Patent for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

Infectious Diseases – Kent B. Thudium, Mark J. Selby, Kyra D. Zens, Mark Yamanaka, Alan J. Korman, Heidi N. LeBlanc, ER Squibb and Sons LLC

Search Patent for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

Abstract for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

“The present disclosure contains isolated monoclonal monoclonal antibody that binds to LAG-3 with high affinity. It is particularly useful for monoclonal human antibodies. The antibodies should bind to LAG-3 from human beings. The antibodies may bind LAG-3 to both monkey and human LAG-3 in certain embodiments. However, they do not bind LAG-3 to mice. Anti-LAG-3 antibodies are provided by the invention that inhibit LAG-3 binding to WIC Class II molecules. They can also stimulate antigen-specific T cells responses. The invention also provides nucleic acid molecules that encode the antibodies, expression vectors and host cells for the invention. The invention also provides bispecific molecules, immunoconjugates and pharmaceutical compositions containing the antibodies. This disclosure provides methods for detecting LAG-3 and methods for treating immune reactions using anti-LAG-3 antibodies of the invention. Combination therapy is where an anti-LAG-3 anti- antibody is administered in combination with at least one other immunostimulatory antibody.

Background for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

LAG-3, also known as CD223, is a member the immunoglobulin supergene and structurally and genetically related with CD4. LAG-3 does not express on resting peripheral blood lymphocytes, but it is expressed on activated T and NK cells. LAG-3, a membrane protein encoded in a gene on the distal end of the short arm chromosome 12, close to the CD4 gene suggests that LAG-3 may have evolved by gene duplication (Triebel and al. (1990) J. Exp. Med. 171:1393-1405).”

“LAG-3 is similar to CD4, but unlike CD4, it has been shown to interact with MHC Class II molecules. However, LAG-3 does NOT interact with the human immune deficiency virus (HIV) gp120 protein (Baixeras and al. (1992) J. Exp. Med. 176:327-337). Huard et. al. demonstrated that LAG-3 can be bound directly to MHC class II cells by using a soluble LAG-3 immunoglobulin-fusion protein (sLAG-3Ig). (1996) Eur. J. Immunol. 26:1180-1186).”

“In vitro studies of antigen specific T cell responses showed that the addition of anti?LAG-3 antibodies led increased T cell proliferation, higher activation antigens like CD25, and higher levels of cytokines, such as interferon gamma or interleukin-4. This supports a role of the LAG/MHC class 2 interaction in downregulating antigen-dependent stimulation (Huard and al. (1994) Eur. J. Immunol. 24:3216-3221). LAG-3’s intra-cytoplasmic domain has been shown to interact with LAP (Iouzalen and al. (2001) Eur. J. Immunol. 31:2885-2891). LAG-3 has been demonstrated to be expressed by CD4+CD25+ regulatory cells (Treg), and that antibodies to LAG-3 can inhibit suppression of induced Treg cell (Huang, C., et al. (2004) Immunity 21.503-513. Further, LAG-3 was shown to negatively regulate the homeostasis of T cells by regulatory T-cells in both independent and dependent ways (Workman, C. J., and Vignali D. A. (2005) J. Immunol. 174:688-695).”

LAG-3 has also been shown to be immunostimulatory in certain situations. LAG-3-transfected tumor cells were transplanted into syngeneic mice and showed significant growth reductions or complete regression compared to untransfected cells. This suggests that LAG-3 expression on tumor cells stimulated an antitumor response via activating antigen presenting cells via MHC Class II molecules (Prigent et. al. (1999) Eur. J. Immunol. 29:3867-3876). In addition, the soluble LAG-3 Ig protein fusion protein stimulates both humoral as well as cellular immune responses in mice when given with an antigen (El Mir and Triebel, 2000 J. Immunol. 164:5583-5589). In vitro generation and maintenance of type I tumor-specific immunity has been enhanced by soluble human LAG-3Ig (Casati, et al. (2006) Cancer Res. 66:4450-4460). Triebel (2003) Trends Immunol. 24:619-622. A number of additional agents that can modulate the activity of LAG-3 may be of interest in light of the above.

The present disclosure contains isolated monoclonal monoclonal antibody, particularly human monoclonal antibody, that specifically bind LAG-3. It also has desirable functional properties. These properties include high affinity binding, binding to LAG-3 in human, monkey, and rhesus monkey LAG-3 (e.g. cynomolgus LAG-3 and/or rhesus LAG-3), the ability to inhibit LAG-3 binding to major histocompatibility Class II molecules, and/or the ability stimulate antigen-specific T cells responses. The invention’s antibodies can be used to detect LAG-3 proteins or stimulate antigen-specific cell responses in tumor-bearing subjects or virus-bearing subjects.

“In one aspect, this invention pertains to an isolate human monoclonal antibody or an antigen binding portion thereof wherein the antibody binds to human LAG-3 and exhibits at minimum one of these properties:

“In an alternative aspect, the invention refers to an isolated monoclonal antibody from a human being, or the antigen binding portion thereof. The antibody crosses-competes with a reference antibody to bind to human LAG-3.

