EA039718B1 - Anti-lag3 antibodies and uses thereof - Google Patents

Abstract

The invention relates to antibodies that bind to the T cell co-inhibitor lymphocyte activation gene 3 (LAG3) protein, and methods of use thereof. In various embodiments of the invention, the antibodies are fully human antibodies that specifically bind to LAG3. In some embodiments, the antibodies of the invention are useful for inhibiting or neutralizing LAG3 activity, thus providing a means of treating a disease or disorder such as cancer or viral infection.

Description

The field of technology to which the present invention relates

The invention relates to antibodies and antigen-binding antibody fragments that specifically bind to an immunomodulatory receptor encoded by lymphocyte activation gene-3 (LAG3), and therapeutic and diagnostic methods for using such antibodies.

Sequence listing

An official copy of the sequence listing is filed simultaneously with this disclosure electronically via EFS-Web as an ASCII sequence listing with the file name 2016_10_07_10176WO01_SEQ_LIST_ST25.txt, created on October 7, 2016, and approximately 240 kilobytes in size. The sequence listing contained in this ASCII document is part of this specification and is incorporated herein by reference in its entirety.

Background of the Invention T cell co-stimulatory and co-inhibitory molecules (collectively referred to as co-signaling molecules) play a key role in the regulation of T cell activation, subpopulation differentiation, effector function and survival (Chen et al 2013, Nature Rev. Immunol. 13 227-242). After T cell receptor recognition of peptide-MHC cognate complexes on the surface of antigen presenting cells, co-signaling receptors co-localize with T cell receptors at the immune synapse where they act synergistically with TCR signaling to promote or inhibit T cell activation and function. (Flies et al 2011, Yale J. Biol. Med. 84. 409-421). The final immune response is regulated by the ratio of costimulatory and coinhibitory signals (immunologic checkpoints) (Pardoll 2012, Nature Reviews Cancer 12: 252-264). The protein encoded by lymphocyte activation gene-3 (LAG3) functions as such an immunological checkpoint in mediating peripheral T cell tolerance. LAG3 (also referred to as CD223) is a 503 amino acid transmembrane protein receptor expressed on the surface of activated CD4 and CD8 T cells, γδ T cells, natural killer T cells, B cells, natural killer cells, plasmacytoid dendritic cells, and regulatory T cells. LAG3 is a member of the immunoglobulin (Ig) superfamily. The main function of LAG3 is to attenuate the immune response. Binding of LAG3 to MHC class II molecules results in negative signaling to LAG3 expressing cells and causes suppression of antigen-dependent CD4 and CD8 T cell responses. LAG3 down-regulates the ability of T cells to proliferate, produce cytokines, and lyse target cells, which is referred to as T cell depletion. LAG3 has also been reported to play a role in enhancing regulatory T cell (Treg) function (Pardoll 2012, Nature Reviews Cancer 12: 252-264).

Since LAG3 plays an important role in antitumor immunity and anti-infective immunity, it is an ideal target for immunotherapy. Blocking of LAG3 by antagonists, including monoclonal antibodies, has been studied in the treatment of cancer and chronic viral infections (Turnis et al 2015, Eur. J. Immunol. 45. 1892-1905).

Monoclonal antibodies for LAG3 are known in this area, and they were described, for example, in the US Patent/Publications No. 5976877, 6143273, 6197524, 85551481, 20110070238, 20110150892, 20130095114, 20140093511, 2014012726, 20140286935, and 20140286935, and in WO95/307 /03695, WO98/58059, WO2004/078928, WO2008/132601, Wo2010/019570, Wo2014/008218, EP0510079V1, EP0843557V1, EP0977856, and ER1897548V2, ER2148V2, ER2148V2, ER2114A

Within the development of immunotherapy for human treatment, there is a need for antibodies having certain properties such as low immunogenicity, suitable kinetic binding parameters, cross-reactivity with the monkey target, suitable in vitro activity and/or suitable in vivo activity.

Brief summary of the present invention

The present invention relates to antibodies and antigen-binding fragments thereof that bind LAG3. The antibodies of the present invention are useful, inter alia, for targeting LAG3 expressing immune cells and for modulating LAG3 activity. In certain embodiments, the antibodies of the present invention are useful for inhibiting or neutralizing LAG3 activity and/or for promoting T cell activation, eg, where T cell-mediated killing is beneficial or desirable. In certain embodiments, the antibodies are useful for inhibiting the regulatory function of T cells and/or for reversing the anergic state of depleted T cells. Anti-LAG3 antibodies of the present invention, or antigen-binding portions thereof, can be incorporated as part of a multispecific antigen-binding molecule, e.g., to modulate an immune response and/or to target antibodies to a specific cell type, such as a tumor cell or a virus-infected cell. The antibodies are useful in the treatment of a disease or disorder such as cancer (cancerous neoplasm, cancer) and viral infection.

The antibodies of the present invention may be full-length (eg, an IgG1- or IgG4-1 antibody) or may contain only an antigen-binding portion (eg, a Fab-, F(ab')2-, or scFv fragment) and may be modified to affect functional properties, for example, to eliminate other effector functions (Reddy et al., 2000, J. Immunol. 164:19251933). In certain embodiments, the antibodies may be bispecific.

According to a first aspect, the present invention relates to isolated recombinant monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to LAG3. In certain embodiments, the antibodies are fully human.

Exemplary anti-LAG3 antibodies of the present invention are shown in Tables 1-3 herein. Table 1 provides amino acid sequence identifiers for heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) exemplary antibodies to LAG3. Table 2 lists the nucleic acid sequence identifiers HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-LAG3 antibodies. Table 3 provides amino acid sequence identifiers for the heavy chain and light chain sequences of exemplary anti-LAG3 antibodies.

The present invention relates to antibodies or antigen-binding fragments containing HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a sequence substantially similar to it, characterized with it at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments containing an LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a sequence substantially similar to it, characterized with it at least 90%, at least at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or their antigennegative fragments containing a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), providing any of those given in table. 1 HCVR amino acid sequences paired with any of the LCVR amino acid sequences shown in Table 1. In accordance with certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-LAG3 antibodies shown in Table 1. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/ 122 130/138 146/154 162/170 178/186 194/202 210/218 226/234 242/250 258/266 274/282 290/298 306/314 322/330, 338/346, 354/362, 370/378, 386/394, 402/410, 418/426, 434/442, 450/522, 458/522, 466/522, 474/522, 482/ 522, 490/522, 498/530, 506/530, 514/530, 538/546 and 554/562. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 386/394 (eg H4sH15479P), 418/426 (eg H4sH15482P) or 538/546 (eg H4sH14813N). In certain embodiments, the present invention relates to anti-LAG3 antibodies or antigen-binding fragments thereof, comprising HCVR and LCVR, said HCVR comprising the amino acid sequence shown in Table 1 with no more than five amino acid substitutions, and said LCVR comprising the one shown in Table 1 amino acid sequence with no more than five amino acid substitutions. For example, the present invention relates to antibodies to LAG3 or their antigen-binding fragments containing HCVR and LCVR, and the specified HCVR contains the amino acid sequence of SEQ GO NO: 418 with no more than five amino acid substitutions, and the specified LCVR contains the amino acid sequence of SEQ GO NO: 426 more than five amino acid substitutions.

According to another illustrative embodiment, the present invention provides antibodies to LAG3 or antigen-binding fragments thereof, comprising HCVR and LCVR, said HCVR comprising the amino acid sequence of SEQ ID NO: 418 with at least one amino acid substitution, and said LCVR comprising the amino acid sequence of SEQ ID NO : 426 with one amino acid substitution.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a sequence substantially similar to it, characterized by at least 90% , at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of those listed in Table 2. 1 amino acid sequences of HCDR2, or a sequence substantially similar to it, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a sequence substantially similar to it, characterized by at least 90% , at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a light chain CDR1 (LCDR1) containing an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a sequence substantially similar to it, characterized by at least 90% , at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a sequence substantially similar to it, characterized by at least 90% , at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a sequence substantially similar to it, characterized by at least 90% , at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or their antigennegative fragments containing a pair of amino acid sequences HCDR3 and LCDR3 (HCDR3/LCDR3), providing any of those given in table. 1 HCDR3 amino acid sequences paired with any of the LCDR3 amino acid sequences shown in Table 1. In accordance with certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-LAG3 antibodies in Table 1. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 392/400 (e.g. H4sH15479P), 424/432 (e.g. H4sH15482P) and 544/552 (e.g. H4sH14813N).

The present invention also relates to antibodies or antigen-binding fragments containing HCVR and LCVR, and the specified HCVR contains HCDR1 containing an amino acid sequence that differs from the amino acid sequence shown in Table 1 by 1 amino acid, HCDR2 containing an amino acid sequence that differs from that shown in Table .1 amino acid sequence per 1 amino acid, and HCDR3 containing an amino acid sequence that differs from that shown in table. 1 amino acid sequence per 1 amino acid. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, said LCVR comprising an LCDR1 containing an amino acid sequence that differs from the amino acid sequence shown in Table 1 by 1 amino acid, an LCDR2 containing an amino acid sequence that differs from the amino acid sequence shown in Table 1 by 1 amino acid, and an LCDR3 containing an amino acid sequence that differs from the amino acid sequence shown in Table 1 by 1 amino acid. For example, the present invention relates to antibodies to LAG3 or antigen-binding fragments thereof, containing HCVR and LCVR, and said HCVR contains HCDR1 containing the amino acid sequence of SEQ ID NO: 420 or an amino acid sequence that differs from SEQ ID NO: 420 by 1 amino acid, HCDR2, containing the amino acid sequence of SEQ ID NO: 422 or an amino acid sequence that differs from SEQ ID NO: 422 by 1 amino acid, and HCDR3 containing the amino acid sequence of SEQ ID NO: 424 or an amino acid sequence that differs from SEQ ID NO: 424 by 1 amino acid. According to another exemplary embodiment, the present invention relates to antibodies or antigen-binding fragments thereof, comprising HCVR and LCVR, said LCVR comprising an LCDR1 containing the amino acid sequence of SEQ ID NO: 428 or an amino acid sequence that differs from SEQ ID NO: 428 by 1 amino acid, LCDR2 , containing the amino acid sequence of SEQ ID NO: 430 or an amino acid sequence that differs from SEQ ID NO: 430 by 1 amino acid, and LCDR3 containing the amino acid sequence of SEQ ID NO: 432 or an amino acid sequence that differs from SEQ ID NO: 432 by 1 amino acid.

The present invention relates to antibodies or antigen-binding fragments thereof, containing a heavy chain containing an amino acid sequence selected from any of the HC amino acid sequences listed in Table 3, or a sequence substantially similar to it, characterized with it at least 90 %, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a light chain containing an amino acid sequence selected from any of the LC amino acid sequences listed in Table 3, or a sequence substantially similar to it, characterized with it at least 90%, by at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a pair of HC and LC amino acid sequences (HC/LC), providing any of the HC amino acid sequences shown in Table 3 in a pair with any of the LC amino acid sequences shown in Table 3. In accordance with certain embodiments, the present invention relates to antibodies, or antigen-binding fragments thereof, comprising an HC/LC amino acid sequence pair contained within any of the exemplary anti-LAG3 antibodies shown in Table 3. In certain embodiments, the HC/LC amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 577/578, 579/578, and 580/581.

The present invention also relates to antibodies or antigen-binding fragments thereof, containing a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-LAG3 antibodies shown in Table 1. In certain embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1lCdR2-LCDR3 is selected from the group consisting of SEQ ID NO: 388-390-392-396-398-400 (e.g., H4sH15479P), 420-422-424-428 -430-432 (for example, H4sH15482P) and 540-542-544-548-550-552 (for example, H4sH14813N).

According to a similar embodiment, the present invention relates to antibodies or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined for any of shown in table.1 illustrative antibodies to LAG3. For example, the present invention includes antibodies, or antigen-binding fragments thereof, comprising a set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 386/394 (eg H4sH15479P), 418/426 (eg H4sH15482P) and 538/546 (eg H4sH14813N). Methods and techniques for identifying CDRs within the amino acid sequences of HCVRs and LCVRs are well known in the art and can be used to identify CDRs within the amino acid sequences of certain HCVRs and/or LCVRs disclosed herein. Illustrative conventional approaches that can be used to identify CDR boundaries include, for example, Kabat detection, Chothia detection, and antibody model detection. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the antibody model definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al, J. Mol. Biol. 273:927-948 (1997); and Martin et al, Proc. Natl. Acad. sci. USA 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within an antibody.

The present invention includes antibodies to LAG3 with a modified glycosylation profile. In some embodiments, modification to remove unwanted glycosylation sites or lack of a fucose moiety present in the oligosaccharide chain may be useful, e.g., to enhance the function of antibody-dependent cellular cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277:26733). According to other applications, in order to modify complement-dependent cytotoxicity (CDC), modification can be performed in relation to galactosylation.

The present invention includes antibodies to LAG3 containing an Fc domain, where the Fc domain provides for IgG1 or IgG4 isotypes, as described elsewhere herein. In certain embodiments, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 569, 570, 571, 572, and 573.

The present invention also relates to antibodies and antigen-binding fragments thereof that compete for specific binding to LAG3 with an antibody or antigen-binding fragment containing an HCVR CDR and an LCVR CDR, where each of the HCVR and LCVR contains an amino acid sequence selected from the sequences shown in Table 1 HCVR and LCVR.

The present invention also relates to isolated antibodies and antigen-binding fragments thereof that block the binding of LAG3 to MHC class II. According to some options

An antibody or antigen-binding fragment thereof that blocks LAG3 binding may bind to the same epitope on LAG3 as MHC class II, or may bind to a different epitope on LAG3 than MHC class II.

The present invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to LAG3 of a human or other species. In certain embodiments, antibodies can bind to human LAG3 and/or monkey LAG3.

