above in the description of anti-LAG-3 antibody monotherapy. In some embodiments, the combination of therapeutic antibodies described herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier or simultaneously as separate compositions with each antibody contained in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies can be administered sequentially. For example, an anti-CTLA-4 antibody and an anti-LAG-3 antibody may be administered sequentially, such that the anti-CTLA-4 antibody is administered first and the anti-LAG-3 antibody is administered second, or the anti-LAG-3 antibody is administered first and the anti-LAG-3 antibody is administered first and the anti-LAG-3 antibody is administered first. CTLA-4 antibody is administered second. Additionally or alternatively, the anti-PD-1 antibody and the anti-LAG-3 antibody may be administered sequentially, such that the anti-PD-1 antibody is administered first and the anti-LAG-3 antibody is administered second, or the anti-LAG-3 antibody is administered first, and the anti-PD-1 antibody is administered second. Additionally or alternatively, the anti-PD-L1 antibody and anti-LAG-3 antibody may be administered sequentially, such that the anti-PD-L1 antibody is administered first and the anti-LAG-3 antibody is administered second, or the anti-L AG-3 antibody is administered first. , and the anti-PD-L1 antibody is administered second. In addition, if more than one dose of combination therapy is administered sequentially, the order of sequential administration may be reversed or the same order of administration may be maintained at each administration time point, sequential administrations may be combined with co-administrations, or any combination thereof. For example, the first administration of a combination of anti-CTLA-4 antibody and anti-LAG-3 antibody may be simultaneous, the second administration may be sequential with the administration of anti-CTLA-4 and then anti-LAG-3, and the third administration may be sequential with the administration of anti-LAG-3 and then anti-CTLA-4, etc. Additionally or alternatively, the first administration of the combination of anti-PD-1 antibody and anti-LAG-3 antibody may be simultaneous, the second administration may be sequential with the administration of anti-PD-1 followed by anti-LAG-3, and the third administration may be sequential. with the introduction of anti-LAG-3 and then anti-PD-1, etc. Additionally or alternatively, the first administration of the combination of αntu-PD-L1 antibody and anti-LAG-3 antibody may be simultaneous, the second administration may be sequential with anti-PD-L1 followed by anti-LAG-3, and the third administration may be sequential. with the introduction of anti-LAG-3 and then anti-PD-L1, etc. Another exemplary dosing schedule may include a first administration that is sequential, with anti-LAG-3 administered first and anti-CTLA-4 antibody (and/or anti-PD-1 and/or anti-PD-L1) administered second, followed by administrations can be simultaneous.

Optionally, a combination of anti-LAG-3 and one or more additional antibodies (eg, anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies) may be further combined with an immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100 peptides, MAGE, Trp-2, MART1 and/or tyrosinase antigens or tumor cells transfected to express the cytokine GM-CSF (described further below). Combination blockade of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 can also be combined with a vaccination protocol, such as any of the vaccination protocols detailed above, including anti-LAG-3 antibody monotherapy.

Combination blockade of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 can also be combined with standard cancer therapies. For example, combination blockade of LAG-3 and CTLA-4 and/or PD1 and/or PD-L1 can be effectively combined with chemotherapeutic treatment regimens. In these cases, it is possible to reduce the dosage of the other chemotherapeutic agent administered with the combination of the present invention (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is a combination of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies also in combination with decarbazine for the treatment of melanoma. Another example is a combination of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies also in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale for the combined use of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade with chemotherapy is that cell death, which is a consequence of the cytotoxic effects of most chemotherapeutic compounds, should result in increased levels of tumor antigen in pathways of antigen presentation. Other combination therapies that may synergize with combined blockade of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 through cell death include radiation, surgery, and endocrine shutdown. Each of these protocols allows the creation of a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with combined blockade of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1. Inhibition of angiogenesis leads to the death of tumor cells, which may represent a source of tumor antigen supplied to the host antigen presentation pathway.

A combination of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blocking antibodies may also be used

- 28 044665 to be used in combination with bispecific antibodies that target Fca or Fcy receptor expressing effector cells to tumor cells (see, for example, US patents 5922845 and 5837243). Bispecific antibodies can be used to target two separate antigens. The effectiveness of T cells in these reactions will be increased by the use of combined blockade of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1.