“In a preferred embodiment, a reference antibody includes a heavy-chain variable region that contains the amino acids sequence of SEQID NO: 37 and a lighter chain variable region that comprises the amino acid sequences of SEQID NO: 43. A preferred embodiment of the reference antibody includes a heavy-chain variable region that contains the amino acids sequence of SEQID NO: 38 and a lighter chain variable region that contains the amino sequence of SEQ ID NO: 44. A preferred embodiment of the reference antibody includes a heavy-chain variable region that contains the amino acids sequence of SEQID NO: 39, and a light-chain variable region that comprises the amino sequence of SEQ ID NO: 45. A preferred embodiment of the reference antibody includes a heavy-chain variable region that contains the amino acids sequence of SEQID NO: 40, and a light-chain variable region that contains the amino sequence of SEQ ID NO 46. A preferred embodiment of the reference antibody includes a heavy-chain variable region that contains the amino sequence of SEQID NO: 41, and a light-chain variable region that comprises the amino sequence of SEQ ID NO: 47. A preferred embodiment of the reference antibody includes a heavy-chain variable region that contains the amino sequence of SEQID NO: 42, and a light-chain variable region that comprises the amino sequence of SEQ ID NO: 48.

“Another aspect of the invention is an isolated monoclonal anti-body, or an antigen binding portion thereof. It comprises a heavy chain variable area that is either the product or derived from a VH 3-20, VH 4-34, VH 1-23, or VH 3-33 genes. The antibody specifically binds to human LAG-3. Another aspect of the invention is an isolated monoclonal anti-body, or an antigen binding portion thereof. It comprises a light chain variable area that is either the product or derived form a VK L18 human gene, a VK L6 human gene or a VK A27 human gene. The antibody specifically binds to human LAG-3. Preferably, the invention is an isolated monoclonal antibody or an antigen binding portion of it.

“In another aspect of the invention, it pertains to an isolated monoclonal anti-body, or antigen binding section thereof.

“A preferred combination includes:

“Another preferred combination includes:

“The invention’s antibodies can include full-length antibodies such as IgG1, IgG2, or IgG4 isotypes. Preferably, the antibody is an IgG4 type. Another preferred embodiment is an IgG4 antibody with a serine-to-proline mutation in the heavy chains constant region hinge region (at position 241 according to Angal et. al. (1993) Mol. Immunol. 30:105-108), so that the inter-heavy-chain disulfide bridge heterogeneity can be reduced or eliminated. Alternately, antibodies can also be fragments of antibody, such as Fab or Fab? You can also use Fab?2 fragments or single-chain antibodies.

“This disclosure also contains an immunoconjugate that includes an antibody, or antigen binding portion thereof, and linked to a therapeutic drug, e.g. a cytotoxin, or radioactive isotope. This disclosure also includes a bispecific moiety that contains an antigen-binding or antibody portion of the invention linked to a second functional molecule with a different binding affinity than the antibody or antigen binding portion.

“Compositions that include an antibody or antigen-binding component thereof or an immunoconjugate, bispecific molecule, of the invention, and a pharmaceutically acceptable carrier” are also available.

This disclosure also includes nucleic acid molecules that encode the antibodies or antigen-binding parts thereof. Expression vectors comprising such nucleic Acids and host cells comprising these expression vectors are also included. Methods of preparing anti-LAG-3 antibodies using host cells that have such expression vectors are also described. These methods can include (i) expressing anti-LAG-3 antibody in the host cell, and (ii). isolating anti-LAG-3 antibody from the host.

“Another aspect of the invention relates to methods of stimulating immune reactions using anti-LAG-3 antibody of the invention. In one embodiment, the invention allows for stimulation of an antigen specific T cell response by contacting the T cell with an anti-LAG-3 antibody. Preferably, the antigen-specific cell stimulates interleukin-2 production. Another embodiment of the invention provides a method for stimulating an immune reaction (e.g. an antigen specific T cell response in a subject). This involves administering an antibody according to the invention to the subject so that the immune response (e.g. an antigen?specific T cell response in the subject) is stimulated. Preferably, the subject is a tumor-bearing person and the immune response is stimulated. Another preferred embodiment is that the subject is a virus-bearing person and the immune response is stimulated.

“In another aspect, the invention provides a method of inhibiting the growth of tumor cells within a subject. This involves administering an antibody of invention to the subject so that the growth of the tumor in the subject is stopped.” Another aspect of the invention is a method to treat viral infection in a subject. This involves administering an antibody from the invention to the subject so that the viral infection can be treated.

“Another aspect of the invention is that it provides a method to stimulate an immune response in a subject. This includes administering an anti?LAG-3 antibody to the subject and at least one additional antibody such as an anti?PD-1 antibody or anti?PD-L1 antibodies to the subject. The aim is to stimulate the immune system in the subject to instigate a response to prevent tumor growth and/or stimulate an antiviral response. One embodiment administers an anti-LAG-3 and anti-PD-1 antibodies to the subject. Another embodiment administers an anti-LAG-3 and anti-PD-1 antibodies to the subject. Another embodiment gives the subject an anti-LAG-3 and anti-CTLA-4 antibodies. One embodiment of the anti-LAG-3 antibodies is a human antibody such as the disclosed antibody. Alternately, the anti-LAG-3 antibodies can be, for instance, a humanized or chimeric antibody. Another embodiment includes at least one additional immunostimulatory antibodies (e.g. anti-PD-1, anti?PD-L1 or anti?CTLA-4 antibodies). This antibody can be a human antibody. Alternately, at least one additional immunostimulatory antibodies can be, for instance, a humanized or chimeric antibody.

“In another aspect, the invention relates to a method of preparing anti-LAG-3 antibodies. This method includes:

“A number of other features and benefits of the instant disclosure will become apparent from the detailed description and examples. These should not be taken as limiting. This application contains all references, Genbank entries and patents cited in this application. These are incorporated by reference.