The present invention also relates to antibodies and antigen-binding fragments thereof that cross-compete for binding to LAG3 with a reference antibody or antigen-binding fragment containing an HCVR CDR and an LCVR CDR, where each of the HCVR and LCVR contains an amino acid sequence selected from those given in Table 1 HCVR and LCVR sequences.

The present invention also relates to antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment containing an HCVR CDR and an LCVR CDR, where each of the HCVR and LCVR contains an amino acid sequence selected from those given in table. 1 sequences HCVR and LCVR. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment thereof, comprising an HCVR CDR and an LCVR CDR, wherein the HCVR/LCVR amino acid sequence pair comprises SEQ ID NO: 418 /426.

The present invention also includes anti-LAG3 antibodies that interact with one or more amino acids contained within the extracellular domain of human LAG3 (SEQ ID NO: 588). In certain embodiments, the present invention provides anti-LAG3 antibodies and antigen-binding fragments thereof that interact with an amino acid sequence selected from the group consisting of (a) amino acids 28-69 of SEQ ID NO: 588; (b) amino acids 28-71 of SEQ ID NO: 588; (c) amino acids 31-52 of SEQ ID NO: 588; and (d) amino acids 32-69 of SEQ ID NO: 588. In certain embodiments, the present invention provides anti-LAG3 antibodies and antigen-binding fragments thereof that interact with one or more amino acids, contained within SEQ ID NO: 589, for example, the present invention relates to antibodies to LAG3 and antigen-binding fragments that interact with at least 5 amino acids, at least 10 amino acids, or at least 20 amino acids contained within SEQ ID NO : 589. In certain embodiments, the present invention provides anti-LAG3 antibodies and antigen-binding fragments thereof that interact with the amino acid sequence of SEQ ID NO: 589 (corresponding to amino acids 28-71 of SEQ ID NO: 588).

In one embodiment, the present invention provides a recombinant human monoclonal antibody, or antigen-binding fragment thereof, that has one or more of the following characteristics: (a) binds specifically to human LAG3 and/or cynomolgus monkey LAG3; (b) blocks the binding of LAG3 to MHC class II; (c) blocks LAG3-induced downregulation of T cells and allows resumption of signaling involving the T cell; and (d) provides suppression of tumor growth and increased survival of a subject with a malignant tumor.

In some embodiments, the antibody, or antigen-binding fragment thereof, can specifically bind to LAG3 as an agonist, i. they can enhance or stimulate the binding and/or activity of LAG3; in other embodiments, the antibody may specifically bind to LAG3 as an antagonist, ie, it may block the binding of LAG3 to its ligand.

In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are bispecific, wherein they provide a first binding specificity for LAG3 and a second binding specificity for a second target epitope. The second target epitope may be a different epitope on LAG3 or a different protein. In certain embodiments, the second target epitope may be on the surface of another cell, including another T cell, B cell, tumor cell, or virus-infected cell.

According to a second aspect, the present invention relates to nucleic acid molecules encoding anti-LAG3 antibodies or parts thereof. For example, the present invention relates to nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCVR-encoding nucleic acid sequences listed in Table 2, or a sequence substantially similar to it, characterized with it at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; according to certain options

- 5 039718 embodiment, the nucleic acid molecule contains a polynucleotide sequence selected from any of the nucleic acid sequences listed in Table 2 encoding

LCVR, or a sequence substantially similar to it, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

The present invention also relates to nucleic acid molecules encoding any of those listed in table. 1 amino acid sequences of HCDR1; according to certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR1-encoding nucleic acid sequences listed in Table 2, or a sequence substantially similar to it, characterized with it at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR2-encoding nucleic acid sequences listed in Table 2, or a sequence substantially similar to it, characterized with it at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR3-encoding nucleic acid sequences listed in Table 2, or a sequence substantially similar to it, characterized with it at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR1-encoding nucleic acid sequences listed in Table 2, or a sequence substantially similar to it, characterized with it at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR2-encoding nucleic acid sequences listed in Table 2, or a sequence substantially similar to it, characterized with it at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR3-encoding nucleic acid sequences listed in Table 2, or a sequence substantially similar to it, characterized with it at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding HCVR, where HCVR contains a set of three CDRs (i.e. HCDR1-HCDR2-HCDR3), where the set of amino acid sequences HCDR1-HCDR2-HCDR3 is as defined for any of those given in Table 1 exemplary anti-LAG3 antibodies.

The present invention also relates to nucleic acid molecules encoding LCVR, where the LCVR contains a set of three CDRs (i.e. LCDR1-LCDR2-LCDR3), where the set of amino acid sequences LCDR1-LCDR2-LCDR3 is as defined for any of those given in tab. 1 illustrative antibodies to LAG3.

The present invention also relates to nucleic acid molecules encoding both HCVR and LCVR, where HCVR contains the amino acid sequence from any of those given in table. 1 amino acid sequences of HCVR, and where LCVR contains an amino acid sequence from any of those given in table. 1 amino acid sequences of LCVR.

In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR-encoding nucleic acid sequences listed in Table 2, or a sequence substantially similar to it,

- 6 039718 characterized with it at least 90%, at least 95%, at least 98% or at least 99% sequence identity, and a polynucleotide sequence selected from any of the nucleic acid sequences listed in Table 2 encoding LCVR, or a substantially similar sequence, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it. In certain embodiments of this aspect of the present invention, the nucleic acid molecule encodes HCVR and LCVR, wherein both HCVR and LCVR are derived from the same anti-LAG3 antibody shown in Table 1.

The present invention relates to nucleic acid molecules encoding any of the heavy chain amino acid sequences shown in Table 3. The present invention also relates to nucleic acid molecules encoding any of the light chain amino acid sequences shown in Table 3.

The present invention also relates to nucleic acid molecules encoding both a heavy chain (HC) and a light chain (LC), where HC contains an amino acid sequence from any of the HC amino acid sequences listed in Table 3, and where LC contains an amino acid sequence from any from the LC amino acid sequences shown in Table 3.

In a similar aspect, the present invention relates to recombinant expression vectors capable of expressing a polypeptide containing an anti-LAG3 antibody heavy or light chain variable region. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, ie. nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences shown in Table 1. The present invention also relates to recombinant expression vectors capable of expressing a polypeptide containing the heavy or light chain of an anti-LAG3 antibody. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, ie. nucleic acid molecules encoding any of the heavy chain or light chain sequences shown in Table 3. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods for producing antibodies or parts thereof by culturing host cells under conditions that allow the production of antibodies or antibody fragments, and isolating the antibodies and antibody fragments thus obtained.

According to a third aspect, the present invention relates to a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds LAG3 and a pharmaceutically acceptable carrier. In a similar aspect, the present invention provides a composition that is a combination of an anti-LAG3 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-LAG3 antibody. Illustrative agents that can advantageously be combined with an anti-LAG3 antibody include, without limitation, other agents that bind and/or modulate LAG3 signaling (including other antibodies or antigen-binding fragments thereof, etc.), and/or agents that do not directly bind LAG3 but modulate immune cell activation. Additional combination therapeutics and formulations comprising the anti-LAG3 antibodies of the present invention are disclosed elsewhere herein.

In a fourth aspect, the present invention relates to methods for modulating an immune response in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LAG3 antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the present invention relates to methods for enhancing an immune response in a subject, the methods comprising administering to the subject an effective amount of an antibody or fragment thereof of the present invention that binds LAG3. According to one embodiment, the present invention relates to a method for stimulating or enhancing T cell activation in a subject. In certain embodiments, the present invention relates to methods for reactivating a T cell, comprising bringing the T cell into contact with an effective amount of an antibody of the present invention, thereby reactivating the T cell. In one embodiment, the present invention relates to methods for inhibiting a regulatory T cell (Treg) in a subject, the methods comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the subject in need may be suffering from a disease or disorder, such as cancer or a viral infection. In certain embodiments, the present invention relates to methods for reversing LAG3-mediated inhibition of T cell activity, comprising contacting the T cell with an effective amount of an antibody of the present invention.

- 7 039718

According to a fifth aspect, the present invention provides therapeutic methods for treating a disease or disorder, such as cancer or a viral infection, in a subject using an anti-LAG3 antibody or an antigen-binding portion of an antibody of the present invention, wherein the therapeutic methods comprise administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical compositions containing the antibody or antibody fragment of the present invention. A treatable disorder is any disease or condition that is ameliorated, ameliorated, inhibited or prevented by stimulating or inhibiting LAG3 activity or signaling. In certain embodiments, an antibody or antigen-binding fragment thereof of the present invention is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of an antibody to another T cell co-inhibitor, an antibody to a tumor cell antigen, an antibody to a T cell receptor, an antibody to an epitope on a virus-infected cell, a cytotoxic agent, an anticancer drug, an antiviral drug, an anti-inflammatory a drug (eg, corticosteroids), a chemotherapeutic agent, radiation therapy, an immunosuppressant, and any other drug or therapy known in the art. According to certain embodiments, the second therapeutic agent may be an agent that helps to neutralize or reduce the severity of any(s) side(s) associated(s) with an antibody or antigen-binding fragment of the present invention, if such(- e) side effect(s) suddenly occur.

In certain embodiments, the present invention relates to methods for suppressing tumor growth. For example, the present invention relates to the suppression of tumor growth in the case of a primary tumor or a metastatic tumor in a subject. In certain embodiments, the present invention relates to methods for improving survival (eg, progression-free survival or overall survival) of a subject with cancer. Examples of cancer include, without limitation, primary and/or recurrent cancer, including hematopoietic tissue cancer (e.g., hematopoietic cancer such as lymphoma, myeloma, or leukemia), brain cancer (e.g., glioblastoma multiforme), lung cancer (e.g., , non-small cell lung cancer), head and neck squamous cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, melanoma, mesothelioma, ovarian cancer, bladder cancer, breast cancer, bone cancer, colon and rectal cancer, prostate cancer, and colon cancer intestines. In certain embodiments, the present invention relates to methods for inhibiting or suppressing the growth of established tumors. The methods include administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an anti-LAG3 antibody of the present invention. In certain embodiments, the antibody is administered in combination with a second therapeutic agent selected from the group consisting of a programmed cell death (PD-1) protein-1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab or REGN2810), a protein ligand inhibitor 1 programmed cell death (PD-L1) (eg, anti-PD-L1 antibodies), vascular endothelial growth factor (VEGF) antagonist (eg, aflibercept, bevacizumab), angiopoietin-2 (Ang2) inhibitor (eg, anti-Ang2 antibody, such such as nesvacumab), a cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor (eg, ipilimumab), an anti-CD20xCD3 bispecific antibody, a cytotoxin, a chemotherapeutic agent, and radiation therapy. Additional examples of additional therapies/therapeutic agents that can be used in combination with an anti-LAG3 antibody of the present invention for use in the treatment of cancer are described elsewhere herein.

The antibody or fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. The antibody or fragment thereof may be administered at a dose of about 0.1 mg/kg body weight to about 100 mg/kg body weight of the subject.

The present invention also includes the use of an anti-LAG3 antibody or antigen-binding fragment thereof of the present invention in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from blocking LAG3 binding and/or signaling, such as cancer.

Other embodiments will be apparent from a review of the following detailed description.

Brief description of the drawings

In FIG. 1 is a schematic representation of the LAG3 bioassay based on the determination of luciferase activity described in Example 8 herein. Panel (A): Bispecific antibody method: Inactive Jurkat-derived T cells were activated by T cell receptor (TCR) clustering with an anti-CD3xCD20 bispecific antibody. Panel (B): Peptide primed method: In this method, inactive Jurkat-derived T cells were activated with a specific MHC/peptide complex (clone Ob2F3 TCR heterodimer with MHCII protein, HLA-DR2AB, complexed with MBP85- 99). Binding of LAG3 to

DR2 attenuates the response of Jurkat-derived activated T cells. The blocking antibody to LAG3 reactivates the response of Jurkat-derived activated T cells.

In FIG. 2A-2B show the in vivo efficacy of anti-mouse LAG3 antibody alone and in combination with anti-mouse PD-1 antibody against established Colon 26 tumors (described in Example 10). Fig. 2A. The mean tumor volume (mm ³ ±SEM) in each treatment group was measured at several time points after tumor cell implantation. Treatment was started on day 14 when the average tumor volume reached 50 mm ³ . Treatment days are indicated by arrows. All antibodies were administered intraperitoneally (ip) at a concentration of 10 mg/kg. Fig. 2B. Individual tumor volume measurements in each treatment group were measured on day 35 post-implantation. Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test for multiple comparison (*p<0.05). Fig. 2A-2B. Treatment groups: rat IgG2a isotype controls + mouse IgG1 control antibody: (·); anti-mouse PD-1 + control mouse IgG1 (A); mouse LAG3 antibody + control rat IgG2a (); anti-mouse PD-1 antibody + anti-mouse LAG3 antibody (▼).

In FIG. 3A-3B show the in vivo efficacy of anti-mouse LAG3 antibody alone and in combination with anti-mouse PD-1 antibody against established MC38 tumors (described in Example 11). Fig. 3A. The mean tumor volume (mm ³ ±SEM) in each treatment group was measured at several time points after tumor cell implantation. Treatment was started on day 8 when the average tumor volume reached 45 mm ³ . Treatment days are indicated by arrows. Fig. 3B. Individual tumor volume measurements in each treatment group were measured on day 23 post-implantation. Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test for multiple comparison (****p<0.0001). Fig. 3A-3B. Treatment groups: isotype controls rat IgG2a + mouse IgG1 (·); anti-mouse PD-1 + control mIgG1 (A); mouse LAG3 antibody + control rat IgG2a (); and anti-mouse PD-1 + anti-mouse LAG3 (▼).