As another example, combinations of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 antibodies and/or antiPD-L1 antibodies can be used in combination with anti-cancer antibodies such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexxar® (tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab), Lymphocide® (eprtuzumab), Avastin® (bevacizumab) and Tarceva® (erlotinib) and the like. By way of example and without being bound by theory, treatment with an anticancer antibody and an anticancer antibody conjugated to a toxin may result in the death of cancer cells (eg, tumor cells), which may enhance the immune response mediated by CTLA-4, PD-1, PD- L1 or LAG-3. In another embodiment, treatment of a hyperproliferative disease (eg, cancer) may include an anti-cancer antibody in combination with anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies, simultaneously or sequentially or in any combination thereof, which can enhance the host's antitumor immune responses. Tumors evade immune control through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins that are expressed by tumors and that are immunosuppressive. These include, among others, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200) and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). In another example, antibodies to each of these agents may also be combined with a combination of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies to counteract the effects of immunosuppressive agents and promote host immune responses against the tumor.

Other antibodies that can be used to activate the host immunological response can further be used in combination with a combination of anti-LAG-3 and anti-CTLA4 and/or anti-PD-1 and/or anti-PD-L1 antibodies. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Ahtu-CD40 antibodies (Ridge et al., supra) can be used in combination with a combination of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-LI antibodies (Ito et al. ., higher). Other activating antibodies against T cell costimulatory molecules (Weinberg et al. supra, Melero et al. supra, Hutloff et al. supra) may also provide increased levels of T cell activation. As described above, bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. Combined blockade of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 can be used to increase the efficacy of transplanted donor tumor-specific T cells.

Several experimental treatment protocols involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to recipients to stimulate antigen-specific T cells against the tumor (Greenberg & Riddell, supra). Such methods can be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies is expected to increase the occurrence and activity of adoptively transferred T cells.

In some embodiments, the present invention provides a method of modifying a side effect associated with treatment of a hyperproliferative disease with an immunostimulating agent, comprising administering to the subject an anti-LAG-3 antibody and a subtherapeutic dose of anti-CTLA4 and/or anti-PD-1 and/or anti-PD -L1 antibodies. For example, the methods of the present invention relate to methods of reducing the incidence of immunostimulating antibody-induced colitis or diarrhea by administering a non-absorbable steroid to a patient. Since any patient who will receive an immunostimulatory therapeutic antibody is at risk of developing colitis or diarrhea induced by such antibody, the entire patient population is suitable for therapy in accordance with the methods of the present invention. Although steroids have been administered to treat inflammatory digestive disease (IBD) and prevent exacerbations of IBD, they have not been used to prevent (reduce the incidence of) IBD in patients who have not been diagnosed with IBD. Significant side effects associated with steroids, even nonabsorbable steroids, have been considered a barrier to prophylactic use.

In other embodiments, combination blockade of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 (i.e., anti-LAG-3 and anti-CTLA-4 and/or antiPD-1 immunostimulatory therapeutic antibodies antibodies and/or anti-PD-L1 antibodies) can be further combined with the use of any non-absorbable steroid. As used herein, the term non-absorbable steroid means a glucocorticoid that exhibits extensive first-pass metabolism such that once metabolism in the liver is complete, the bioavailability of the steroid is low, i.e. less than about 20%. In one embodiment of the invention, the non-absorbable steroid

- 29 044665 is budesonide. Budesonide is a locally acting glucocorticosteroid that is extensively metabolized, primarily by the liver, after oral administration. ENTOCORT EC® (Astra-Zeneca) is a pH- and time-dependent oral formulation of budesonide designed to optimize drug delivery to the ileum and colon. ENTOCORT EU® is approved in the United States for the treatment of mild to moderate Crohn's disease involving the ileum and/or ascending colon. The usual oral dose of ENTOCORT EC® for the treatment of Crohn's disease is 6-9 mg/day. ENTOCORT EC® is released in the intestine before absorption and is stored in the intestinal mucosa. After passing through the target tissue of the intestinal mucosa, ENTOCORT EC® is extensively metabolized by the cytochrome P450 system in the liver to metabolites with negligible glucocorticoid activity. Thus, bioavailability is low (approximately 10%). This low bioavailability of budesonide results in an improved therapeutic index compared to other glucocorticoids with less extensive first-pass metabolism. Budesonide results in fewer side effects, including less hypothalamic-pituitary suppression, than systemically acting corticosteroids. However, prolonged administration of ENTOCORT EC® may result in systemic glucocorticoid effects such as hypercortisolism and adrenal suppression. See PDR 58th ed. 2004; 608-610.