The present disclosure concerns monoclonal monoclonal antibody, especially human monoclonal antibodies that bind to human LAG-3, and have desirable functional properties. The antibodies of the invention may be derived from specific heavy or light chain germline sequences or include particular structural features, such as CDR regions that contain particular amino acid sequences. This disclosure discloses isolated antibodies, methods for making them, immunoconjugates, bispecific molecules that contain such antibodies, and pharmaceutical compositions containing these antibodies, immunoconjugates, or bispecific molecules. This disclosure also covers methods of using antibodies to detect LAG-3 proteins, and methods of using anti-LAG-3 antibody of the invention to stimulate immune reactions, either alone or with other immunostimulatory agents. This disclosure also includes methods for using anti-LAG-3 antibodies of the invention to, among other things, treat viral infections or inhibit tumor growth.

“The present disclosure is easier to understand if certain terms are defined first. The detailed description also contains additional definitions.

“The term ‘LAG-3’ is an abbreviation of Lymphocyte Activation Gene-3. Lymphocyte activation gene-3 is the term. The term “LAG-3” is used. The term “LAG-3” includes variants, isoforms as well as orthologs, homologs, paralogs, and orthologs. In some cases, antibodies that are specific for the human LAG-3 proteins may cross-react with LAG-3 proteins from other species. Other embodiments of the antibody specific for a human LAG-3 proteins may only be specific for that LAG-3 protein. They may not show species or other types cross-reactivity or cross-react to LAG-3 from other species, but not all species. (e.g. cross-reacting with LAG-3 from monkey LAG-3, but not with LAG-3 in mice). “Human LAG-3” is the term. The term “human LAG-3” refers to the human sequence LAG-3. This includes the entire amino acid sequence for human LAG-3 with Genbank Accession No. NP_002277. The term “mouse LAG-3” is used. Refers to the mouse sequence LAG-3. This includes the complete amino-acid sequence of mouse LAG-3 with Genbank Accession No. NP_032505. LAG-3 is also known as CD223. LAG-3 sequences for humans may be different from the LAG-3 sequences for human LAG-3 in Genbank Accession No. NP_002277 may have, e.g. conserved mutations, or mutations in unconserved areas. The LAG-3 functions substantially the same as the Genbank Accession Number. NP_002277. One example of a biological function for human LAG-3 may be having an epitope within the extracellular domain LAG-3 that is bound by an antibody according to the instant disclosure. Another example of a biological function for human LAG-3 might be binding to MHC Class II molecules.

“Monkey LAG-3” is a term that refers to monkey LAG-3. LAG-3 proteins are expressed by Old World and New World monkeys. This includes cynomolgus and rhesus monkey LAG-3. FIG. shows a representative sequence of amino acids for monkey LAG-3. 19 and SEQ ID No: 85. This is also deposited under Genbank Accession Number. XM_001108923. FIG. 2 shows another representative sequence of amino acids for monkey LAG-3. 19, and SEQ ID NO. 84, as described in Example 3A. The Genbank-deposited sequence has a single amino acids difference in this alternative rhesus sequence, which is at position 419.

“A specific human LAG-3 sequence will usually be at least 90% identical in amino acid sequence to the human LAG-3 of Genbank Accession Number. NP_002277 contains amino acid residues which identify the amino acids sequence as human, when compared with LAG-3 amino sequences from other species (e.g. murine). A human LAG-3 can have a minimum 95% or 96%, 97% or 98% amino acid sequence, and be 99% identical to LAG-3 from Genbank Accession Number. NP_002277. A human LAG-3 sequence may not differ from Genbank Accession Number No. 10 in certain instances. NP_002277. The human LAG-3 may not display more than 5, or even 4, 2, or 1 amino acids difference from the LAG-3 sequence in Genbank Accession Number. NP_002277. As described in this article, you can determine percent identity.

“Immune response” is a term that refers to the body’s immune system. “Immune response” refers to the actions of lymphocytes and antigen-presenting cells, phagocytic and granulocytes as well as soluble macromolecules (including antibodies, complement, cytokines and complement) that cause selective damage to, destruction or elimination of the human body of pathogens, cells and tissues infected by pathogens, cancerous or normal cells and tissues.

“An ?antigen-specific T cell response? A T cell’s response to stimulation with an antigen-specific antigen is what it refers too. A T cell’s responses to antigen-specific stimulation are not limited to proliferation and cytokine (e.g. IL-2 production).

“Antibody” is a generic term that refers to whole antibodies and antigen binding fragments. “Antibody” as used herein refers to whole antibodies and antigen binding fragments (i.e., the?antigen-binding part). or single chains. Glycoproteins that contain at least two heavy (H), and two light (L), chains, interconnected by disulfide bond are called whole antibodies. Each heavy chain is composed of a heavy-chain variable region (abbreviated VH) as well as a heavy-chain constant region. The three domains that make up the heavy chain constant area are CH1, CH2 or CH3. Each light chain is composed of a light-chain variable region (abbreviated as VL) along with a light-chain constant region. One domain, CL, is the light chain constant region. You can further subdivide the VH and VL into regions of hypervariability (CDR), as well as regions that are conserved (FR). Each VH or VL contains three CDRs (and four FRs), arranged in the following order from amino-terminus through carboxy-terminus: FR1, CDR1, CDR2, CDR2, CDR2, CDR3, CDR3, and FR4. Variable regions of heavy and light chains have a binding domain that interacts to an antigen. The binding domains of antibodies in the constant regions can mediate the interaction of immunoglobulin with host tissues or factors. This includes various cells of immune system (e.g. effector cells) as well as the first component (Clq), of the classical complement system.