In FIG. 4A-4B show mouse IFNy and CD8b expression levels (normalized to mouse cyclophilin B expression levels) determined by Taqman assay in draining lymph nodes (DLN) (A) and spleen (B) isolated at the end of the experiment (described in example 11) from tumor-bearing mice. *P<0.05, **P<0.01, ***P<0.001.

In FIG. 5A-5B show the in vivo efficacy results of an anti-human LAG3 antibody (mAb1) and an anti-human PD-1 antibody (REGN2810) against MC38 tumors (described in Example 12). Fig. 5A. Measures mean tumor volume (mm ³ ±SEM) in each treatment group at multiple time points after tumor cell implantation. Treatment days are indicated by arrows. Fig. 5V. Individual tumor volume measurements in each group were monitored for 24 days. The number of tumor-free mice in each group by day 24 is shown. FIG. 5A-5B. Treatment groups: mAb1 (antibody to hLAG3) at 25 mg/kg ( ^and ); REGN2810 (anti-hPD-1 antibody) at 10 mg/kg (A) and human isotype control antibody at 25 mg/kg (·).

In FIG. 6A-6C show the in vivo efficacy of anti-human LAG3 antibody (mAb1) alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors (described in Example 13). Fig. 6A. Measures mean tumor volume (mm ³ ±SEM) in each treatment group at multiple time points after tumor cell implantation. Treatment days are indicated by arrows. Fig. 6B. Individual tumor volume measurements in each treatment group were measured on day 22 post-implantation. Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test for multiple comparison (*<0.05;**p<0.01). Fig. 6A-6B. Treatment groups: mAb1 (anti-hLAG3 antibody) at 25 mg/kg (♦); REGN2810 (antibody to hPD-1) at a concentration of 10 mg/kg (); combination of mAb1 (antibodies to hLAG3) at a concentration of 25 mg/kg and REGN2810 (antibodies to hPD-1) at a concentration of 10 mg/kg (A); human isotype control antibody at a concentration of 25 mg/kg (·). Fig. 6C. Percentage of tumor-free survival among treated mice. Statistical significance was determined using the logarithmic rank test (Mantel-Cox) (****p<0.0001). Treatment groups: mAb1 (anti-hLAG3 antibody) at 25 mg/kg (♦); REGN2810 (antibody to hPD-1) at 10 mg/kg (▼); combination of mAb1 (antibodies to hLAG3) at a concentration of 25 mg/kg and REGN2810 (antibodies to hPD-1) at a concentration of 10 mg/kg (A); human isotype control antibody at a concentration of 25 mg/kg (·).

In FIG. 7A-7C show the in vivo efficacy of anti-human LAG3 antibody (mAb1) alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors in the first experiment (described in Example 14). Fig. 7A. Measures mean tumor volume (mm ³ ±SEM) in each treatment group at multiple time points after tumor cell implantation. Treatment days are indicated by arrows. Fig. 7B. Individual tumor volume measurements in each treatment group were measured on day 22 post-implantation. Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test for multiple comparison (*p<0.05) FIG. 7C. Percentage of tumor-free survival among treated mice. Statistical significance was determined using the logarithmic rank test (Mantel-Cox) (*p<0.0197). Treatment groups: mAb1 (antibody to hLAG3) at 25 mg/kg (♦); REGN2810 (anti-hPD-1 antibody) at 10 mg/kg (*); REGN2810 (anti-hPD-1 antibody) at 1 mg/kg (A); combination of mAb1 (antibodies to hLAG3) at a concentration of 25 mg/kg and REGN2810 (antibodies to hPD-1) at a concentration of 10 mg/kg (); a combination of mAb1 (anti-hLAG3 antibodies) at 25 mg/kg and REGN2810 (anti-hPD-1 antibodies) at 1 mg/kg (▼) and a human isotype control antibody at 25 mg/kg (·).

In FIG. 8A-8C show the results of in vivo efficacy of anti-human LAG3 antibody (mAb1) alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors in the second experiment (described in Example 14). Fig. 8A. Measures mean tumor volume (mm ³ ±SEM) in each treatment group at multiple time points after tumor cell implantation. Treatment days are indicated by arrows. Statistical significance was determined using two-way ANOVA with Dunnett's post hoc test for multiple comparison (*p<0.05). Fig. 8B. Individual tumor volume measurements in each treatment group measured on day 23 post-implantation. Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test for multiple comparison (**p<0.01). Fig. 8C. Percentage of tumor-free survival among treated mice. Statistical significance was determined using a log-rank test (Mantel-Cox) with a Bonferroni correction for multiple comparisons (***p<0.001, **p<0.01). Treatment groups: mAb1 (anti-hLAG3 antibody) at 25 mg/kg (♦); mAb1 (antibody to hLAG3) at a concentration of 5 mg/kg (·); REGN2810 (antibody to hPD-1) at 10 mg/kg (^); combination of mAb1 (antibodies to hLAG3) at a concentration of 25 mg/kg and REGN2810 (antibodies to hPD-1) at a concentration of 10 mg/kg (); a combination of mAb1 (anti-hLAG3 antibodies) at 5 mg/kg and REGN2810 (anti-hPD-1 antibodies) at 10 mg/kg (▼) and a human isotype control antibody at 25 mg/kg (·).

In FIG. 9 shows mean tumor volume (mm ³ ±SEM) in each treatment group at several time points after tumor cell implantation in an experiment to evaluate the efficacy of anti-human LAG3 antibody (mAb1) alone and in combination with anti-human PD-1 antibody (REGN2810) against mature MC38 tumors (described in Example 15 herein). Treatment days are indicated by arrows.

In FIG. 10 shows individual tumor volume measurements in each treatment group measured on day 22 post-implantation in the experiment described in Example 15. Statistical significance was determined by one-way analysis of variance with Dunnett's post hoc test for multiple comparison.

In FIG. 11 shows the percent tumor-free survival among treated mice in the experiment described in Example 15 herein. Statistical significance was determined using a log-rank test (Mantel-Cox) with a Bonferroni correction for multiple comparisons (**p<0.01).

In FIG. 12 is a graph of mean (+SD) functional mAb1 serum concentrations versus time following a single intravenous infusion of mAb1 in a female cynomolgus monkey (described in Example 16).

Detailed disclosure of the present invention

Before the methods of the present invention are described, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is only intended to describe specific embodiments, and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which the present invention pertains. While any methods and materials similar or equivalent to those described herein may be used to practice or test the present invention, the methods and materials described herein are preferred. All publications cited herein are incorporated herein by reference in their entirety.

The term LAG3 refers to a protein encoded by the lymphocyte activation gene-3, a receptor that is part of an immunological checkpoint, or a T cell co-inhibitor, also known as CD223. The amino acid sequence of full-length LAG3 is listed in GenBank under accession number NP_002277.4, and is also referred to herein as SEQ ID NO: 582. The term LAG3 also includes variants of the LAG3 protein with the amino acid sequence of SEQ ID NO: 574, 575, or 576. The term LAG3 includes recombinant LAG3 or fragment thereof. The term is also coverage

- 10 039718 LAG3 or a fragment thereof linked, for example, to a histidine tag, a mouse or human Fc, or a signal sequence, for example, ROR1. For example, the term includes sequences exemplified by SEQ ID NO: 575 containing at the C-terminus a mouse Fc (mIgG2a) fused to amino acid residues 29-450 of full-length LAG3. Protein variants, exemplified by SEQ ID NO: 574, contain a C-terminal histidine tag attached to amino acid residues 29-450 of full-length LAG3. The term LAG3 means human LAG3 unless indicated to be of a non-human species.

LAG3 is a member of the immunoglobulin (Ig) superfamily. LAG3 is a 503 amino acid type 1 transmembrane protein with four extracellular Ig-like domains D1-D4 and is expressed on the surface of activated T cells, natural killer cells, B cells, plasmacytoid dendritic cells, and regulatory T cells. The LAG3 receptor binds to class II MHC molecules present on the surface of antigen presenting cells (APCs).

As used herein, the term T cell co-inhibitor refers to a ligand and/or receptor that modulates an immune response by activating or suppressing a T cell. The term T-cell co-inhibitor, also known as T-cell co-signaling molecule, includes, but is not limited to, programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), CD-28, 2B4, LY108, T-cell molecule-3 containing immunoglobulin and mucin domain (TIM3), T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT; also known as VSIG9) associated with leukocyte immunoglobulin-like receptor-1 (LAIR1; also known as CD305), inducible T-cell costimulator (ICOS; also known as CD278), suppressor of T-cell activation containing V-Ig domain (VISTA) and CD 160.

In the context of this document, the term antibody is understood to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains, and two light (L) chains connected to each other by disulfide bonds (i.e., complete antibody molecules), as well as their multimers (eg, IgM) or their antigen-binding fragments. Each heavy chain is composed of a heavy chain variable region (HCVR or VH) and a heavy chain constant region (consisting of the CH1, C _H 2 and CH3 domains). Each light chain is composed of a light chain variable region (LCVR or V _L ) and a light chain constant region (C _L ). The V _H and VL regions can further be divided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged, from amino to carboxy, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FR of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences, or may be naturally or artificially modified. Amino acid consensus sequence can be determined based on a comparative analysis of two or more CDRs.

It is also possible to replace one or more CDR residues or remove one or more CDR residues. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with to enable binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions of antibodies with their antigens based on published crystal structure data and concluded that in reality only about one fifth to one third of the CDR residues contact the antigen. According to Padlan, a variety of antibodies have also been found in which one or two of the CDRs lacked amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not in contact with antigen can be identified based on previous studies (e.g., H60-H65 residues in CDRH2 are often unnecessary), based on Kabat CDR regions outside the Chothia CDR, using molecular modeling and/ or experimentally. If the CDR or residue(s) thereof are deleted, they are typically replaced with an amino acid at the appropriate position in another human antibody sequence, or a consensus of such sequences. The positions within the CDR at which substitutions will be made and the amino acids to be replaced can also be chosen experimentally. Experimental substitutions may be conservative or non-conservative substitutions.

The fully human anti-LAG3 monoclonal antibodies disclosed herein, when compared to the corresponding germline sequences, may include one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or

- 11 039718 several amino acids within one or more frame and/or CDR regions are mutated with the corresponding(s) residue(s) of the germline sequence from which the antibody originates, or the corresponding(s) residue(s) i) a different germline sequence, or are subject to a conservative amino acid substitution of the corresponding germline residue(s) (such changes from a sequence are collectively referred to herein as germline mutations). One skilled in the art, based on the heavy and light chain variable region sequences disclosed herein, will readily be able to generate a variety of antibodies and antigen-binding fragments that contain one or more single germline mutations, or combinations thereof. In certain embodiments, all of the framework and/or CDR region residues within the V _H and/or VL domains are back-mutated to residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and/or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody is originally derived). In addition, the antibodies of the present invention may include any combination of two or more germline mutations within the framework and/or CDR regions, for example, where certain individual residues are mutated with the corresponding residue of a particular germline sequence, while certain other residues, that differ from those in the original germline sequence are retained or mutated to the corresponding residue of a different germline sequence. Once generated, antibodies and antigen-binding fragments that carry one or more germline mutations can be readily tested for one or more of the desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as appropriate). ), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by such a general method are covered by the scope of the present invention.

The present invention also includes fully human anti-LAG3 monoclonal antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes antibodies to LAG3 containing the amino acid sequences of HCVR, LCVR and/or CDR, for example, with 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions compared to any from the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

As used herein, the term human antibody is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., due to mutations introduced by in vitro random or site-directed mutagenesis or due to in vivo somatic mutation), for example, in CDRs and, in particular, CDR3. However, in the context of this document, the term human antibody is intended not to include mAbs in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human FR sequences. The term includes antibodies produced recombinantly in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies produced in or taken from a human subject.

The term recombinant, as used herein, refers to antibodies or antigen-binding fragments thereof of the present invention, created, expressed, isolated, or produced using technologies or methods known in the art, such as recombinant DNA technology, which include, for example, DNA splicing and transgenic expression. . The term refers to antibodies expressed in an expression system using a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice) or cells (e.g., CHO cells), or isolated from a recombinant combinatorial human antibody library.

The term multispecific antigen-binding molecules in the context of this document refers to bispecific, trispecific or multispecific antigen-binding molecules and antigen-binding fragments. Multispecific antigen-binding molecules may be specific for different epitopes of the same target polypeptide or may contain

- 12 039718 reap antigen-binding domains specific for epitopes of more than one target polypeptide. The polyspecific antigen binding molecule may be a single polyfunctional polypeptide, or it may be a multimeric complex of two or more polypeptides that are covalently or non-covalently linked to each other. The term multispecific antigen-binding molecules includes antibodies of the present invention that can be co-expressed with, or linked to, another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be operably linked (e.g., by chemical bonding, splicing, non-covalent bonding, or otherwise) to one or more other compounds, such as a protein or fragment thereof, to form a bispecific or polyspecific antigen-binding molecule with a second binding specificity. . In accordance with the present invention, the term multispecific antigen-binding molecules also includes bispecific, trispecific or polyspecific antibodies or antigen-binding fragments thereof. In certain embodiments, an antibody of the present invention is operably linked to another antibody or antigen-binding fragment thereof to form a bispecific antibody with a second binding specificity. The bispecific and polyspecific antibodies of the present invention are described elsewhere in this document.

The term specifically binds or specifically binds to or so on means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may have an equilibrium dissociation constant of at least about 1±10' ⁸ M or less (eg, lower K _D means stronger binding). Methods for determining whether specific binding of two molecules occurs are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies that specifically bind to LAG3 have been identified using the surface plasmon resonance method, for example, within the BIACORE™ system. In addition, multispecific antibodies that bind to one LAG3 domain and one or more additional antigens, or bispecific antibodies that bind to two different regions of LAG3, are considered antibodies that specifically bind in the context of this document.