In other embodiments, combination blockade of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 (i.e., anti-LAG-3 and anti-CTLA-4 and/or antiPD-1 immunostimulatory therapeutic antibodies and/or anti-PD-L1 antibodies) in combination with a non-absorbable steroid may additionally be combined with a salicylate. Salicylates include 5-ASA agents such as: sulfasalazine (AZULFIDINE®, Pharmacia &UpJohn); olsalazine (DIPENTUM®, Pharmacia &UpJohn); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay). In accordance with the methods of the present invention, the salicylate administered in combination with anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies and a non-absorbable steroid may include any overlapping or sequential administration of a salicylate and a nonabsorbable steroid to reduce the incidence of immunostimulatory antibody-induced colitis. Thus, for example, methods of reducing the incidence of colitis induced by the immunostimulatory antibodies of the present invention include administering salicylate and a non-absorbable steroid simultaneously or sequentially (eg, salicylate is administered 6 hours after administration of the non-absorbable steroid) or any combination thereof. In addition, in accordance with the present invention, the salicylate and the non-absorbable steroid may be administered by the same route (eg, both are administered orally) or by different routes (eg, the salicylate is administered orally and the non-absorbable steroid is administered rectally), which may differ from the route ( ov) used to administer anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or αntu-PD-L1 antibodies.

This description is further illustrated by the following examples, which should not be construed as limiting. The contents of all figures and references, sequences from the Genbank database, patents and published patent applications referenced herein are incorporated herein by reference in their entirety. In particular, the disclosures of PCT publications WO 09/045957, WO 09/073533, WO 09/073546 and WO 09/054863 are incorporated herein by reference in their entirety.

Examples

Example 1: Design of LAG3.1 variants (antibody 25F7)

Variants of the anti-LAG-3 antibody described above, 25F7, referred to here as LAG3.1, were generated based on initial analysis of the antibody's amino acid sequence for potential decay sites. Site-directed mutagenesis expression of the VH region of LAG3.1 was performed using the QuikChange II XL® Site-Directed Mutagenesis Kit (Agilent Technologies). The modified V _H regions were then subcloned into UCOE® vectors (EMD Millipore), which contain the human IgG4-S228P constant region. Each of the different heavy chain vectors was cotransfected with a vector expressing the LAG3.1 kappa chain into CHO-S cells, and stable pools were screened for expression.

Five potential deamidation motifs were identified in the heavy chain variable region of CDR2. These sites were located at positions 52, 54, 56, 58 and 60 of the LAG3.1 heavy chain variable region (SEQ ID NO: 2) (see Fig. 1A). In particular, deamidation of the NG sequence in CDR2 V _H (SEQ ID NO: 6) was observed under all conditions, as was additional isomerization of the sequence. Deamidation of the starting material was about 10%. In addition, it was found that this NG sequence does not correspond to the germline sequence (see Fig. 3). However, the germline consensus sequence represented a potential glycosylation site and thus was not present among the antibody variants.

Four variants (referred to here as LAG3.5, LAG3.6, LAG3.7, and LAG3.8) were obtained that corresponded to two potential deamidation motifs (positions 54 and 56), as shown

- 30 044665 in Fig. 3. These options were subjected to a state matrix, as indicated in table. 1 below, the following characteristics were analyzed: chemical and thermal stability (physical stability); (b) size exclusion chromatography (aggregation); (c) isoelectric focusing (IEF) gel (charge heterogeneity); (d) activity by Biacore assay (binding and functional activity) and (e) peptide mapping by mass spectrometry (chemical modifications/molecular stability).

Table 1

Buffer Acetate (100 nM NaCl, 3% w/v mannitol, 0.03% Tween 20) Citrate (100 nM NaCl, 3% w/v mannitol, 0.03% Tween-20) rn 5.5, 6.0, 6.5, 7.0 5.5, 6.0, 6.5, 7.0 Temperature 4°C and 37°C 4°C and 37°C Time 0, 4, 8, 12 weeks 0, 4, 8, 12 weeks

Example 2. Characteristics of LAG-3 options

1. Binding of activated human CP4 ⁺ T cells

To test the ability of antibody variants to bind to native human LAG-3 on the surface of activated human T cells, mononuclear cells from the peripheral blood of a normal healthy donor were stimulated on 15 cm tissue culture plates at a density of 2 x 10 ⁶ cells/ml with a combination of anti-CD3 (eBioscience, catalog number 16-0037-85) and anti-CD28 (BD Bioscience, catalog number 555725) antibodies present in solution at 5 μg/ml and 3 μg/ml, respectively. After three days of stimulation, cells were collected, washed 1x with 1x PFAE buffer (1x PBS + 2% FBS, 0.02% sodium azide, 2 mM Na EDTA) and resuspended in 1x PFAE staining buffer.

For the binding reaction, LAG3.1 variants were serially diluted with cold 1x PFAE buffer, then mixed 50 μl of the antibody dilution with 50 μl of Fitc-labeled CD4 anti-human antibody (BD Bioscience, catalog no. 555346) diluted 1:16 in 1x PFAE buffer. For the binding reaction, 100 μl of this diluted antibody mixture was added to 2 x 10 ⁵ cells, and the mixture was cultured at 4°C for 30 minutes. Cells were then washed twice with 1x PFAE buffer. A 1:200 solution of PE-labeled goat anti-human FcY-specific antibody (Jackson ImmunoResearch, catalog no. 109-116-170) was added and the mixture was cultured for 30 min at 4°C, followed by washing twice with cold 1x PFAE buffer. After the final wash, 150 μl of cold 1x PFAE was added to each solution and antibody binding analysis was performed by flow cytometry using a FACSCanto flow cytometer (BD Bioscience).