“The term ‘antigen-binding part? An antibody (or simply the?antibody section) is a portion of an antibody. As used herein, an antibody fragment (or simply?antibody portion) is one or more fragments that can bind specifically to an antigen (e.g. a LAG-3-protein). A fragment of a full length antibody can perform the antigen-binding function. The term “antigen-binding section” includes examples of binding fragments. A Fab fragment is a monovalent fragment that includes the VL and VH domains, CL, CH1 domains, and CL domains. (iii) A F(ab?) Fab2 fragment is a bivalent fragment that consists of two Fab fragments linked together by a disulfide link at the hinge region. (iii). A Fab? fragment, which is essentially a Fab with a portion of the hinge area (see FUNDAMENTAL IMMUNOLOGY, Paul ed.). 1993; (iv) A Fd fragment comprising the VH, CH1 and VH domains; and (v) A Fv fragment consisting only of the VL, VH, and CDR domains of an antibody. Although VL, and VH are distinct domains in the Fv fragment’s code, they can be combined using recombinant techniques to create a single protein chain. In this case, the VL-VH regions will pair to form monovalent molecules. This is known as single chain Fv (scFv); see Bird et. al. (1988) Science 242(4):423-426. Huston et. al. (1988) Proc. Natl. Acad. Sci. USA 85:5879?5883. These single-chain antibodies are also included in the term “antigen binding portion?” An antibody. These fragments of antibody are obtained using standard techniques that are well-known to those skilled in the art. They are then screened for utility in much the same way as intact antibodies.

An “isolated antibody” is an antibody that is substantially unbound by other antibodies with different antigenic specificities. For example, an antibody that binds to a LAG-3 protein is significantly free from antibodies that bind to other antigens than LAG-3 proteins. However, an isolated antibody that binds to a specific LAG-3 human protein could also cross-react with other antigens such as LAG-3 proteins of other species. An isolated antibody may also be free from other cellular material or chemicals.

“Monoclonal antibody” is a term that refers to monoclonal antibodies. Monoclonal antibody composition or monoclonal antibody? The term “monoclonal antibody composition” is used herein to refer to a preparation containing monoclonal antibodies molecules with a single molecular structure. Monoclonal antibody compositions have a single binding affinity and specificity for a particular epitope.

“Human antibody” is a term that refers to antibodies with variable regions. Both the CDR and framework regions are derived form human germline immunoglobulin sequencings. If the antibody has a constant area, that region is also derived from human germline immuneglobulin sequences. The invention allows human antibodies to include amino acid residues that are not encoded in human germline immunoglobulin sequencings. This includes mutations introduced either by random or site-specific mutation in vitro or in vivo. The term “human antibody” as used in this invention does not include antibodies in which CDR-derived sequences from another mammalian species (e.g., a mouse) have been grafted onto human frame sequences.

“Human monoclonal antibodies” is a term that refers to an antibody that is monoclonal. A single-binding specificity antibody is one that has variable regions. Both the CDR and framework regions are derived directly from human germline immunoglobulins sequences. One embodiment of the human monoclonal antibody is produced from a hybridoma that includes a human monoclonal antibody made from a human monoclonal antibody. This hybridoma may include a B cell derived from a transgenic nonhuman species, such as a transgenic mouse. It has a genome that contains a human heavy-chain transgene and light chain transgene, which are fused to immortalized cells.

Recombinant human antibodies, as they are called, include all antibodies that have been prepared, created, or expressed by recombinant methods. These recombinant human antibody have variable regions where the framework and CDR regions were derived from human germline immuneglobulin sequences. However, certain embodiments allow for in vitro mutation (or when an animal transgenic to human Ig sequences has been used, in-vivo somatic mutation) of such recombinant antibody recombinants. The VH and L regions of these recombinant antibodies contain sequences that are derived from the human germline VH or VL sequences.

“The term “isotype” refers to the antibody class (e.g. IgM or IgG1) that is encoded by the heavy chain constant region genes.” The antibody class (e.g. IgM or IgG1) encoded by heavy chain constant gene genes is called “isotype”.

“The expressions?an antigen-recognizing antibody? An antibody that recognizes an antigen and an antibody specific for it? These terms are interchangeable with the term “an antibody that binds specifically for an antigen?”

“Human antibody derivatives” is a term that refers to any modified form of the human antibody. Any modified form of the human antibodies, such as a conjugate of the antibody with another agent or antibody, is called “human antibody derivatives”.

“Humanized antibody” is a term that refers to antibodies that have been grafted onto human framework sequences. “Humanized antibody” refers to antibodies in which CDR-sequences derived from another mammalian species (e.g., a mouse) have been grafted onto human frame sequences. You can also modify the framework regions of the human framework sequences.

“Chimeric antibody” is a term that refers to an anti-microbial that can be chimed. “Chimeric antibodies” is an acronym that refers to antibodies in which variable region sequences come from one species and constant region sequences from another. For example, an antibody in the variable area sequences of a mouse antibody are derived while constant region sequences from a human antibody are derived.

“Subject” is a term that refers to any human or nonhuman animal. Any human or nonhuman animal is included in the definition of?subject? “Nonhuman animal” is a broad term. All vertebrates are included, e.g. mammals and non-mammals.

“Various aspects of the invention will be described in detail in the subsections below.”

Anti-LAG-3 Antibodies with Particular Functional Properties

The invention’s antibodies are distinguished by specific functional features or properties. The antibodies can bind specifically to human LAG-3, but may also bind LAG-3 of certain species such as monkey LAG-3 (e.g. cynomolgus monkey, or rhesus monkey), but not enough to bind LAG-3 form certain species such as mouse LAG-3. The invention prefers that an antibody binds to LAG-3 from human with high affinity.