The term high affinity antibody refers to those mAbs that have a binding affinity for LAG3, expressed in K _D , of at least 10' ⁸ M; preferably 10' ⁹ M; more preferably 10' ¹⁰ M, even more preferably 10' ¹¹ M, even more preferably 10' ¹² M, as measured by surface plasmon resonance, for example, within the BIACORE™ system, or ELISA with determination of affinity in solution.

By the term low dissociation rate, Koff” or kd is meant an antibody that dissociates from LAG3 with a rate constant of 1x10'3 s ^-1 or less, preferably 1x10' ⁴ s ^-1 or less, as measured by surface plasmon resonance, for example, within the BIACORE™ system.

The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, etc., as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms antigen-binding antibody fragment or antibody fragment as used herein refer to one or more antibody fragments that retain the ability to bind to LAG3.

In specific embodiments, an antibody or antibody fragments of the present invention may be conjugated to a compound, such as a ligand, or a therapeutic compound (immunoconjugate), such as a cytotoxin, a second anti-LAG3 antibody, an antibody to a tumor-specific antigen, an anticancer drug, or any other therapeutic compound. suitable for the treatment of a disease or condition, including cancer or viral infection, including chronic viral infection.

As used herein, an isolated antibody is meant to refer to an antibody that is substantially free of other antibodies (Abs) with other types of antigen specificity (e.g., an isolated antibody that specifically binds LAG3, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than LAG3.

As used herein, a blocking antibody or neutralizing antibody (or an antibody that neutralizes LAG3 activity or an antagonist antibody) is intended to refer to an antibody whose binding to LAG3 results in inhibition of at least one type of LAG3 biological activity. For example, an antibody of the present invention can prevent or block the binding of LAG3 to MHC class II.

As used herein, an activating antibody or enhancing antibody (or agonist antibody) is intended to refer to an antibody whose binding to LAG3 results in an increase or stimulation of at least one type of LAG3 biological activity.

For example, an antibody of the present invention can increase the degree of binding of LAG3 to MHC class II.

The term surface plasmon resonance in the context of this document refers to an optical phenomenon that makes it possible to analyze the interactions of biological molecules in real time by detecting changes in protein concentrations on a matrix in the form of a biosensor, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, New Jersey).

As used herein, the term K _D is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as a paratope. One antigen may have more than one epitope. Thus, different antibodies can bind to different regions of the antigen and can have different biological effects. The term epitope also refers to a site on an antigen to which B and/or T cells respond. It also refers to the region of an antigen that an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are usually a variety of structural epitopes, and contain residues that directly affect the affinity of the interaction. Epitopes can also be conformational, that is, consisting of non-linear amino acids. In certain embodiments, epitopes may include determinants that are reactive groups on the surface of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structure characteristics and/or charge characteristics.

The term "cross-compete" as used herein means that an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both directions, i.e. the first antibody binds and blocks the binding of the second antibody, and vice versa. In certain embodiments, the first antibody and the second antibody can bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes such that binding of one antibody inhibits or blocks binding of the second antibody, such as through steric mismatch. Cross-competition between antibodies can be measured using methods known in the art, for example, real-time biolayer interferometric analysis without the use of a label. Cross-competition between two antibodies can be expressed as the binding of the second antibody, which is less than the background signal due to self-binding (where the first and second antibodies are the same antibody). Cross-competition between 2 antibodies can be expressed, for example, as % binding of the second antibody, which is less than the original background signal due to self-binding (where the first and second antibodies are the same antibody).

The term significant identity, or substantially identical, when referring to a nucleic acid or fragment thereof, means that when optimally aligned, with appropriate nucleotide insertions or deletions, with another nucleic acid (or its complementary strand), there is nucleotide sequence identity. at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide bases, as measured by any well known sequence identity algorithm, as discussed below. A nucleic acid molecule that has a significant degree of identity with a reference nucleic acid molecule may, in certain instances, encode a polypeptide with the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

Sequence identity can be calculated using an algorithm such as the Needleman and Wunsch (1970, J. Mol. Biol. 48. 443-453) algorithm for global alignment or the Smith and Waterman (1981, J. Mol. Biol. 147: 195-197) for local alignment. Another preferred algorithm is described by Dufresne et al in Nature Biotechnology in 2002 (vol. 20, pp. 1269-71) and is used in the GenePAST software package (GQ Life Sciences, Inc., Boston, Massachusetts).

In relation to polypeptides, the term significant similarity or substantially similar means that two peptide sequences, when optimally aligned, for example, using the GAP or BESTFIT programs using standard gap opening penalties, have at least 90% sequence identity, even more preferably on

- 14 039718 at least 95, 98 or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced with another amino acid residue with a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be increased to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to the person skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic side chains with a hydroxyl group: series and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups for conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change with a positive value in the PAM250 log probability table disclosed in Gonnet et al. (1992) Science 256: 1443 45, incorporated herein by reference. Average conservative substitution is any change with a non-negative value in the PAM250 log probability table.

Sequence similarity for polypeptides is typically determined using sequence analysis software. Protein analysis software allows similar sequences to be matched by similarity determination for various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as GAP and BESTFIT that can be used using default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutant variant. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percent sequence identity of regions of best overlap between given and target sequences (Pearson (2000), supra). Another preferred algorithm for comparing a sequence of the present invention with a database containing multiple sequences from different organisms is the BLAST computer program, in particular BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

By the phrase "therapeutically effective amount" is meant an amount that provides the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be determined by one of skill in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the term subject refers to an animal, preferably a mammal, in need of the alleviation, prevention and/or treatment of a disease or disorder such as a viral infection or cancer. The term includes human subjects who are at risk of developing or who are observed to have cancer, metastatic cancer, or viral infection.

In the context of this document, an anticancer drug means any agent useful for treating or attenuating or inhibiting the growth of a malignant tumor, including, without limitation, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase , corticosteroids, cyclophosphamide, mitotane (O,P'-(DDD)), biologics (eg antibodies and interferons), and radioactive substances. In the context of this document, a cytotoxin or cytotoxic agent also refers to a chemotherapeutic agent, and means any agent that is detrimental to cells. Examples include Taxol® (paclitaxel), temozolomide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1- dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues.

In the context of this document, the term antiviral drug refers to

- 15 039718 any drug or therapeutic agent used to treat, prevent or attenuate a viral infection in a host subject. The term antiviral drug includes, without limitation, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics, and corticosteroids. In the context of the present invention, viral infections include prolonged or chronic infections caused by viruses, including, without limitation, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), lymphocytic choriomeningitis virus ( LCMV) and simian immunodeficiency virus (SIV).

As used herein, the term enhance the immune response refers to increasing the activity of an immune cell, such as a T cell or NK cell, against a tumor cell or a virus-infected cell. In the context of the present invention, the term includes blocking LAG3-mediated inhibition of T cell activity or reactivation or reversal of T cell depletion. It also includes inhibition of the activity of regulatory T cells. An enhanced immune response in the context of the present invention results in an increased degree of tumor cell killing and/or inhibition of tumor growth.

The antibodies and antigen binding fragments of the present invention bind specifically to LAG3 and enhance T cell activation. Anti-LAG3 antibodies can bind to LAG3 with high affinity or low affinity. In certain embodiments, the antibodies of the present invention may be blocking antibodies, wherein the antibodies may bind to LAG3 and inhibit LAG3 signaling. In some embodiments, the antibodies of the present invention block LAG3 binding to MHC class II and/or stimulate or enhance T cell activation. In some embodiments, the antibodies bind to LAG3 and reverse the anergic state of depleted T cells. In certain embodiments, the antibodies bind to LAG3 and inhibit the activity of regulatory T cells. In some embodiments, antibodies may be useful in stimulating or enhancing an immune response and/or in treating a subject suffering from cancer or a viral infection. The antibodies, when administered to a subject in need thereof, may provide suppression in the subject of a chronic infection caused by a virus such as HIV, LCMV, or HBV. They can be used to inhibit the growth of tumor cells in a subject. They can be used as monotherapy or as adjuvant therapy with other therapeutic compounds or methods known in the art for the treatment of cancer or viral infection.

In certain embodiments, anti-LAG3 antibodies may be multispecific antigen-binding molecules, wherein they have a first binding specificity for LAG3 and a second binding specificity for an antigen selected from the group consisting of another T cell co-inhibitor and another LAG3 epitope.

An immunogen comprising any of the following can be used to generate antibodies to LAG3. In certain embodiments, antibodies of the present invention are obtained from mice immunized with full-length native LAG3 (see NCBI accession number NP_002277.4) (SEQ ID NO: 582) or recombinant LAG3 peptide. Alternatively, LAG3 or a fragment thereof can be obtained using standard biochemical methods and modified (SEQ ID NO: 574-576) and used as an immunogen.

In certain embodiments, the immunogen is one or more extracellular domains of LAG3. According to one embodiment of the present invention, the immunogen is a LAG3 fragment that is within approximately amino acid residues 29-450 of SEQ ID NO: 582.

In some embodiments, the immunogen may be a recombinant LAG3 peptide expressed in E. coli or other eukaryotic or mammalian cells, such as Chinese hamster ovary (CHO) cells.

In certain embodiments, antibodies that specifically bind to LAG3 can be generated using fragments of the above regions or peptides that extend from the defined regions by about 5 to about 20 amino acid residues from each or both of the N- or C-terminus. areas described in this document. In certain embodiments, any combination of the above regions or fragments thereof can be used to produce LAG3-specific antibodies.

Peptides can be modified to include the addition or substitution of certain residues for labeling or for conjugation to carrier molecules such as KLH. For example, cysteine can be added to either the N-terminus or the C-terminus of the peptide, or a linker sequence can be added to provide a peptide for conjugation to, for example, KLH, for immunization.

Certain anti-LAG3 antibodies of the present invention are capable of binding to and neutralizing LAG3 activity as determined by in vitro or in vivo assays. The ability of the antibodies of the present invention to bind to LAG3 and neutralize its activity can be measured using any standard method known to those skilled in the art, including the binding assays or activity assays described herein.

Non-limiting illustrative in vitro assays for measuring binding activity are provided in the Examples section of this document. According to Example 3, the binding affinities and rate constants of human anti-LAG3 antibodies against human LAG3 were determined using the surface plasmon resonance method, and measurements were performed on a Biacore 4000 or T200 instrument. According to Example 4, blocking assays were used to determine cross-competition between anti-LAG3 antibodies. Examples 5 and 6 describe the binding of antibodies to cells overexpressing LAG3. According to Example 7, binding assays were used to determine the ability of anti-LAG3 antibodies to block the ability of LAG3 to bind MHC class II in vitro. According to Example 8, a luciferase gene activity assay was used to determine the ability of anti-LAG3 antibodies to interfere with LAG3 signaling in T cells. According to Example 9, a fluorescence assay was used to determine the ability of anti-LAG3 antibodies to bind to activated monkey CD4+ and CD8+ T cells.

In certain embodiments, the antibodies of the present invention are capable of enhancing or stimulating T cell activity in vitro, in a subject with a cancer, or in a subject infected with a virus, such as LCMV. In certain embodiments, the antibodies of the present invention are used in combination with a second therapeutic agent, such as an antibody to a second T cell co-inhibitor, to enhance an immune response and inhibit tumor growth in a subject.

Antibodies specific for LAG3 may not contain additional labels or compounds, or they may contain an N-terminal or C-terminal label or compound. In one embodiment, the label or compound is biotin. In a binding assay, the location of the label (if any) may determine the orientation of the peptide with respect to the surface on which the peptide is bound. For example, if the surface is coated with avidin, the peptide containing the N-terminal biotin will be positioned such that the C-terminal portion of the peptide is away from the surface. In one embodiment, the label may be a radioisotope, a fluorescent dye, or an MRI-detectable label. In certain embodiments, such labeled antibodies can be used in diagnostic assays, including imaging assays.

Exemplary embodiments of the present invention In certain embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to a protein encoded by human lymphocyte activation gene-3 (LAG3), wherein the antibody or antigen-binding fragment thereof has a property selected from the group, consisting of: (a) binding monomeric human LAG3 with a dissociation constant (KD) for equilibrium binding of less than about 10 nM as measured in a surface plasmon resonance assay at 25°C; (b) bind monomeric human LAG3 with a KD of less than about 8 nM as measured in a surface plasmon resonance assay at 37°C; (c) bind dimeric human LAG3 with a KD of less than about 1.1 nM as measured in a surface plasmon resonance assay at 25°C; (d) bind dimeric human LAG3 with a KD of less than about 1 nM as measured in a surface plasmon resonance assay at 37°C; (e) bind to an hLAG3-expressing cell with an EC _{50 of} less than about 8 nM as measured in a flow cytometry assay; (f) bind to an mfLAG3 expressing cell with an EC _{50 of} less than about 2.3 nM as measured in a flow cytometry assay; (g) block hLAG3 binding to human MHC class II with an IC _{50 of} less than about 32 nM as determined by cell adhesion assay; (h) block hLAG3 binding to mouse MHC class II with an IC _{50 of} less than about 30 nM as determined by cell adhesion assay; (i) block hLAG3 binding to MHC class II by more than 90% as determined by cell adhesion assay; (j) provide relief from LAG3 mediated inhibition of T cell activity with an EC _{50 of} less than about 9 nM as determined by a luciferase reporter gene assay; and (k) bind to activated CD4+ and CD8+ T cells with an EC _{50 of} less than about 1.2 nM, as determined by fluorescent assay.

In certain embodiments, the isolated antibody or antigen-binding fragment thereof of the present invention comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within any of the heavy chain variable region (HCVR) sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any of the light chain variable region (LCVR) sequences shown in Table 1. In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises an HCVR with an amino acid sequence selected from the group consisting of the HCVR sequences shown in Table 1.