The results of the flow cytometry analysis are summarized in FIG. 4A, which is a graph showing the EC ₅₀ value for antibody binding to activated human CD4+ T cells. Fig. 4B is a graph showing antibody binding to soluble human LAG-3/Fc antigen using BIACORE. As shown, the binding affinities of LAG3.5 and LAG3.8 are slightly lower compared to LAG3.1, while their dissociation constants are slightly higher compared to LAG3.1.

2. Physical stability

Thermal stability and thermal denaturation of the variants were determined using Microcal VP-DSC. Specifically, each variant was diluted with PBS (Mediatech, catalog number 21-040-CV, lot 21040139). The final sample concentration was 250 μg/mL after dilution with PBS. The sample was heated to 74°C, cooled to 25°C and reheated to 74°C. PBS buffer was used as a null control. The data was fitted to a model without 2 states and the curve was fitted using Origin software.

As indicated in table. 2 and shown in FIG. 5, LAG3.5 had a higher melting point TM2 than LAG3.1, indicating higher overall stability.

- 31 044665

table 2

MAH Tml (°C) Corresponds to CH2 and/or Fab domains Tt2 (°C) Corresponds to CH3 and/or Fab domains LAG3.1 70.7 75.7 LAG3.5 70.5 76.3 LAG3.6 67.8 70.8 LAG3.7 69.4 73.5 LAG3.8 70.3 75.4

Antibody refolding after denaturation is the inverse of long-term aggregation potential. Thus, LAG-3 variants were also tested and compared in terms of thermal reversibility. Specifically, the antibodies were heated to 74°C and cooled to room temperature before being reheated to 74°C. The ratio of the area under the curves of the second and first thermograms allows us to estimate thermal reversibility, which is directly proportional to conformational reversibility.

As indicated in table. 3 and shown in FIG. 6, LAG3.5 had significantly higher thermal reversibility than all other variants. It should be noted that the percentage reversibility of LAG3.5 (47%) was more than two times higher than the percentage reversibility of LAG3.1 (20%). Thermal reversibility was strongly correlated with long-term aggregation potential. Lower reversibility corresponds to higher aggregation potential. Based on this observation, LAG3.1 would potentially have significantly higher aggregation over time compared to LAG3.5. Likewise, all other variants could potentially exhibit higher aggregation over time compared to LAG3.5.

Table 3

MAH Thermal reversibility (%) LAG3.1 20 LAG3.5 47 LAG3.6 0 LAG3.7 AND LAG3.8 26

3. Aggregation

The variants were also tested for stability as protein aggregation values using standard size exclusion HPLC (SEC-HPLC) according to the following protocol: antibody samples tested were diluted to 1.0 mg/mL with phosphate-buffered saline (PBS) and subjected to 10 μL HPLC ( Waters, model 2795). Separation was performed on a gel filtration column (TOSOH Bioscience, TSKgel G3000 SWxl, 7.8 mm x 300 mm, product 08541) using a mobile phase of 0.1 M sodium phosphate, 0.15 M sodium chloride, 0.1 M sulfate sodium, pH 7.2. The assay was detected by UV absorbance monitoring at 280 nm, and the percentage peak area of the antibody composition was determined using Empower software. As shown in table. 4, LAG3.5 had significantly reduced aggregation compared to LAG3.1.

Table 4

Sample IgG monomer (% peak area) IgG aggregate (% peak area) LAG3.1 90 10 LAG3.5 96 4 LAG3.6 96 4 LAG3.7 95 5 LAG3.8 95 5

Example 3. Selection of options

Based on the studies described above, the LAG3.5 antibody variant was selected for further analysis in light of its significantly increased physical and chemical stability compared to its unmodified form (LAG3.1), in particular its high conformational refolding capacity (thermal reversibility) . This analysis included a two-stage

- 32 044665 stroke with (a) increased load, (b) and real-time stability assessment for 12 weeks. Specifically, LAG3.5 was cultured at 1.0 mg/ml in pH 8.0, 50 mM ammonium bicarbonate for 5 days at 40°C. The extent of modifications after 5 days was analyzed, as well as the effect on activity and stability. The LAG3.5 variant was then subjected to real-time stability analysis in PBS for 12 weeks and further analysis. The results of these studies are described below.