The ability of an antibody to stimulate an immune reaction, such as an antigen specific T cell response, can be shown by, for instance, its ability to stimulate interleukin-2 production in an antigen?specific T cell reaction. An antibody of the invention can bind to LAG-3 in human cells and stimulate antigen-specific T cells. An antibody of the invention can bind to LAG-3 in human but not stimulate an antigen-specific response from T cells. Another way to assess the ability the antibody stimulates an immune response is to determine whether it inhibits tumor growth (see, for example, Example 6), or whether the antibody stimulates an autoimmune response (such as the ability to promote diabetes in the NOD mouse model).

One or more of the techniques that are well-known in the art can be used to determine whether an antibody of the invention binds to LAG-3. In a preferred embodiment, the antibody can be tested using a flow-cytometry assay. This is where the antibody is react with a cell that expresses LAG-3. Anti-CD3-stimulated CD4+ activated cells T cells that express native LAG-3 are also suitable for flow cytometry. You can also test the binding of the antibody and the binding kinetics (e.g. KD value) in BIAcore binding tests (see Example 3B). Other suitable binding assays include ELISA tests, such as using a recombinant LAG-3 proteins (see, e.g.., Example 1)

“Typically, an antibody of the invention binds to LAG-3 in lymphoid tissues, such as tonsil, spleen or thymus, which can be detected by immunohistochemistry. Additionally, as described further in Example 8, certain anti-LAG-3 antibodies of the invention stain pituitary tissue (e.g., are retained in the pituitary) as measured by immunohistochemistry, whereas other anti-LAG-3 antibodies of the invention do not stain pituitary tissue (e.g., are not retained in the pituitary) as measured by immunohistochemistry. Thus, in one embodiment, the invention provides a human anti-LAG-3 antibody that stains pituitary tissue by immunohistochemistry, whereas in another embodiment, the invention provides a human anti-LAG-3 antibody that does not stain pituitary tissue by immunohistochemistry.”

“Human monoclonal antibodies are the preferred antibodies of the invention.” The antibodies can also be chimeric, humanized, or a combination of both.

“Monoclonal Antibodies 25F7 and 26H10, 25E3, 25E3, 8B7 and 11F2”

“Preferred antibodies are the human monoclonal antibody 25F7 and 26H10, 25E3, 25E3, 8B7 and 11F2 respectively. These antibodies have been structurally characterized and isolated as per Examples 1 and 2. SEQ ID NOs 37-42 show the VH amino acids sequences of 25F7 and 26H10, 25E3, 8,B7, 11F2 & 17E5 respectively. SEQ ID NOs 43-48 show the VK amino acids sequences of 25F7 and 26H10, 25E3, 25E3, 8B7 and 11F2 respectively.

“As each of these antibodies can bind human LAG-3, it is possible to mix and match the VH/VL sequences. To create additional anti-LAG-3 binding molecules, the invention can be?mixed and matched? When VH and L chains are mixed and matched together, a VH sequence of a particular VH/VL pair is preferred to be replaced by a structurally identical VH sequence. A VL sequence from a specific VH/VL pairing should be replaced by a structurally identical VL sequence.

“Accordingly, this disclosure provides an isolate monoclonal antibody or antigen binding section of the antigen-binding portion.

“Preferred heavy- and light-chain combinations include:

“In an alternative aspect, this disclosure discloses antibodies that contain the heavy chain and light-chain CDR1s, CDR2s, and CDR3s of 25F7 and 26H10, 25E3, CDR2s and CDR3s at 25F7 and 26H10, CDR2s and CDR3s at 25F7 and 25E3, 8B7 and 11F2 respectively, or combinations thereof. SEQ ID NOs 37-42 show the amino acid sequences for the VH CDR1s 25F7 and 26H10, 25E3, 8,B7, 11F2 or 17E5 respectively. SEQ ID NOs 43-48 show the amino acid sequences for VH CDR2s 25F7 and 26H10, 25E3, 8,B7, 11F2 & 17E5 respectively. SEQ ID NOs 13-14, GGY, 16-18 and 17-18 respectively show the amino acid sequences for the VH CDR3s 25F7 and 26H10, 25E3, 25E3, 8B7 and 11F2 respectively. SEQ ID NOs 19-24 show the amino acid sequences for VK CDR1s 25F7 and 26H10, 25E3, 25E3, 8B7 11F2 & 17E5 respectively. SEQ ID NOs 25-30 show the amino acid sequences for VK CDR2s 25F7 and 26H10, 25E3, 25E3, 8B7 11F2 & 17E5 respectively. SEQ ID NOs 31-36 show the amino acid sequences for VK CDR3s 25F7 and 26H10, 25E3, 25E3, 8B7 11F2 & 17E5. Kabat et al. delineate the CDR regions using the Kabat method. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. 91-3242).”

“Accordingly, in another embodiment, this disclosure provides an isolate monoclonal antibody or antigen binding section thereof consisting of:

“In a preferred embodiment of the antibody, it comprises:

“In an alternative preferred embodiment, the antibody includes:

“It is well-known that the CDR3 domain can be used to determine the binding specificity for an antibody against a cognate antigen. Multiple antibodies can be predicted to have the same binding affinity if they are derived from a common CDR3 sequence. See, e.g., 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, Stoller and J. Immunol. 152:5218-55329 (1993); Polymenis and Stoller, J. Immunol. U.S. Pat. Nos. Nos. Each of these references are hereby included by reference in their entirety.

“Accordingly, monoclonal antibodies are provided by the present disclosure that contain one or more heavy or light chain CDR3 domains derived from an antibody derived either from a non-human or human animal. The monoclonal antibody can be specifically bound to human LAG-3. The present disclosure includes monoclonal monoclonal antibodies that contain one or more heavy or light chain CDR3 domains from non-human antibodies, such as mouse or rat antibodies. These monoclonal antibodies are capable of binding specifically to LAG-3 in certain aspects. In some instances, inventive antibodies that contain one or more heavy-or light-chain CDR3 domains from a nonhuman antibody are capable of competing with; (b), retain the functional characteristics; and (c) bind at the same epitope.