In certain embodiments, the isolated antibody or antigen-binding fragment thereof contains an LCVR with an amino acid sequence selected from the group consisting of those shown in Table 1. 1 LCVR sequences.

In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148 164 180 196 212 228 244 260 276 292 308 324 340 356 372 388 404 420 436 452 460 468 476 484 492 500, 508, 516, 540 and 556; (b) an HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 462, 470, 478, 486, 494, 502, 510, 518, 542 and 558; (c) an HCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 464, 472, 480, 488, 496, 504, 512, 520, 544 and 560; (d) an LCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 524, 532, 548 and 564; (e) an LCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 398, 414, 430, 446, 526, 534, 550 and 566; and (f) an LCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240 , 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 528, 536, 552 and 568.

In certain embodiments, the isolated antibody or antigen-binding fragment thereof of the present invention comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74 , 82/90 98/106 114/122 130/138 146/154 162/170 178/186 194/202 210/218 226/234 242/250 258/266 274 /282, 290/298, 306/314, 322/330, 338/346, 354/362, 370/378, 386/394, 402/410, 418/426, 434/442, 450/522, 458/522 , 466/522, 474/522, 482/522, 490/522, 498/530, 506/530, 514/530, 538/546 and 554/562.

In certain embodiments, the isolated antibody or antigen-binding fragment is as claimed in claim 7, wherein the antibody or antigen-binding fragment thereof comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID nO: 386/394, 418 /426 and 538/546.

In certain embodiments, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that blocks the binding of LAG3 to an MHC class II containing three HCVR CDRs, wherein the HCVR contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34 , 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434 , 450, 458, 466, 474, 482, 490, 498, 506, 514, 538 and 554; and three LCVR CDRs, wherein the LCVR contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234 , 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546 and 562.

In certain embodiments, the isolated antibody or antigen binding fragment comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 386/394, 418/426, and 538/546.

In certain embodiments, the present invention provides an antibody or antigen-binding fragment thereof that competes for binding with an antibody or antigen-binding fragment thereof comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 18/26 , 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226 /234, 242/250, 258/266, 274/282, 290/298, 306/314, 322/330, 338/346, 354/362, 370/378, 386/394, 402/410, 418/426 .

In certain embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody or antigen-binding fragment thereof, comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 2/10 18/26 34/42 50/58 66/74 82/90 98/106 114/122 130/138 146/154 162/170 178/186 194/202 210 /218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 322/330, 338/346, 354/362, 370/378, 386/394, 402/410 , 418/426 434/442 450/522 458/522 466/522 /562.

In certain embodiments, the present invention provides an antibody, or antigen-binding fragment thereof, that is a human, humanized, or chimeric antibody. Said antibody, or antigen-binding fragment thereof, may be, for example, an IgG1 or IgG4 antibody, such as, for example, a human IgG1 or IgG4 antibody. The constant regions of such antibodies may correspond to wild-type or mutated constant regions.

- 18 039718

In certain embodiments, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 577, 579, and 580. In certain embodiments, the present invention provides an isolated an antibody comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 578 and 581. chain/light chain selected from the group consisting of SEQ ID NOs: 577/578, 579/578 and 580/581.

In one aspect, the present invention provides a multispecific antigen-binding molecule, comprising a first antigen-binding specific element that specifically binds to LAG3 and a second antigen-binding specific element that specifically binds to a second target epitope.

In one aspect, the present invention relates to a pharmaceutical composition comprising an anti-LAG3 antibody or antigen-binding fragment thereof according to any of the above embodiments and a pharmaceutically acceptable carrier or diluent.

In one aspect, the present invention relates to isolated polynucleotide molecules and vectors containing polynucleotide sequences encoding the antibodies or antigen-binding fragment thereof disclosed herein. In certain embodiments, the present invention provides an isolated polynucleotide molecule and/or a vector containing a polynucleotide sequence that encodes for the HCVR of an antibody provided herein. In certain embodiments, the present invention provides an isolated polynucleotide molecule and/or a vector containing a polynucleotide sequence that encodes for the LCVR of an antibody provided herein.

In one aspect, the present invention relates to methods for enhancing an immune response in a subject, the method comprising administering a pharmaceutical composition comprising an isolated anti-LAG3 antibody or antigen-binding fragment disclosed herein. In certain embodiments, the present invention relates to methods for inhibiting a regulatory T cell (Treg) in a subject, comprising administering a pharmaceutical composition comprising the isolated anti-LAG3 antibody or antigen-binding fragment disclosed herein. In certain embodiments, the present invention relates to methods for enhancing T cell activation in a subject, the method comprising administering a pharmaceutical composition comprising the isolated anti-LAG3 antibody or antigen-binding fragment disclosed herein. In certain embodiments, the subject has a disease or disorder selected from the group consisting of hematopoietic malignancy, brain cancer, renal cell carcinoma (e.g., clear cell carcinoma of the kidney), ovarian cancer, bladder cancer, prostate cancer, cancer breast cancer (eg, triple-negative breast cancer), hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colon and rectal cancer, mesothelioma, lymphoma (eg, B-cell lymphoma, diffuse B-large cell lymphoma) and melanoma. In certain embodiments, the subject has a chronic viral infection caused by a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), human papillomavirus (HPV), lymphocytic virus choriomeningitis (LCMV) and simian immunodeficiency virus (SIV). In certain embodiments, an anti-LAG3 antibody is administered to a subject in combination with a second therapeutic agent selected from the group consisting of a PD-1 inhibitor, a CTLA inhibitor, an antibody to a tumor-specific antigen, an antibody to a virus-infected cell antigen, a PD-L1 inhibitor, a CD20 inhibitor, an anti-CD20 and CD3 bispecific antibody, a dietary supplement such as an antioxidant, a VEGF antagonist, a chemotherapeutic agent, a cytotoxic agent, radiation, an NSAID, a corticosteroid, and any other therapy suitable for alleviating at least one symptom associated with a disease or disorder.

In one aspect, the present invention relates to methods for inhibiting the growth of a tumor or tumor cell in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LAG3 antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the tumor is primary or recurrent. In certain embodiments, the tumor is an advanced tumor. In certain embodiments, the subject has a metastatic tumor and/or has been treated with prior therapy. In certain embodiments, the tumor is present in a subject with a disease or disorder selected from the group consisting of hematopoietic tissue cancer, brain cancer, renal cell cancer, ovarian cancer, bladder cancer, prostate cancer.

- 19 039718 cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colon and rectal cancer, mesothelioma, lymphoma and melanoma. In certain embodiments, the anti-LAG3 antibody or antigen-binding fragment thereof is administered in one or more doses, with each dose administered 1 to 4 weeks after the nearest prior dose. In certain embodiments, the anti-LAG3 antibody, or antigen-binding fragment thereof, is administered at a dose of about 0.1 mg/kg body weight to about 100 mg/kg body weight of the subject. In certain embodiments, an anti-LAG3 antibody is administered to a subject in combination with a second therapeutic agent selected from the group consisting of a PD-1 inhibitor, a CTLA inhibitor, a tumor-specific antigen antibody, a PD-L1 inhibitor, a CD20 inhibitor, an anti-CD20 and CD3 bispecific antibody, a dietary supplement such as an antioxidant, a VEGF antagonist, a chemotherapeutic agent, a cytotoxic agent, radiation, an NSAID, a corticosteroid, and any other therapy suitable for alleviating at least one symptom associated with a disease or disorder. In one embodiment, the second therapeutic agent is a PD-1 inhibitor, wherein the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to PD-1. In one embodiment, the PD-1 inhibitor is REGN2810. In certain embodiments, the anti-LAG3 antibody, or antigen-binding fragment thereof, is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

In one aspect, the present invention relates to methods for relieving LAG3-mediated inhibition of T cell activity, comprising contacting the T cell with an anti-LAG3 antibody or antigen-binding fragment thereof disclosed herein. In one embodiment, the T cell is contacted with an anti-LAG3 antibody of the present invention in combination with an anti-PD-1 antibody (eg, REGN2810).

Antigen-binding antibody fragments

Unless specifically stated otherwise, the term antibody, as used herein, is to be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., complete antibody molecules), as well as antigen-binding fragments thereof. The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, etc., as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms antigen-binding antibody fragment or antibody fragment as used herein refer to one or more antibody fragments that retain the ability to specifically bind to LAG3. An antibody fragment may include a Fab fragment, a F(ab') ₂ fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term antigen-binding fragment refers to a polypeptide fragment of a polyspecific antigen-binding molecule. In such embodiments, the term antigen-binding moiety includes, for example, an MHC class II molecule that specifically binds to LAG3. Antigen-binding fragments of an antibody can be generated, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic cleavage techniques or recombinant DNA genetic engineering techniques to manipulate and express DNA encoding the variable and (optionally) constant domains of antibodies. . Such DNA is known and/or readily available, for example from commercial sources, DNA libraries (including, for example, antibody phage libraries), or it can be synthesized. The DNA can be sequenced and chemically manipulated using molecular biology techniques, for example, to place one or more variable and/or constant domains in an appropriate configuration, or to include codons, introduce cysteine residues, modify, add or remove amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (eg, a distinguished complementarity determining region (CDR), such as a CDR3 peptide), or a sterically restricted FR3- CDR3-FR4. Other engineered molecules such as domain specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanoantibodies (e.g. monovalent nanobodies, divalent nanobodies, etc.) , small modular protein (SMIP) immunopharmaceuticals, and shark IgNAR variable domains are also encompassed within the context of this document by the expression antigen-binding fragment.

An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition, and

- 20 039718 will typically contain at least one CDR that is adjacent to one or more frame sequences or is in the same reading frame with them. In antigen-binding fragments with a VH domain associated with a VL domain, the VH and V _L domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be dimeric and contain V _H - V _H , V _H - V _L or V _L - V _L dimers. Alternatively, the antigen binding fragment of an antibody may contain a monomeric VH or VL domain.

In certain embodiments, an antigen-binding antibody fragment may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary variable and constant domain configurations that can be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of the variable and constant domains, including any of the above illustrative configurations, the variable and constant domains can either be directly connected to each other, or can be connected through a full or partial hinge region or a linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connection of adjacent variable and/or constant domains within a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the above variable and constant domain configurations non-covalently linked to each other and/or to one or more monomeric VH or VL domains (e.g. , via disulfide(s) bond(s)).

As with full antibody molecules, antigen binding fragments can be monospecific or polyspecific (eg, bispecific). A polyspecific antigen binding fragment of an antibody will typically contain at least two different variable domains, where each variable domain is capable of specifically binding to a different antigen or a different epitope on the same antigen. Any multispecific antibody format, including exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using conventional techniques known in the art.

Obtaining human antibodies

Methods for producing human antibodies in transgenic mice are known in the art. Any of these known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to LAG3.

Using VELOCIMMUNE® technology (see, for example, US 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for producing monoclonal antibodies, high affinity chimeric anti-LAG3 antibodies with a human variable region and a mouse constant region are first isolated. The VELOCIMMUNE® technology provides for the production of a transgenic mouse with a genome containing sequences encoding human heavy and light chain variable regions operably linked to endogenous loci encoding mouse constant regions, whereby in response to antigen stimulation, the mouse produces an antibody containing a human variable region and mouse constant region. The DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to the DNA encoding the constant regions of the human heavy and light chains. The DNA is then allowed to be expressed in a cell capable of expressing the fully human antibody.

In general, a VELOCIMMUNE® derived mouse is immunized with an antigen of interest, and lymphocytes (such as B cells) are recovered from mice expressing antibodies. Lymphocytes can be fused to a myeloma cell line to form immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired isotypic heavy chain and light chain constant regions. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

First, high-affinity chimeric antibodies with a human variable region and a mouse constant region are isolated. As described below in the experiments section, antibodies are characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant region to produce a fully human antibody of the present invention. eg wild-type IgG1 or IgG4 or modified IgG1 or IgG4. While the constant region chosen may vary according to the particular application, the characteristics of high affinity antigen binding and target specificity are inherent in the variable region.

Bioequivalents

Anti-LAG3 antibodies and antibody fragments of the present invention encompass proteins with amino acid sequences that differ from those of the described antibodies, but retain the ability to bind LAG3. Such variant antibodies and antibody fragments have one or more amino acid additions, deletions, or substitutions from the original sequence, but have biological activity that is substantially equivalent to that of the described antibodies. Similarly, the antibody-encoding DNA sequences of the present invention encompass sequences that include one or more nucleotide additions, deletions, or substitutions compared to the disclosed sequence, but that encode an antibody or antibody fragment that is, in essence, bioequivalent to the antibody or antibody fragment of the present invention. invention.

Two antigen-binding proteins or antibodies are considered to be bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical variants, the rate and extent of absorption of which is not significantly different when administered at the same molar dose under similar experimental conditions, either as a single dose or in multiple dose form. Some antibodies will be considered equivalents or pharmaceutical options if they are equivalent in their extent of absorption, but not in their rate of absorption, and may still be considered bioequivalent because such differences in absorption rate are intentional and are reflected in the instructions for use of the medicinal product, not are essential to achieve effective drug concentrations in the body, for example, with long-term use, and are not considered medically significant for a particular investigational drug.

In one embodiment, two antigen-binding proteins are considered bioequivalent if there are no clinically relevant differences in their safety, purity, or potency.

In one embodiment, two antigen-binding proteins are bioequivalent if the patient can be switched one or more times from treatment with a reference drug to treatment with a biological drug, and vice versa, without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduced efficacy, compared with ongoing therapy without such a transfer.

In one embodiment, two antigen-binding proteins are bioequivalent if they both function by a common mechanism or mechanisms of action for a condition or conditions, provided that such mechanisms are known.