1. Antigen binding

As shown in FIG. 7 and in table. 5, no change in antigen binding was observed after 5 days. As also shown in FIG. 10A and B, LAG3.5 showed no change in antigen binding or physical stability after 12 weeks. In particular, LAG3.5 maintained higher affinity than LAG3.8 over the entire 12-week period at 4°C and 40°C.

Table 5

Clone ID Antigen K _D xlO' ⁹ (Μ) k _on x 10 ⁴ (1/Ms) Kir'IO' ⁴ (1/s) Lag3.1 PBS 0.21 166 3.44 pH8 0.20 184 3.61 Lag3.5 PBS 0.25 130 3.22 pH8 0.20 148 2.98 Lag3.8 PBS 0.25 147 3.68 pH8 0.25 162 4.02

2. Chemical modifications/Molecular stability

Peptide mapping by mass spectrometry was used to analyze the chemical/molecular stability of LAG3.5 compared to LAG3.1. Specifically, the purified body was fragmented, alkylated, dialyzed and digested with trypsin (Promega cat. V5111) and GluC (Roche Cat. 11047817001). Digestion products were analyzed using nano-LC tandem mass spectrometry (Thermo Fisher LTQ Orbitrap).

As shown in FIG. 8, LAG3.1 had increased heterogeneity in VH compared to LAG3.5 when assayed for increased stability at higher pH, resulting in deamidation of aspartic residues (step 1). The change in mass due to isomerization could not be determined under the current experimental conditions. The percentage change is expressed as the ratio of all changes to the original peak.

Moreover, as shown in FIG. 11, LAG3.1 had increased heterogeneity in V _H compared to LAG3.5 in a long-term real-time stability assay for 12 weeks at 4°C and 40°C (step 2).

3. Physical stability

Thermal reversibility was measured in PBS and at pH 8.0. Under both conditions, LAG3.5 again showed approximately twice the level of folding compared to LAG3.1. In particular, as shown in table. 6-8, LAG3.5 exhibited a refolding rate of 43% compared to 18% for LAG3.1 in PBS. LAG3.5 also exhibited a refolding rate of 48% compared to a refolding rate of 29% for LAG3.1 at pH 8.0.

Table 6. DSC:melting

MAb Condition Tml Tm2 Lag3.1 PBS 70.7 75.7 Lag3.1 pH8 70.4 75.6 Lag3.5 PBS 70.8 76.4 Lag3.5 pH8 70.5 76.3

Table 7 - Fluorolog-2:unfolding

MAb/mutants Midpoint (Μ) Aggregation (Μ) Lag3.1 PBS 1.99 — Lag3.1 pH8 2.08 — Lag3.5PBS 1.86 — Lag3.5 pH8 2.00 —

- 33 044665

Table 8. DSC:refolding

MAH % reversibility, PBS % reversibility, pH 8 Lag3.1 18 29 Lag3.5 43 48

4. Heterogeneity of charges

To analyze charge heterogeneity, variants were analyzed using isoelectric focusing (IEF) with standard markers with protein isoelectric point values of 5.5 and 10.0 compared to LAG3.1. Briefly, antibody solutions were applied to a prefabricated 1 mm thick isoelectric focusing gel with a protein isoelectric point value of 3-7 (Invitrogen, catalog number EC6648BOX) along with markers with a protein isoelectric point value of 3-10 (SERVA, catalog number 39212). Electrophoresis was carried out using IEF 3-7 cathodic buffer (Invitrogen, catalog number LC5370) and IEF anodic buffer (Invitrogen, catalog number LC5370) and using electric current with a constant voltage of 100 V for 1 hour, a constant voltage of 200 V in for 1 hour and a constant voltage of 500 V for 30 minutes. IEF gels were stained with Coomassie blue to detect protein bands and destained with a solution of methanol and acetic acid. IEF gels were then analyzed using ImageQuant TL software. Based on this analysis (data not shown), LAG3.5 showed significantly less heterogeneity compared to LAG3.1.

5. CHB-HPLC

For solubility analysis, variants were analyzed using standard hydrophobic interaction chromatography (CHB-HPLC) according to the following protocol: 50 μL of 2 M ammonium sulfate was added to 50 μL of antibody test sample at 1 mg/mL, then 80 μL of test sample was applied to the HPLC column (Waters, model 2795) connected in series with a CHB column (TOSOH Bioscience, Ether-5PW TSK-gel, 7.5 mm x 75 mm, product number 07573). The sample was eluted at a flow rate of 1.0 ml/min with a gradient of 100% buffer A (2 M ammonium sulfate, 0.1 M sodium phosphate, pH 7.0) to 100% buffer B (0.1 M sodium phosphate, pH 7.0) within 50 min. Antibody was detected by monitoring UV absorbance at 280 nm and data analyzed using Empower software. As shown in FIG. 9, the hydrophilicity of LAG3.5 ensured solubility at high concentrations of ammonium sulfate.