“The present disclosure also provides monoclonal antibody that contains one or more heavy-or light-chain CDR3 domains from a human antigen, such as an antibody from a nonhuman animal. The human antibody can specifically bind to LAG-3. The present disclosure also provides monoclonal antibodies that contain one or more heavy-or light-chain CDR3 domains from a first human antigen. This is, for example, an antibody obtained from a nonhuman animal that can specifically bind to human LAG-3. In other words, the CDR3domain from the first antibody replaces the CDR3domain from a human antigen that lacks binding specificity for LAG-3. To create a second antibody that specifically binds to LAG-3, In some instances, inventive antibodies that contain one or more heavy- and/or lighter chain CDR3 domains from the first human antigen (a) can compete for binding with (b), retain functional characteristics (c) bind at the same epitope (d) and/or have a similar binding affinity to the parent first human antigen (e).

“Antibodies with Particular Germline Sequences”

“In some embodiments, an antigen of the invention includes a heavy-chain variable region from one germline heavy-chain immunoglobulin genetic gene and/or light chain variable regions from another germline light-chain immunoglobulin genetic gene.”

“For example, the preferred embodiment provides an isolated monoclonal anti-body, or an antigen binding portion thereof. It includes a heavy chain variable area that is either the product or derived of a VH 3-20, VH 4-34, VH 333, VH 333, or VH 1-24 genes. The antibody specifically binds to human LAG-3. Another preferred embodiment of this disclosure is an isolated monoclonal anti-body, or an antigen binding portion thereof. It comprises a light chain variable area that is either the product or derived form a VK L18, VK L6 or VK A27 genes, and specifically binds to human LAG-3. Another preferred embodiment of this disclosure is an isolated monoclonal antibody or antigen-binding part thereof. The antibody comprises a heavy chains variable region that is either the product or derived form a human VH 3-20 genes and a light chain variable area that is the product or derived form a human VK L18 genetic. The antibody specifically binds to human LAG-3. Another preferred embodiment of this disclosure is an isolated monoclonal antibody or antigen-binding section. The antibody contains a heavy chain variable area that is either the product or derived form a human VH-434 gene. It also includes a light chain variable area that is either the product or derived form a human VK L6 genes. This antibody specifically binds to human LAG-3. Another preferred embodiment of this disclosure is an isolated monoclonal antibody or antigen-binding section. The antibody comprises a heavy chains variable region that is either the product or derived form a human VH-333 gene. It also contains a light chain variable area that is either the product or derived form a human VK A27 genes. This antibody specifically binds to human LAG-3. Another preferred embodiment of this disclosure is an isolated monoclonal antibody or antigen-binding section. The antibody comprises a heavy chains variable region that is either the product or derived of a human VH 1-24 genes and a light chain variable area that is either the product or derived of a human VK L6 genetic. The antibody specifically binds to human LAG-3. Another preferred embodiment is an isolated monoclonal antibody or antigen-binding section. The antibody comprises a heavy chains variable region which is either the product or derived form a human VH 333 gene. It also contains a light chain variable area that is either the product or derived form a VK L6 human gene. This antibody binds specifically to human LAG-3.

“Such antibodies may also have one or more of these functional characteristics, including high affinity binding to LAG-3 in human, monkey, and mouse LAG-3, as well as the ability to inhibit LAG-3 binding to MHC Class II molecules, and/or the ability stimulate antigen-specific T cells responses.”

The 25E3 antibody is an example of an antibody with VH and L of VH 3-20, VK L18 respectively. The 25F7 and 8B7 are examples of antibodies that have VH and VL respectively of VH 4-34, VK L6 and VK A27. The 26H10 antibody is an example of an antibody that has VH and VL from VH 333 and VK A27. The 11F2 antibody is an example of an antibody that has VH and L of VH 1-24, VK L6, and VK A27, respectively. The 17E5 antibody is an example of an antibody that has VH and VL for VH 333 and VK 6 respectively.

“A human antibody is defined as a combination of heavy or light-chain variable regions in which the product of? It is derived from? If the variable regions of the antibody were obtained from a system that uses human genetic immunoglobulin genes, it will be considered a specific germline sequence. These systems include immunizing transgenic mice with human immunoglobulin genes or screening a human immuneglobulin gene collection displayed on phage containing the antigen. A human antibody that is?the product? A human antibody that is?the product of? A human germline immuneglobulin sequence can easily be identified by comparing its amino acid sequence to that of human germline antibodies. Then, select the human germline sequence closest to the sequence of the antibody (i.e. the highest % identity). A human antibody that is?the product? A human antibody that is?the product of? A particular sequence of human germline immunoglobulin can have amino acid differences compared to the germline sequence. This could be due to naturally occurring somatic mutations, or intentional introductions of site-directed mutation. A selected human antibody is usually at least 90% identical in its amino acids sequence to the sequence encoded by the human germline immuneglobulin gene. It also contains amino acid residues which identify the antibody as human, when compared with the sequences of germline immunoglobulin amino acids from other species (e.g. murine germline sequences). A human antibody may be as high as 95% or higher, but it can also be 96%, 97% or 98% identical in amino acids sequence to the sequence encoded by the germline immuneglobulin gene. A human antibody that is derived from a specific human germline sequence will typically not differ more than 10 amino acids from the sequence encoded in the human germline immuneglobulin gene. The human antibody may not display more than 5 amino acids, or even 4, 3, 2, or 1 amino difference from the sequence encoded in the germline immunoglobulin genes.