Bioequivalence can be demonstrated using in vivo and/or in vitro methods. Determining bioequivalence includes, for example, (a) an in vivo test in humans or other animals that measures the concentration of an antibody or its metabolites in blood, plasma, serum, or body fluid as a function of time; (b) an in vitro test that correlates with in vivo bioavailability data in humans and is sufficiently predictive of such data; (c) an in vivo test in humans or other animals in which the corresponding immediate pharmacological effect of an antibody (or its target) is measured as a function of time; and (d) a clinical trial under highly controlled conditions that establishes the safety, efficacy, or bioavailability or bioequivalence of the antibody.

Bioequivalent variants of the antibodies of the present invention can be constructed, for example, by making various residue or sequence substitutions, or deleting terminal or internal residues or sequences not necessary for biological activity. For example, cysteine residues not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of extra or inappropriate intramolecular disulfide bridges upon renaturation. In other instances, bioequivalent antibodies may include antibody variants that include amino acid changes that modify the glycosylation characteristics of the antibodies, such as mutations that exclude or abolish glycosylation.

Anti-LAG3 antibodies containing Fc variants

In accordance with certain embodiments of the present invention, anti-LAG3 antibodies are provided that contain an Fc domain containing one or more mutations that increase or decrease the binding of the antibody to the FcRn receptor, for example, at acidic pH versus neutral pH. For example, the present invention includes anti-LAG3 antibodies comprising a mutation in the CH2 or C _H 3 region of an Fc domain, wherein the mutation(s) increase(s) the affinity of the Fc domain for FcRn in an acidic environment (e.g., in the endosome where the pH is ranging from approx.

- 22 039718

5.5 to about 6.0). Such mutations can lead to an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (eg L/Y/F/W or T), 254 (eg S or T) and 256 (eg S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g. H/L/R/S/P/Q or K) and/or 434 (e.g. A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (eg 308F, V308F) and 434. In one embodiment, the modification includes modification 428L (eg M428L) and 434S (eg N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 433K (for example, H433K) and modification 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (eg, 308F or 308P). In yet another embodiment, the modification includes modification 265A (eg, D265A) and/or 297A (eg, N297A).

For example, the present invention includes antibodies to LAG3 containing an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (eg, T250Q and M248L); 252Y, 254T and 256E (eg M252Y, S254T and T256E); 428L and 434S (eg M428L and N434S); 257I and 311I (eg P257I and Q311I); 257I and 434H (eg P257I and N434H); 376V and 434H (for example, D376V and N434H); 307A, 380A and 434A (for example, T307A, E380A and N434A); and 433K and 434F (eg H433K and N434F). In one embodiment, the present invention includes anti-LAG3 antibodies comprising an Fc domain containing an S108P mutation in the IgG4 hinge region to help stabilize the dimer. All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are included within the scope of the present invention.

The present invention also includes anti- _LAG3 antibodies comprising a chimeric heavy chain constant (CH) region, wherein the chimeric CH region contains segments derived from _CH regions of more than one immunoglobulin isotype. For example, antibodies of the present invention may comprise a chimeric C _H region comprising part or all of the C _H 2 domains derived from a human IgG1, human IgG2, or human IgG4 molecule, in combination with part or all of the C _H 3 domains, derived from a human IgG1, human IgG2 or human IgG4 molecule. In accordance with certain embodiments, the antibodies of the present invention comprise a chimeric CH region with a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge amino acid sequence (amino acid residues corresponding to positions 216-227 according to EU numbering) derived from the hinge region of human IgG1, human IgG2, or human IgG4, in combination with a lower hinge sequence (amino acid residues, corresponding to provisions 228-236 according to the EU numbering) derived from the hinge region of human IgG1, human IgG2 or human IgG4. In certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. An antibody comprising a chimeric C _H region as described herein can, in certain embodiments, exhibit the effector functions of a modified Fc without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, US Patent Application Publication No. 20140243504, the disclosure of which is incorporated herein by reference in its entirety). In certain embodiments, the Fc region contains a sequence selected from the group consisting of SEQ ID NOs: 569, 570, 571, 572, and 573.

Biological characteristics of antibodies

In general, the function of the antibodies of the present invention is to bind LAG3. The present invention includes anti-LAG3 antibodies and antigen-binding fragments thereof that bind soluble monomeric or dimeric LAG3 molecules with high affinity. For example, the present invention includes antibodies and antigen-binding antibody fragments that bind monomeric LAG3 (e.g., at 25° C. or 37° C.) with a K _D of less than about 10 nM, as measured by a surface plasmon resonance method, e.g. the analysis format defined in Example 3 herein. In certain embodiments, the antibodies, or antigen-binding fragments thereof, bind monomeric LAG3 with a K _D of less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.1 nM, less than less than about 0.05 nM, or less than about 0.04 nM, as measured by surface plasmon resonance, for example, using the assay format defined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding fragments thereof that bind dimeric LAG3 (e.g., at 25°C or 37°C) with a K _D of less than about 1.1 nM,

- 23 039718 as measured using the method of surface plasmon resonance, for example, using the analysis format defined in example 3 herein. In certain embodiments, the antibodies, or antigen-binding fragments thereof, bind dimeric LAG3 with a K _D of less than about 0.5 nM, less than about 0.25 nM, less than about 0.1 nM, less than about 0.05 nM, or less than about 0.01 M as measured by surface plasmon resonance, for example, using the assay format defined in Example 3 herein, or a substantially similar assay.

The invention also includes antibodies and antigen-binding fragments thereof that bind LAG3 with a dissociation half-life (t ¹ /2) of greater than about 1.6 min as measured by surface plasmon resonance at 25°C or 37°C, for example using the assay format defined in Example 3 herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind LAG3 to t ¹ /2 for more than about 5 minutes, more than about 10 minutes, more than about 30 minutes, more than about 50 minutes, more than about 60 minutes, more than more than about 70 minutes, more than about 80 minutes, more than about 90 minutes, more than about 100 minutes, more than about 200 minutes, more than about 300 minutes, more than about 400 minutes, more than about 500 minutes, more than about 600 min, more than about 700 min, more than about 800 min, more than about 900 min, more than about 1000 min, or more than about 1100 min, as measured by surface plasmon resonance at 25°C or 37°C, for example , using the assay format defined in Example 3 herein (e.g., mAb immobilization format or and antigen immobilization), or a largely similar assay.

The present invention also includes antibodies, or antigen-binding fragments thereof, that bind to a human LAG3 expressing cell with an EC _{50 of} less than about 8 nM, as measured by the flow cytometric assay defined in Example 5 herein, or a substantially similar assay. . In certain embodiments, the antibodies, or antigen-binding fragments thereof, bind to an hLAG3-expressing cell with an EC _{50 of} less than about 5 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by assay using flow cytometry, for example, using the assay format of Example 5 herein, or a substantially similar assay.

The present invention also includes antibodies, or antigen-binding fragments thereof, that bind to a cynomolgus monkey LAG3-expressing cell with an EC _{50 of} less than about 2.5 nM, as measured by the flow cytometry assay defined in Example 5 herein, or substantially degree of such analysis. In certain embodiments, the antibodies, or antigen-binding fragments thereof, bind to an mfLAG3-expressing cell with an EC _{50 of} less than about 2 nM, or less than about 1 nM, as measured by a flow cytometric assay, e.g., using the assay format defined in Example 5 in this document, or a largely similar analysis.

The present invention also includes antibodies, or antigen-binding fragments thereof, that block the binding of LAG3 to MHC class II (HLA-DR2 human) with an IC _{50 of} less than about 32 nM, as determined using a cell adhesion assay, such as that provided in Example 7, or in much of the same analysis. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block the binding of LAG3 to human MHC class II with an IC _{50 of} less than about 25 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, as measured by cell assay. adhesion, for example, using the assay format defined in Example 7 herein, or a substantially similar assay.

The present invention also includes antibodies, or antigen-binding fragments thereof, that block binding of LAG3 to MHC class II (mouse HLA-DR2) with an IC _{50 of} less than about 30 nM, as determined using a cell adhesion assay, such as that provided in Example 7, or in much of the same analysis. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block binding of mouse LAG3 to human MHC class II with an IC _{50 of} less than about 25 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, as measured by assay cell adhesion, for example, using the assay format defined in Example 7 herein, or a substantially similar assay.

The present invention also includes antibodies, or an antigen-binding fragment thereof, that block LAG3 binding to human or mouse MHC class II by more than 90% as measured by the cell adhesion assay defined in Example 7 herein, or by a significant amount. degree of such analysis.

The present invention also includes antibodies, or antigen-binding fragments thereof, that block LAG-induced downregulation of T cells with an EC _{50 of} less than 9 nM as measured by a luciferase reporter gene assay using T cells/APC as defined in Example 8 herein. document, or a largely similar analysis. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block _LAG3 -induced downregulation of T cells with an EC50 of less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by using a luciferase reporter gene assay using T cells/APC, for example using the assay format defined in Example 8 herein, or a substantially similar assay.

The present invention also includes antibodies, or antigen-binding fragments thereof, that bind to activated CD4+ and CD8+ cynomolgus monkey T cells with an EC _{50 of} less than about 1.2 nM, as measured by the fluorescent assay defined in Example 9 herein, or in much of the same analysis. In certain embodiments, the antibodies, or antigen-binding fragments thereof, bind to activated CD4+ and CD8+ cynomolgus monkey T cells with an EC _{50 of} less than about 1.1 nM, less than about 1 nM, less than about 0.5 nM, less than about 0, 2 nM, or less than about 0.1 nM, as measured by a fluorescent assay, eg, using the assay format defined in Example 9 herein, or a substantially similar assay.

In certain embodiments, the function of the antibodies of the present invention may be to block or inhibit the binding activity of MHC class II associated with LAG3 by binding to any other region or fragment of the full-length protein, the amino acid sequence of which is represented by SEQ ID NO: 582.

In certain embodiments, the antibodies of the present invention are useful in inhibiting tumor growth or delaying cancer progression when administered prophylactically to a subject in need, and may provide improved survival of the subject. For example, administration of an antibody of the present invention may result in shrinkage of the primary tumor and may prevent metastasis or development of secondary tumors. In certain embodiments, the antibodies of the present invention are useful in inhibiting tumor growth when therapeutically administered to a subject in need thereof, and may provide improved survival of the subject. For example, administration to a subject of a therapeutically effective amount of an antibody of the present invention may result in the reduction and disappearance of a developed tumor in the subject.

In one embodiment, the present invention provides an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that binds to LAG3, wherein the antibody or fragment thereof has one or more of the following: (i) comprises an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554, or a sequence substantially similar to it, characterized by at least 90%, 95%, at least 98%, or at least 99% sequence identity; (ii) contains an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250 , 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546 and 562, or a sequence substantially similar to it, characterized by at least 90%, at least at least 95%, at least 98%, or at least 99% sequence identity; (iii) contains an HCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 528, 536, 552 and 568, or a sequence substantially similar to it, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) contains an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228 , 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 460, 468, 476, 484, 492, 500, 508, 516, 540 and 556, or substantially similar sequence, characterized by at least 90%, at least

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95%, at least 98%, or at least 99% sequence identity; HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278 , 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 462, 470, 478, 486, 494, 502, 510, 518, 542, and 558, or a substantially similar sequence , characterized by at least 90%, at least 95%, at least 98% or at least 99% sequence identity; LCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268 , 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 524, 532, 548 and 564, or a sequence substantially similar to it, characterized by at least 90%, at least 95 %, at least 98% or at least 99% sequence identity; and an LCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 398, 414, 430, 446, 526, 534, 550 and 566, or a sequence substantially similar to it, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) bind monomeric human LAG3 with a dissociation constant (KD) for equilibrium binding of less than about 10 nM as measured in a surface plasmon resonance assay at 25°C; (vi) bind monomeric human LAG3 with a KD of less than about 8 nM as measured in a surface plasmon resonance assay at 37°C; (vii) bind dimeric human LAG3 with a K _D of less than about 1.1 nM as measured in a surface plasmon resonance assay at 25°C; (viii) bind dimeric human LAG3 with a K _D of less than about 1 nM as measured in a surface plasmon resonance assay at 37°C; (ix) bind to an hLAG3-expressing cell with an EC50 of less than about 8 nM as measured in a flow cytometry assay; (x) bind to an mfLAG3 expressing cell with an EC50 of less than about 2.3 nM as measured in a flow cytometry assay; (xi) block hLAG3 binding to human MHC class II with an IC50 of less than about 32 nM as determined by cell adhesion assay; (xii) block hLAG3 binding to mouse MHC class II with an IC50 of less than about 30 nM as determined by cell adhesion assay; (xiii) block hLAG3 binding to MHC class II by more than 90% as determined by cell adhesion assay; (xiv) provide relief from LAG3 mediated inhibition of T cell activity with an EC _{50 of} less than about 9 nM as determined by a luciferase reporter gene assay; (xv) bind to activated CD4+ and CD8+ T cells with an EC50 of less than about 1.2 nM, as determined by fluorescent assay; (xvi) provide suppression of tumor growth and increase the survival of a subject with a malignant tumor; and (xvii) are fully human.

The antibodies of the present invention may have one or more of the aforementioned biological characteristics, or any combination thereof. Other biological characteristics of the antibodies of the present invention will be apparent to those skilled in the art from the overview of the present disclosure, including the demonstration examples herein.

Cross-Species Selectivity and Cross-Species Reactivity In accordance with certain embodiments of the present invention, anti-LAG3 antibodies bind to human LAG3 but not to LAG3 of other species. Alternatively, anti-LAG3 antibodies of the present invention, in certain embodiments, bind to human LAG3 and to LAG3 of one or more non-human species. For example, anti-LAG3 antibodies of the present invention may bind to human LAG3, and may or may not bind, as appropriate, one or more of mouse, rat, guinea pig, hamster, gerbil, porcine, cat, dog, rabbit, goat LAG3. , sheep, cow, horse, camel, cynomolgus monkey, monkey, rhesus monkey or chimpanzee. In certain embodiments, the anti-LAG3 antibodies of the present invention can bind to human and cynomolgus monkey LAG3 with similar affinities or different affinities, but do not bind to rat and mouse LAG3.