Example 4 Reversing T Cell-Mediated Immune Response Inhibition

LAG3.5 activity was determined using a functional assay using an antigen-specific mouse T-cell hybridoma (3A9). Hybridoma 3A9 expresses a T-cell receptor specific for a peptide from chicken egg lysozyme (HEL48-62) and secretes IL-2 when co-cultured with peptide-matched MHC antigen-presenting cells (LK35.2). Because huLAG-3-Fc is able to bind to murine MHC class II B cell lines, expression of huLAG-3 in the 3A9 lineage may have an inhibitory effect through interaction with class II in the presenting mouse line. Comparison of the peptide response profile of the 3A9 source with that of LAG-3 transduced 3A9 cells co-cultured with MHC-matched antigen-presenting cells showed that expression of human LAG-3 inhibited peptide responses compared with control 3A9 cells. This inhibition was reversed by LAG-3 blockade using LAG3.5. Thus, blockade of LAG-3-mediated inhibition was shown for LAG3.5.

Example 5 Activation of T Cells by LAG3.5

The functional effect of LAG3.5 on primary T cells was analyzed using human peripheral blood mononuclear cell (PBMC) cultures stimulated with SEB superantigen. The entire volume of PBMCs was isolated from the blood of eighteen human donors and stimulated for 72 hours in either of two assay formats:

(i) fixed amount of antibody (20 μg/ml) and serial dilutions of SEB or (ii) fixed amount of SEB (85 ng/ml) and serial dilutions of antibody. The level of secreted IL-2, as a measure of T cell activity, was monitored using ELISA. Anti-PD-I antibody and ipilimumab were used as positive controls, and the activity of LAG3.5 in combination with anti-PD-1 or anti-CTLA-4 was also assessed in a subset of donors.

Increased IL-2 secretion was observed across a range of SEB concentrations in fifteen of eighteen donors treated with LAG3.5 alone compared with isotype control antibody treatment.

In most cases, the level of stimulation was less than that observed with anti-PD treatment! or ipilimumab. Considering LAG3.5, the results of the two analysis formats (described above) were consistent with each other. Moreover, in 5 of 6 donors studied, combining LAG3.5 with anti-PD-1 or ipilimumab resulted in higher levels of stimulation than those observed for the isotype control antibody combined with anti-PD-1 or ipilimumab.

- 34 044665

These data indicated that LAG3.5 can function in normal human T cell assays and can also activate responses mediated by inhibition of PD-1 and CTLA-4 function.

Brief list of sequences

SEP ID NO: DESCRIPTION SUBSEQUENCE 1 _VH na 25F7 (LAG3.1)

>1408_LAG-3_403_25F7.1_VHl_NT

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTC CCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGATTACTACTGGAACTGGATCCG CCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAATGGAAACA CCAACTCCAACCCGTCCCTCAAGAGTCGAGTCACCCTATCACTAGACAGTCCAAGA ACCAGTTCTCCCTGA AGCTGAGGTCTGTGACCGCCGCGGACACGGCTGTGTATTACT GTGCGTTTGGATATAGTGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGA ACCCTGGTCACCGTCTCCTCA | 2 | U _n a.a. 25F7 | | >1408_LAG-3_403_25F7.1_VHl_AA

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHNGNTN SNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLV TVSS | 3 | U _k pa. 25F7 | | >1408_LAG-3_403_25F7.1_VKl_NT

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTGGTACCAACAG AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGC ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTAGAGCC TGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCT CTCACTTTTGGCCAGGGGACCAACCTGGAGATCAAA

[4 I Criminal Code a.a. 25F7 I I >1408_LAG-3_403_25F7.1_VKl_AA

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPAR

FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK

5 V _H CDR1 aa 25F7 DYYWN 6 V _H CDR2 aa 25F7 EINHNGNTNSNPSLKS 7 V _H CDR3 aa 25F7 GYSDYEYNWFDP 8 V _K CDR1 aa 25F7 RASQSISSYLA 9 V _K CDR2 aa 25F7 DASNRAT 10 V _K CDR3 aa 25F7 QQRSNWPLT eleven V _H na LAG3.5

Un n.a. LAG3.5 caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcagtg attactactggaactggatccgccagcccccagggaaggggctggagtggattggggaaatcaatcatcgtggaagcaccaactccaacc cgtccctcaagagtcgag tcaccctatcactagacacgtccaagaaccagttctccctgaagctgaggtctgtgaccgccgcggacacggc tgtgtattactgtgcgtttggatatagtgactacgagtacaactggttcgacccctggggccagggaaccctggtcaccgtctcctca

I 12 | V _H aa LAG3.5 | |

V _H aa LAG3.5

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGE INHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYS D YEYNWFDPWGQGTL VTVS S