“Homologous Antibodies”

“Another embodiment of an antibody of this invention includes heavy and light-chain variable regions that contain amino acid sequences homologous with the amino acids sequences of the prefer antibodies. The antibodies retain the desired functional characteristics of the anti?LAG-3 antibodies. This disclosure, for example, provides an isolated monoclonal anti-LAG-3 antibody or antigen binding section that includes a heavy and light chain variable regions.

“Additionally, or alternatively, an antibody may possess one or more the following functional properties: high affinity binding to LAG-3 in human, binding to LAG-3 in monkey LAG-3 or lack of binding LAG-3 to LAG-3 in mouse LAG-3; the ability to inhibit LAG-3 binding to WIC Class II molecules; and/or the ability stimulate antigen-specific T cells responses.”

“The antibody can be, in various embodiments, a human antibody or a humanized antibody.

“In other embodiments the VH or VL amino acids sequences can be 95%, 90%, 96%, 97% and 98% homologous to sequences set forth above. A mutagenesis procedure can produce an antibody with VH or VL regions that are homologous to the VH or VL regions of the sequences. This is done using nucleic acids molecules encoding SEQ ID Nos 49-54 and 55-60. Then, the altered antibody can be tested for retained function (i.e. the functions described above) using the functional assays discussed herein.

“The algorithm of E. Meyers (Comput.) can determine the percent identity between two amino acids sequences. Appl. Appl. The Needleman-Wish (J. Mol.) method can also be used to determine the percent identity of two amino acid sequences. Biol. 48:444-453 (1970). This algorithm has been incorporated into GAP program in GCG software package (available on http://www.gcg.com), using either Blossum 62 or PAM250 matrixes, with a gap weight 16-14, 12, 10, 8, 6 or 4, and a length weight 1, 2, 3, 4, 5, 6, or 6.

“Additionally, or alternatively, you can use the protein sequences in the present disclosure as a query sequence?” To search public databases, such as the Internet, to find related sequences. These searches can be done using the)(BLAST program (version2.0) by Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain sequences of amino acids homologous with the invention’s antibody molecules, BLAST protein searches can also be done using the) (BLAST program, score=50 and wordlength=3. Gapped BLAST is a method to obtain gapped alignments that can be used for comparison purposes. Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. 25(17):3389-3402. See www.ncbi.nlm.nih.gov.”

“Antibodies with Conservative Modifications.”

“In some embodiments, the antibody of the invention includes a heavy chain variable area comprising CDR1, CDR2 or CDR3 sequences and an intermediate region comprising CDR1 and CDR2 sequences. One or more of these CDR sequences comprises specified amino acids sequences based upon the preferred antibodies described herein (e.g. 25F7 or 26H10, 25E3, 25E3, 8B7 or 11F2, 17E5) and where the antibodies retain the desired functional characteristics of the anti-LAG-3 anti-LAG-3 anti-LAG-3 anti-LAG-3 antibody. Certain sequence modifications that do not alter antigen binding can be made, it is known in the art. See, e.g., 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, Beers et. al. (2000) Clin. Can. Res. 6:2835-43. This disclosure provides an isolated monoclonal anti-body, or antigen binding section thereof. It comprises a heavy chain variable area comprising CDR1, CDR2, CDR3 and CDR3 sequences as well as a light chain variable regions comprising CDR1, CDR2, CDR3 and CDR1.

“Additionally, or alternatively, an antibody may possess one or more the following functional properties: high affinity binding to LAG-3 in human, binding to LAG-3 in monkey LAG-3 or lack of binding LAG-3 to LAG-3 in mouse LAG-3; the ability to inhibit LAG-3 binding to MHC Class II molecules; and/or the ability stimulate antigen-specific T cells responses.”

“In a preferred embodiment, heavy chain variable area CDR2 contains an amino sequence chosen from the group consisting amino acid series of SEQID NOs 7-12, and conservative mods thereof. The light chain variable regions CDR2 comprises an amino sequence selected from SEQID NOs 25-30, as well as conservative modifications thereof. Another preferred embodiment of the heavy chain variable area CDR1 sequence consists of an amino acids sequence chosen from the group consisting only of SEQID NOs: 1 through 6, and conservative modifications thereof. The light chain variable regions CDR1 and CDR1 respectively consist of an amino sequence chosen from the group consisting only of SEQID NOs 19-24, and conservative modification thereof.

“The antibody can be either human antibodies, humanized antibodies, or chimeric in various embodiments.”

“Conservative sequence modifications” is the term used herein. The term “conservative sequence modifications” is used to describe amino acid modifications that don’t significantly alter or alter the binding properties of the antibody containing the sequence. These modifications can be amino acid additions, substitutions, and deletions. Standard techniques such as site-directed mutagenesis or PCR-mediated mutagenesis can be used to introduce modifications into the antibody of the invention. Conservative amino acid substitutions involve replacing the amino acid residue with one that has a similar sidechain. The art has identified families of amino acid residues with similar side chains. These include amino acids with simple side chains, such as lysine and arginine or glutamine, acidic side chain (e.g. aspartic acid, glutamic), uncharged, polar sidechains (e.g. glycine and asparagine), nonpolar sidechains (e.g. alanine and valine), beta-branched sidechains (e.g. threonine), valine, isoleucine, tryptophanine, tryptophan, tryptophan, tryptophan, tryptophan, tryptophan, tryptophan, tryptopine, tryptophan, tryptophan, tryptophan The invention allows for the replacement of one or more of the CDR regions with amino acids from the same side chains family. This alteration can then be used to test the retained function of the altered antibody (i.e. the functions described above), using the functional assays herein.