Epitope mapping and related technologies

The present invention includes anti-LAG3 antibodies that interact with one or more amino acids located within one or more domains of the LAG3 molecule, including, for example, D1-D4 extracellular domains, a transmembrane domain, and an intracellular domain. The epitope to which antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the aforementioned domains of the LAG3 molecule (eg, a linear domain epitope). Alternatively, an epitope may consist of a plurality of discontinuous amino acids (or amino acid sequences) located within any or all of the aforementioned domains of the LAG3 molecule (eg, a conformational epitope).

To determine whether an antibody interacts with one or more amino acids

within a polypeptide or protein, various techniques known to those skilled in the art can be used. Illustrative techniques include, for example, standard epitope cross-competition assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, New York). Other methods include alanine scanning mutational analysis, peptide blotting (Reineke (2004) Methods Mol. Biol. 248. 443-63), crystallographic studies with peptide digestion assays, and NMR analysis. In addition, methods such as the epitope excision method, the epitope isolation method, and the chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9. 487-496). Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen-deuterium exchange, detected using mass spectrometry. In general terms, the hydrogen-deuterium exchange method involves labeling a protein of interest with deuterium, followed by binding of an antibody to the deuterium labeled protein. The protein/antibody complex is then transferred to water, and the exchangeable protons in amino acids closed when complexed with the antibody undergo deuterium-to-hydrogen exchange more slowly than exchangeable protons in amino acids that are not part of the contact zone. As a result, amino acids that form part of the protein/antibody junction may retain deuterium in their composition and thus have a relatively higher mass compared to amino acids not included in the junction. After dissociation of the antibody, the target protein is subjected to protease digestion and mass spectrometric analysis, thus revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

The term epitope refers to a site on an antigen to which B and/or T cells respond. Epitopes for B cells can be formed by both continuous and discontinuous amino acids located side by side due to protein folding into a tertiary structure. Epitopes formed from continuous amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by folding into a tertiary structure are generally lost upon treatment with denaturing solvents. An epitope typically includes at least 3, and more often at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP), is a method that allows the classification of large numbers of monoclonal antibodies (mAbs) directed against the same antigen, based on the similarity of the binding profiles of each antibody with chemically or enzymatically modified antigen surfaces (see US 2004/0101920, incorporated herein by reference in its entirety). Each category may correspond to a unique epitope that either clearly differs from the epitope corresponding to another category, or partially overlaps with it. This technology allows the exclusion of genetically identical antibodies, so that characterization can be focused on genetically different antibodies. When used for hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to classify the antibodies of the present invention into groups of antibodies that bind different epitopes.

In certain embodiments, anti-LAG3 antibodies, or antigen-binding fragments thereof, bind an epitope within any one or more of the regions represented by LAG3, either in natural form as represented by SEQ ID NO: 582 or recombinantly produced as represented by SEQ ID NO: 574 - 576 , or their fragment. In some embodiments, the antibodies of the present invention bind to an extracellular region containing one or more amino acids selected from the group consisting of LAG3 amino acid residues 29-450. In some embodiments, the antibodies of the present invention bind to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 1-533 of the cynomolgus monkey LAG3 represented by SEQ ID NO: 576.

In certain embodiments, the present invention includes anti-LAG3 antibodies and antigen-binding fragments thereof that interact with one or more epitopes located within the extracellular region of LAG3 (SEQ ID NO: 588). The epitope(s) may consist of one or more contiguous sequences of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within the extracellular region of LAG3. Alternatively, an epitope may consist of a plurality of discontinuous amino acids (or amino acid sequences) located within the extracellular region of LAG3. As shown in Example 18 herein, the LAG3 epitope with which the exemplary antibody of the present invention, H4sH15482P interacts, is defined by the amino acid sequence LRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (seq ID 589), which corresponds to amino acids 28-71 of SEQ ID NO: 588. Therefore, the present invention is 039718 The definition includes anti-LAG3 antibodies that interact with one or more amino acids contained within the region consisting of amino acids 28-71 of SEQ ID NO: 588 (i.e., the sequence LRRAGVTWQHQPDSGPPAAAPGHPLAPGPPHPAAPSSWGPRPRRY [seq id _yes .589]).

The present invention includes antibodies to LAG3 that bind to the same epitope, or part of an epitope, as any of the specific illustrative antibodies described herein in table. 1, or an antibody with CDR sequences from any of the exemplary antibodies described in Table 1. Similarly, the present invention also includes anti-LAG3 antibodies that compete for binding to LAG3 or a fragment of a LAG3 fragment with any of the specific exemplary antibodies described herein in Table 1, or an antibody with CDR sequences from any of the exemplary antibodies described in Table 1. .one. For example, the present invention includes antibodies to LAG3 that cross-compete for binding to LAG3 with one or more of the antibodies defined in Example 4 herein (e.g., H4sH15482P, H4sH15479P, H4sH14813N, H4H14813N, H4H15479P, H4H15482P, H4H15483P, H4sH15498P H4H17828P2, H4H17819P and H4H17823P).

Using standard methods known in the art, it can be readily determined whether an antibody binds to the same epitope as a reference anti-LAG3 antibody or competes with it for binding. For example, to determine if a test antibody binds to the same epitope as a reference anti-LAG3 antibody of the present invention, the reference antibody is provided to bind to the LAG3 protein or peptide under saturation conditions. The ability of the test antibody to bind to the LAG3 molecule is then evaluated. If the test antibody is able to bind to LAG3 after saturation binding using a reference anti-LAG3 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-LAG3 antibody. Conversely, if the test antibody is unable to bind to the LAG3 protein after saturation binding using the reference anti-LAG3 antibody, then the test antibody can bind to the same epitope as the epitope bound by the reference anti-LAG3 antibody of the present invention.

To determine if an antibody competes for binding with a reference anti-LAG3 antibody, the bidirectional binding method described above is performed: In the first direction method, binding of the reference antibody to the LAG3 protein under saturation conditions is carried out, followed by an assessment of the binding of the test antibody to the molecule LAG3. When implementing the method in the second direction, the binding of the test antibody to the LAG3 molecule under saturation conditions is ensured, followed by the evaluation of the binding of the reference antibody to the LAG3 molecule. If, when the method is carried out in both directions, only the first (saturation binding) antibody is able to bind to the LAG3 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to LAG3. As will be appreciated by one of skill in the art, an antibody that competes for binding with a reference antibody need not bind to an identical epitope to that of the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or contiguous epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other to the antigen. Namely, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, and preferably 75%, 90%, or even 99%, as measured in a competitive binding assay ( see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies bind the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies bind overlapping epitopes if certain amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

Additional routine experiments (e.g., peptide mutations and binding assays) can then be performed to confirm whether the observed lack of binding of the antibody under test is indeed due to binding to the same epitope as that of the reference antibody, or whether steric blocking is responsible for the lack of observed binding ( or some other event). Experiments of this type can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay known in the art.

Immunoconjugates

The present invention encompasses a human anti-LAG3 monoclonal antibody conjugated to a therapeutic compound (immunoconjugate), such as a cytotoxin or a chemotherapeutic agent for the treatment of cancer. As used herein, the term immunoconjugate refers to an antibody that is chemically or biologically linked to a cytotoxin, radioactive substance, cytokine, interferon, targeting or reporter compound, enzyme, toxin, peptide or protein, or therapeutic agent. An antibody may be associated with a cytotoxin, radioactive substance, cytokine, interferon, targeting or reporter compound, enzyme, toxin, peptide, or therapeutic agent at any location along the entire molecule, provided that it retains the ability to bind its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second distinct anti-LAG3 antibody. In certain embodiments, the antibody may be conjugated with an agent specific for a tumor cell or virus-infected cell. The type of therapeutic compound that can be conjugated to the anti-LAG3 antibody will depend on the condition being treated and the desired therapeutic effect to be achieved. Examples of suitable agents for the formation of immunoconjugates are known in the art; see, for example, WO 05/103081.

Polyspecific antibodies

The antibodies of the present invention may be monospecific, bispecific or polyspecific. Multispecific antibodies may be specific for different epitopes of the same target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

In one aspect, the present invention includes polyspecific antigen-binding molecules, or antigen-binding fragments thereof, wherein one specific immunoglobulin element is specific for the extracellular domain of LAG3, or a fragment thereof, and another specific immunoglobulin element is specific for binding outside the extracellular domain of LAG3, or a second target. for a therapeutic effect, or conjugated to a therapeutic compound.

Any of the polyspecific antigen-binding molecules of the present invention, or variants thereof, can be designed using standard molecular biology techniques (eg, recombinant DNA technology and protein engineering), as will be known to those skilled in the art.

In some embodiments, LAG3-specific antibodies are produced in a bispecific format (bispecific) in which variable regions that bind to different LAG3 domains are linked together to provide specificity for two domains within a single binding molecule. Appropriately designed bispecific molecules can enhance overall efficacy in LAG3 inhibition by increasing both specificity and binding avidity. Variable regions with specificity for individual domains (eg, segments of the N-terminal domain), or which can bind to different regions within the same domain, are paired on a scaffold, allowing each region to bind simultaneously to separate epitopes or to different regions. within the same domain. In one example, heavy chain variable regions (VH) of a binding molecule with specificity for one domain are recombined with variable light chain regions (V _L ) of a number of binding molecules with specificity for a second domain to identify unrelated VL partners that can be paired. with the original V _H without violating the original specificity for that V _H . Thus, one V _L segment (eg VL1) can be combined with two different VH domains (eg VH1 and VH2) to form a bispecific molecule consisting of binding arms (VH1-VL1 and VH2VL1). The use of a single VL segment simplifies the system and thus facilitates and enhances the cloning, expression and purification processes used to obtain the bispecific molecule (see, for example, USSN13/022759 and US2010/0331527).

Alternatively, antibodies that bind more than one domain and a second target, such as, but not limited to, for example, a second distinct anti-LAG3 antibody, can be generated in a bispecific format using the techniques described herein or other known to those skilled in the art. techniques. Variable regions of an antibody that bind to different regions can be linked together with variable regions that bind to corresponding regions, eg, the extracellular domain of LAG3, to provide specificity for two antigens within a single binding molecule. Appropriately designed bispecific molecules of this type perform a dual function. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity for binding outside the extracellular domain and paired on a scaffold that allows each variable region to bind to separate antigens.

An exemplary bispecific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig C _H 3 domains differ from each other in at least one amino acid. , and where the difference in at least one amino acid causes a decrease in the ability of the bispecific antibody to bind to protein A compared to the bispecific anti

- 29 039718 body, not characterized by such a difference in amino acid. In one embodiment, the first Ig C _H 3 domain binds protein A, and the second Ig C _H 3 domain contains a mutation that reduces or eliminates the ability to bind to protein A, such as an H95R modification (according to the IMGT mutation numbering in exons; H435R according to EU numbering). The second CH3 may additionally provide for the Y96F modification (as per IMGT; Y436F as per EU). Additional modifications that can be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I according to EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (as per IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I as per EU) for IGg4 antibodies. Variations on the format of the bispecific antibody described above are included within the scope of the present invention.

Other illustrative bispecific formats that can be used in the context of the present invention include, without limitation, for example, scFv-based or diabody bispecific formats, IgG-scFv fusion bispecific formats, Ig dual variable domains (DVD), Quadroma, antibodies tab-in-pit, common light chain antibodies (e.g., tab-in-pit common light chain antibodies, etc.), CrossMab, CrossFab, SEED-derived antibody ((SEED)body), antibody, produced by leucine zipper mediated dimerization, Duobody, IgG1/IgG2, IgG with dual acting Fab (DAF) and Mab ² bispecific formats (see e.g. Klein et al. 2012, mAbs 4:6, 1-11 and cited therein links, for an overview of the formats listed above). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, for example, where non-naturally occurring amino acids with cross-reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valence, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Administration and Formulations

The present invention relates to therapeutic compositions containing antibodies to LAG3 or their antigennegative fragments of the present invention. Therapeutic compositions in accordance with the present invention will be administered with suitable carriers, excipients and other means, which are included in the compositions to provide improved transfer, delivery, tolerability, etc. A variety of suitable formulations can be found in the reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipid containing lipid (cationic or anionic) vesicles (e.g. LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and water. in oil, Carbowax in the form of emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax. See also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of the antibody may vary depending on the age and size of the subject to whom it will be administered, the target disease, conditions, route of administration, and so on. When an antibody of the present invention is used to treat a disease or disorder in an adult patient, or to prevent such a disease, it is expedient to administer the antibody of the present invention usually as a single dose of from about 0.1 to about 60 mg/kg of body weight, more preferably about 5 to about 60, about 20 to about 50, about 10 to about 50, about 1 to about 10, or about 0.8 to about 11 mg/kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, an antibody or antigen-binding fragment thereof of the present invention may be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or from about 10 to about 200 mg, to about 100 mg, or to about 50 mg. In certain embodiments, after the initial dose, a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof may be administered in an amount that may be about the same or less than the amount for the initial dose, where administration of subsequent doses is separated by a time span of from to at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks or at least 14 weeks.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, for example, incorporation into liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated

- 30 039718 tori endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Routes of administration include, without limitation, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes of administration. The composition may be administered by any suitable route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and small intestinal mucosa, etc.), and may be administered together with other biologically active agents. The introduction can be systemic or local. The pharmaceutical composition can also be delivered as a vesicle, in particular a liposome (see, for example, Langer (1990) Science 249:1527-1533).