I 13 IV _K naLAG3.5 | |

V _K na LAG3.5 gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtattagcag ctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccagg ttcagtggcagtgggtctgg gacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagca actggcctctcacttttggccaggggaccaacctggagatcaaa

14 V _K aa LAG3.5

V _K aa LAG3.5

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYD

ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQ

- 35 044665

GTNLEIK

15 V _H CDR1 aa LAG3.5 DYYWN 16 V _H CDR2 aa LAG3.5 EINHRGSTNSNPSLKS 17 V _H CDR3 aa LAG3.5 GYSDYEYNWFDP 18 V _K CDR1 aa LAG3.5 RASQSISSYLA 19 V _K CDR2 aa LAG3.5 DASNRAT 20 V _K CDR3 aa LAG3.5 QQRSNWPLT 21 LAG-3 epitope PGHPLAPG 22 LAG-3 epitope HPAAPSSW 23 LAG-3 epitope PAAPSSWG 24 V _H CDR2 aa LAG3.6 EIIHSGSTNSNPSLKS 25 V _H CDR2 aa LAG3.7 EINHGGGTNSNPSLKS 26 V _H CDR2 aa LAG3.8 EINHIGNTNSNPSLKS 27 V _H CDR2 aaHUMAN GERMLINE GEINHSGSTNY 28 (Gly ₄ -Ser) ₃ 29 LAG-3 human a.a.

sequence of human LAG-3 a.a.

MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRR AGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPL QPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTA SPPGSLRASDWVILNC SF SRPDRPAS VHW FRNRGQGRVPVRESPHHHLAESFLFLPQ VSP MDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRS FLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAII TVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLS QPW QCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLTLGVLSLLLLVTGAF GFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEPEQL*

thirty V _H CDR2 aa LAG3.2 VIWYDGSNKYYADSVKG 31 V _H LAG3.1na

LAG3.1HC

CAGGTGCAGCTACAGCAGTGGGGCGCCAGGACTGTTGAAGCCTTCGGAGACCCTGTC

CCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGATTACTACTGGAACTGGATCCG

CCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAATGGAAACA

CCAACTCCAACCCGTCCCTCAAGAGTCGAGTCACCCTATCACTAGACACGTCCAAGA

ACCAGTTCTCCCTGAAGCTGAGGTCTGTGACCGCCGCGGACACGGCTGTGTATTACT

- 36 044665

GTGCGTTTGGATATAGTGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGA ACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCG CCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGA CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACA AGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGC CCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCA AAACCCAAGGACACTCTCATGATC TCCCGGACCCCTGAGGTCACGTGCGTGGTGGTG GACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTG TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGGCCTCCCGTCCTC CATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTG GACAAG AGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA CAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA

32 V _H LAG3.1 aa

TRANSLATION\OF\LAG3.1HC

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHNGNTN SNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

33 V _l LAG3.1 na

LAG3.1LC

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTGGTACCAACAG AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGC ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTAGAGCC TGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCT CTCACTTTTGGCCAGGGGACCAACCTGGAGATCAAACGTACGGTGGCTGCACCATCT GTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC TTCAACAGGGGAGAGTGTTAG

34 V _l LAG3.1 aa

TRANSLATION\OF\LAG3.1LC

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTH QGLSSPVTKSFNRGEC*

35 V _H LAG3.5 aa

-

Claims (23)