Antibodies that Bind to the same Epitope as Anti LAG-3 Antibodies

“In another embodiment, the disclosure provides antibodies that bind the same epitope to LAG-3 as any anti-LAG-3 monoclonal antibody of the invention (i.e. antibodies that can cross-compete for binding human LAG-3 with any monoclonal antibody of the invention). Preferable embodiments allow cross-competition studies to be conducted using monoclonal antibodies 25F7 or 26H10, 8B7, 11F2 and 17E5 as the reference antibody.

Cross-competing antibodies are identified by their ability to compete with standard LAG-3 binding assays 25F7 and 26H10, 8B7, 25E3, 25E3, 25E3, 25E3, 25E3, 25E3, 8B7, 9B7, 11F2 or 17E5 respectively. Standard ELISA assays are able to be used. One of the antibodies is fluorescently marked and one of the antibodies is immobilized onto the plate. The ability of the non-labeled antibody to compete with the labeled antibody can then be evaluated. BIAcore analysis, which can also be used to evaluate cross-competition of antibodies, is an alternative. A test antibody’s ability to inhibit human LAG-3 binding, such as 25F7 or 26H10 and 25E3, 8B7 and/or 11F2, is a sign that it can compete with human LAG-3 binding agents 25F7 and 26H10 and/or 25E3, and/or 11F2 and/or 17.E5 respectively. It binds to the human LAG-3 epitope as either 25F7 or 26H10 and 25E3, and/or 25E3, and/or17E5 and/or 27E3, and/or 7 and/or 8B7 and/or 11F2 or 17E3 and/or 8B7 and/or 11F2 /or 17E3 and/or 25E3 and/or 8B7 and/or 25E3, and/or 25F7 and/or 25E3, and/or 25F7 and/or 26H10 and/or 8B7 and/or 11F7 and/or 25F7 and/or 8B7 and/or 3B7 and/or 3B7 and/or 3B7 and/or 3B7 Preferably, an antibody that binds the same epitope as human LAG-3 25F7 or 8B7, 26H10 and 11F2 is human monoclonal antibody. These human monoclonal antibodies are possible to be prepared and isolated according to the Examples.

“As discussed in Example 3C, the binding strength of 25E3, 25F7, and 8B7 to human LAG-3 was mapped to an “extra loop”. The extracellular domain is the first extracellular region of human LAG-3. The sequence of this extra loop region can be found in SEQ ID NO. 79. A peptide scan experiment revealed that 25E3 bound to the extra region was mapped to a sequence of amino acids: PGHPLAPG (SEQID NO:76), while 25F7 binding to the extra region was mapped using the sequence of amino acids: HPAAPSSW. (SEQID NO:77). The binding of 8B7 is mapped according to the sequence of amino acids: PAAPSSWG. (SEQID NO:78). In a preferred embodiment, an anti-LAG-3 antibody is provided that binds to an epitope in human LAG-3 composed of the amino acid sequence PAAPSSWG (SEQ ID NO. 76). Another preferred embodiment of the invention is an anti-LAG-3 anti- antibody that binds to an epitope human LAG-3 composed of the amino acid sequence HPAAPSSW or PAAPSSWG. (SEQID NO:77)

“Engineered and Modified Antibodies.”

Summary for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

“In another aspect of the invention, it pertains to an isolated monoclonal anti-body, or antigen binding section thereof.

“A preferred combination includes:

“Another preferred combination includes:

“In another aspect, the invention relates to a method of preparing anti-LAG-3 antibodies. This method includes:

“The present disclosure is easier to understand if certain terms are defined first. The detailed description also contains additional definitions.

“Various aspects of the invention will be described in detail in the subsections below.”

Anti-LAG-3 Antibodies with Particular Functional Properties

“Human monoclonal antibodies are the preferred antibodies of the invention.” The antibodies can also be chimeric, humanized, or a combination of both.

“Monoclonal Antibodies 25F7 and 26H10, 25E3, 25E3, 8B7 and 11F2”

“Accordingly, this disclosure provides an isolate monoclonal antibody or antigen binding section of the antigen-binding portion.

“Preferred heavy- and light-chain combinations include:

“Accordingly, in another embodiment, this disclosure provides an isolate monoclonal antibody or antigen binding section thereof consisting of:

“In a preferred embodiment of the antibody, it comprises:

“In an alternative preferred embodiment, the antibody includes:

“Antibodies with Particular Germline Sequences”

“Homologous Antibodies”

“The antibody can be, in various embodiments, a human antibody or a humanized antibody.

“Antibodies with Conservative Modifications.”

“Additionally, or alternatively, an antibody may possess one or more the following functional properties: high affinity binding to LAG-3 in human, binding to LAG-3 in monkey LAG-3 or lack of binding LAG-3 to LAG-3 in mouse LAG-3; the ability to inhibit LAG-3 binding to MHC Class II molecules; and/or the ability stimulate antigen-specific T cells responses.”

“The antibody can be either human antibodies, humanized antibodies, or chimeric in various embodiments.”

Antibodies that Bind to the same Epitope as Anti LAG-3 Antibodies

“Engineered and Modified Antibodies.”

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Abstract for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

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Summary for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

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Software Technology for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

Infectious Diseases – Kent B. Thudium, Mark J. Selby, Kyra D. Zens, Mark Yamanaka, Alan J. Korman, Heidi N. LeBlanc, ER Squibb and Sons LLC

Search Patent for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

Abstract for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

Background for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

Summary for “Human antibodies that bind lymphocyte activation protein-3 (LAG-3), and their uses”

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