LAG3.5 heavy chain sequence - complete QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGE INHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYS DYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* | 36 | V _H LAG3.5 η.a. | | LAG3.5 heavy chain sequence - full caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcagtg attactactggaactggatccgccagcccccagggaaggggctggagtggattggggaaatcaatcatcgtggaagcaccaactccaacc cgtccctca agagtcgagtcaccctatcactagacacgtccaagaaccagttctccctgaagctgaggtctgtgaccgccgcggacacggc tgtgtattactgtgcgtttggatatagtgactacgagtacaactggttcgacccctggggccagggaaccctggtcaccgtctcctcagctag caccaagggcccatccgtcttccccctggcgccctgct ccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggact acttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggact ctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacacct gcaacgtagatcacaagcccagcaacac caaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgagttcctggggggaccatcagtcttcctg ttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccga ggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtg tggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctcca tcgagaaaaccatctccaaagcca aagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaa ccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtggggagagcaatgggcagccggagaacaact acaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaacc gtggacaagagcaggtggcaggagggg aatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatga 37 V _L LAG3.5a.a. LAG3.5 kappa chain sequence - complete EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQ GTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQES VTEQD SKD STYSL S STLTL SKAD YEKHK VY ACEVTHQG LSSPVTKSFNRGEC* I 38 IV _l LAG3.5 na | | LAG3.5 - kappa chain sequence - completely gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtattagcag ctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccagg ttcagtggcagt gggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagca actggcctctcacttttggccaggggaccaacctggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagc agttgaaatctggaactgcctctgttgtg tgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctcc aatcgggtaactcccaggagagtgtcacagagcaggacagcaagggacagcacctacagcctcagcagcaccctgacgctgagcaaagc agactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctc gcccgtcacaaagagcttcaacaggggagagt gttag CLAIM 1. Pharmaceutical composition containing: (a) a monoclonal antibody or antigen-binding portion thereof that binds human lymphocyte activation gene 3 (LAG-3), (b) an anti-PD-1 antibody or antigen-binding portion thereof, and (c) a pharmaceutically acceptable carrier; wherein the monoclonal antibody or antigen-binding portion thereof that binds human LAG-3 comprises heavy chain CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 15, 16 and 17, respectively, and light chain CDR1, CDR2 and CDR3 regions, containing the amino acid sequences SEQ ID NO: 18, 19 and 20, respectively. 2. Pharmaceutical composition according to claim 1, where the monoclonal antibody or its antigen binds - 38 044665 The main portion that binds human LAG-3 contains heavy and light chain variable regions containing the amino acid sequences of SEQ ID NOs: 12 and 14, respectively. 3. The pharmaceutical composition according to claim 1 or 2, wherein the monoclonal antibody or antigen-binding portion thereof that binds human LAG-3 exhibits one or a combination of the following properties: (a) binding to monkey LAG-3; (b) lack of binding to mouse LAG-3; (c) inhibiting the binding of LAG-3 to major histocompatibility complex (MHC) class II molecules or (d) stimulating an immune response. 4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the monoclonal antibody or antigen-binding portion thereof, which binds human LAG-3, stimulates the production of interleukin-2 (IL-2) in an antigen-specific T-cell response and/or stimulates an antitumor immune answer. 5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding portion thereof that binds human LAG-3 is a full-length antibody. 6. Pharmaceutical composition according to any one of claims 1 to 5, which has an isotype of IgG1, IgG2 or IgG4. 7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the monoclonal antibody that binds human LAG-3 is a full-length human IgG4 antibody that binds human LAG-3 with a K _D of 0.27 x 10 ^-9 M or less, as determined by surface plasmon resonance. 8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the monoclonal antibody that binds human LAG-3 contains heavy and light chains containing the amino acid sequences of SEQ ID NOs: 35 and 37, respectively. 9. The pharmaceutical composition according to any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding portion thereof that binds human LAG-3 is an antibody fragment or a single-chain antibody. 10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the monoclonal antibody or antigen-binding portion thereof that binds human LAG-3 is a human antibody, a humanized or chimeric antibody, and the anti-PD-1 antibody or antigen-binding portion thereof is is a human antibody, humanized or chimeric antibody. 11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the monoclonal antibody or antigen-binding portion thereof that binds human LAG-3 and the anti-PD-1 antibody or antigen-binding portion thereof are human antibodies. 12. The pharmaceutical composition according to any one of claims 1 to 11, where the anti-PD-1 antibody or an antigen-binding part thereof contains the CDR1, CDR2 and CDR3 regions of the heavy and light chains of the 5C4 antibody. 13. The pharmaceutical composition according to any one of claims 1 to 12, where the anti-PD-1 antibody or its antigen-binding part contains variable regions of the heavy and light chains of the 5C4 antibody. 14. Pharmaceutical composition according to any one of claims 1 to 13, suitable for parenteral administration. 15. Pharmaceutical composition according to any one of claims 1 to 14, suitable for intravenous administration. 16. Pharmaceutical composition according to any one of claims 1 to 15 for stimulating an immune response in a subject. 17. The pharmaceutical composition according to claim 16, wherein the subject is a subject with a tumor and an immune response against said tumor is stimulated. 18. The pharmaceutical composition according to claim 16, wherein the immune response is an antigen-specific T-cell response such that an antigen-specific T-cell response is stimulated. 19. The pharmaceutical composition according to claim 18, wherein the production of interleukin-2 by an antigen-specific T cell is stimulated. 20. Pharmaceutical composition according to any one of claims 1 to 15 for inhibiting the growth of tumor cells in a subject. 21. The pharmaceutical composition according to any one of claims 1 to 15 for treating cancer in a subject. 22. The pharmaceutical composition according to claim 21, wherein the cancer is melanoma, metastatic malignant melanoma, renal cancer, hypernephroma, prostate cancer, hormone-unresponsive prostate adenocarcinoma, breast cancer, colon cancer, lung cancer or non-small cell lung cancer . 23. The pharmaceutical composition according to claim 21 or 22, wherein the cancer is an intractable or recurrent malignant tumor, or metastatic cancer. -