The use of nanoparticles for delivery of antibodies of the present invention is also included in this document. The antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods for their preparation and use are described in detail in Arruebo, M., et al. 2009 (Antibody-conjugated nanoparticles for biomedical applications in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporated herein by reference. The nanoparticles can be designed and conjugated with antibodies contained in pharmaceutical compositions to target tumor cells, or autoimmune tissue cells, or virus-infected cells. Nanoparticles for drug delivery have also been described, for example, in US 8257740 or US 8246995, each of which is incorporated herein in its entirety.

In certain cases, the pharmaceutical composition may be delivered using a controlled release system. In one embodiment, a pump may be used. According to another embodiment, polymeric substances can be used. According to another embodiment, the controlled release system can be placed near the target of the composition, whereby only a fraction of the systemic dose is required.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. Such injectable preparations can be obtained using well-known methods. For example, injectable preparations can be obtained, for example, by dissolving, suspending or emulsifying the antibody described above, or a salt thereof, in a sterile aqueous or oily medium commonly used for injection. As an aqueous injection medium, there are, for example, physiological saline, an isotonic solution containing glucose and other excipients, etc., which can be used in combination with an appropriate solubilizing agent such as alcohol (for example, ethanol), polyhydric alcohol (e.g. propylene glycol, polyethylene glycol), nonionic surfactant (e.g. polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The thus obtained injection solution is preferably filled in an appropriate ampoule.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen-type delivery device can be easily used to deliver the pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. A refillable pen delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition from the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. The disposable pen delivery device does not have a replaceable cartridge. Instead, a disposable pen-type delivery device comes pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the reservoir is depleted of the pharmaceutical composition, the device is completely discarded.

For subcutaneous delivery of the pharmaceutical composition of the present invention, a variety of delivery devices in the form of a syringe pen and a reusable auto-injector can be used. Examples include, without limitation, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, syringe - HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Indiana), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), syringe pen BD™ (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis,

- 31 039718

Frankfurt, Germany) and many others. Examples of disposable pen delivery devices that can be used to deliver the pharmaceutical composition of the present invention subcutaneously include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), auto-injector SURECLICK™ (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA™ pen (Abbott Labs, Abbott Park, Illinois) and many others.

Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are formulated into unit dosage forms suitable for adjusting the dosage of the active ingredients. Such single dose dosage forms include, for example, tablets, pills, capsules, injectable solutions (ampoules), suppositories, etc. The amount of antibody they contain is typically from about 5 to about 500 mg per single dose dosage form; in particular, in the case of an injection form, it is preferred that the antibody is contained in an amount of from about 5 to about 100 mg, and from about 10 to about 250 mg for other dosage forms.

Therapeutic applications of antibodies

The antibodies of the present invention are useful, inter alia, in the treatment, prevention and/or amelioration of any disease or disorder associated with or caused by the expression, signaling or activity of LAG3, or treatable by blocking the interaction between LAG3 and LAG3 MHC class II ligand. or another way of inhibiting LAG3 activity and/or signaling with its participation. For example, the present invention provides methods for treating cancer (tumor growth inhibition) and/or viral infections by administering an anti-LAG3 antibody (or a pharmaceutical composition comprising an anti-LAG3 antibody) described herein to a patient in need of such treatment, and anti-LAG3 antibodies. (or a pharmaceutical composition containing an anti-LAG3 antibody) for use in the treatment of cancer (inhibition of tumor growth) and/or viral infections. The antibodies of the present invention are useful in the treatment, prevention and/or amelioration of a disease or disorder or condition such as cancer or a viral infection and/or in the amelioration of at least one symptom associated with such disease, disorder or condition. In the context of the treatments described herein, an anti-LAG3 antibody may be administered as monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

In some embodiments, the antibodies described herein are useful in treating subjects suffering from primary or recurrent cancer, including, without limitation, hematopoietic malignancy, brain cancer (e.g., glioblastoma multiforme), renal cell carcinoma (e.g., clear cell kidney cancer), ovarian cancer, bladder cancer, prostate cancer, breast cancer (eg, triple negative breast cancer), hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colon cancer and rectum, mesothelioma and melanoma.

In the context of this document, the term malignant neoplasm of the hematopoietic tissue includes a malignant disease of the blood system that affects the blood, bone marrow, lymph or lymphatic system. Thus, the term includes malignant neoplasms from cells of the lymphoid and myeloid cell lineages. From cells of the myeloid line of differentiation, granulocytes, erythrocytes, platelets, macrophages, and mast cells are usually formed; from cells of the myeloid line of differentiation, B-, T-, NK- and plasma cells are formed. Thus, the term includes malignant neoplasms from the aforementioned cells, namely forms of lymphoma, forms of myeloma, forms of lymphoid leukemia and forms of myeloid leukemia. Examples include, without limitation, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, forms of Hodgkin's lymphoma, forms of non-Hodgkin's lymphoma (e.g., B-cell lymphoma, diffuse large cell lymphoma), and myeloma (including multiple myeloma) .

Antibodies can be used to treat symptoms of early or advanced cancer. In one embodiment, an antibody or fragment thereof of the present invention can be used to treat advanced stage or metastatic cancer. The antibodies are useful in slowing down or inhibiting or reducing the rate of tumor growth in both solid tumors and hematopoietic malignancies. In certain embodiments, treatment with an antibody or antigen-binding fragment thereof of the present invention provides greater than 40% regression, greater than 50% regression, greater than 60% regression, greater than 70% regression, greater than 80% regression, or greater than 90% tumor regression in the subject. According to defined

- 32 039718 In other embodiments, antibodies can be used to prevent tumor recurrence. In certain embodiments, antibodies are useful in increasing progression-free survival or overall survival of a subject with cancer. In some embodiments, antibodies are useful in reducing toxicity associated with chemotherapy or radiation therapy while maintaining long-term survival of a patient suffering from cancer.

In certain embodiments, the subject is a patient suffering from cancer and who has not previously received anti-PD-1/PD-L1 antibody therapy, but is a potential candidate for anti-PD-1 antibody therapy, and/or has previously received anti-PD-1/PD-L1 antibody therapy and had a confirmed objective response (CR or PR) or SD for at least 3 months while on anti-PD-1/PD-L1 antibody therapy, but then there was progression during such therapy, or SD or PR was observed as the best response followed by a sustained response within 6 months; and/or is not a candidate for standard therapy, or where the available therapy is not expected to provide clinical benefit, and is suitable for mAb1 monotherapy; and/or suffering from untreated anti-PD-1/PD-L1 NSCLC stage IIIB or IV, either without prior therapy for metastatic disease, or with progression/recurrence of disease following a platinum drug regimen; and/or suffering from anti-PD-1/PD-L1 antibody-treated stage IIIB or IV NSCLC with no more than 2 prior therapies for metastatic disease; and/or suffering from untreated with antibodies to PD-1/PD-L1 late stage ccRCC or metastatic ccRCC with a clear cell component, and who received 1-2 prior anti-angiogenic therapeutic regimens; and/or suffering from advanced anti-PD-1/PD-L1 antibody-treated ccRCC or metastatic ccRCC with a clear cell component and who has received 1-2 prior anti-angiogenic therapeutic regimens; and/or suffering from untreated PD-1/PD-L1 antibodies, metastatic TNBC (estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 negative) and who has received 5 or fewer previous lines of therapy; and/or suffers from untreated advanced PD-1/PD-L1 antibodies or metastatic melanoma, and who has received no more than 2 prior treatment regimens for metastatic disease; and/or suffering from advanced anti-PD-1/PD-L1 antibody-treated melanoma or metastatic melanoma, and who has received no more than 2 prior treatment regimens for metastatic disease; and/or suffers from untreated with anti-PD-1/PD-L1 antibodies, relapsed/refractory DLBCL, and who either progressed after autologous stem cell transplantation or is not a candidate for it; and/or suffers from PD-1/PD-L1 antibody-treated relapsed/refractory DLBCL and has either progressed following autologous stem cell transplantation or is not a candidate for autologous stem cell transplantation.

In certain embodiments, the antibodies of the present invention are useful in the treatment of subjects suffering from a chronic viral infection. In some embodiments, the antibodies of the present invention are useful in reducing viral titers in the host and/or reactivating depleted T cells. In certain embodiments, an antibody or fragment thereof of the present invention may be used to treat chronic lymphocytic choriomeningitis virus (LCMV) viral infection. In some embodiments, an antibody or antigen-binding fragment thereof of the present invention may be administered at a therapeutic dose to a patient with a human immunodeficiency virus (HIV) or human papillomavirus (HPV) or hepatitis B/C (HBV/HCV) infection. In a similar embodiment, an antibody or antigen-binding fragment thereof of the present invention can be used to treat a simian immunodeficiency virus (SIV) infection in a simian subject, such as a cynomolgus monkey.

In certain embodiments, a blocking antibody of the present invention may be administered in a therapeutically effective amount to a subject suffering from a cancer or viral infection.

One or more antibodies of the present invention may be administered to alleviate or prevent or reduce the severity of one or more of the symptoms or conditions of a disease or disorder.

This document also contemplates the prophylactic use of one or

- 33 039718 several antibodies of the present invention for patients at risk of developing a disease or disorder such as cancer and viral infection.

According to a further embodiment of the present invention, the antibodies of the present invention are used in the preparation of a pharmaceutical composition for the treatment of patients suffering from cancer or a viral infection. According to another embodiment of the present invention, the antibodies of the present invention are used as an adjuvant therapy along with another agent known to those skilled in the art or other therapy suitable for the treatment of cancer or viral infection.

Combined Therapeutic Products and Formulations

Combination therapeutics include an anti-LAG3 antibody of the present invention and any additional therapeutic agent that can advantageously be combined with an antibody of the present invention or with a biologically active fragment of an antibody of the present invention.

The antibodies of the present invention can be combined to produce a synergistic effect with one or more anticancer drugs or therapy used to treat or inhibit cancer, including, for example, hematopoietic cancer, brain cancer (e.g., glioblastoma multiforme), renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colon and rectal cancer, mesothelioma and melanoma. The present document contemplates the use of the anti-LAG3 antibodies of the present invention in combination with immunostimulatory and/or immunoreplacement therapies to inhibit tumor growth and/or improve survival in cancer patients. Immunostimulatory therapies include direct-acting immunostimulatory therapies to increase the activity of immune cells, either by releasing the brake on suppressed immune cells or by adding gas to activate the immune response. Examples include targeting other receptors that are part of immunological checkpoints, vaccination, and adjuvants. Methods of immunoreplacement effects can provide an increase in tumor antigenicity by stimulating immunogenic cell death, inflammation, or have other indirect effects that stimulate an antitumor immune response. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery.

In various embodiments, one or more antibodies of the present invention may be used in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, BGB-A317 or REGN2810), a PD-L1 inhibitor (e.g. , an anti-PD-L1 antibody such as avelumab, atezolizumab, durvalumab, MDX-1105, or REGN3504), CTLA-4 inhibitor (eg, ipilimumab), TIM3 inhibitor, BTLA inhibitor, TIGI inhibitor, CD47 inhibitor, GITR inhibitor, other co-inhibitor antagonist T cell or ligand (eg, anti-CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA antibody), indolamine 2,3 dioxygenase inhibitor (IDO, vascular endothelial growth factor (VEGF) antagonist (eg, trap protein for VEGF, such as aflibercept, or another VEGF-inhibiting fusion protein as set forth in US 7,087,411, or an anti-VEGF antibody or antigen-binding fragment thereof (e.g., bevacizumab or ranibizumab) or a small molecule VEG kinase inhibitor F receptor (eg, sunitinib, sorafenib, or pazopanib)), Ang2 inhibitor (eg, nesvacumab), transforming growth factor beta (TGFP) inhibitor, epidermal growth factor receptor (EGFR) inhibitor (eg, erlotinib, cetuximab), CD20 inhibitor ( for example, an anti-CD20 antibody such as rituximab), an antibody against a tumor-specific antigen (eg, CA9, CA125, melanoma-associated antigen 3 (MAGE3), germline tumor antigen (CEA), vimentin, tumor M2-PK, prostate-specific antigen ( PSA), mucin-1, MART-1, and CA19-9), a vaccine (eg, Bacillus Calmette-Guerin, cancer vaccine), an adjuvant to enhance antigen presentation (eg, granulocyte-monocyte colony stimulating factor), a bispecific antibody (eg, a bispecific anti-CD3xCD20 antibody or anti-PSMAxCD3 bispecific antibody), cytotoxin, chemotherapeutic agent (eg, dacarbazine, temozolomide, cyclophosphamido m, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitor (eg, sarilumab), IL-4R inhibitor (eg, dupilumab), an IL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21 and IL-15, an antibody drug conjugate (ADC) (e.g. anti-CD19-DM4 ADC and anti-DS6-DM4 ADC) , an anti-inflammatory drug (eg, corticosteroids and non-steroidal anti-inflammatory drugs), a dietary supplement such as antioxidants, or any other cancer therapy.

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In certain embodiments, the anti-LAG3 antibodies of the present invention can be used in combination with antitumor vaccines, including dendritic cell based vaccines, oncolytic viruses, tumor cell based vaccines, etc., to enhance the antitumor response. Examples of cancer vaccines that can be used in combination with the anti-LAG3 antibodies of the present invention include MAGE3 vaccine for melanoma and bladder cancer, MUC1 vaccine for breast cancer, EGFRv3 (e.g. rindopepimut) for brain cancer (including glioblastoma multiforme). ) or ALVAC-CEA (for CEA+ forms of malignancy).

In certain embodiments, the anti-LAG3 antibodies of the present invention can be used in combination with radiation therapy in methods for providing long-term antitumor responses and/or improving survival in cancer patients. In some embodiments, the anti-LAG3 antibodies of the present invention may be administered to a cancer patient before, after, or concurrent with radiation therapy. For example, radiation therapy can be applied at one or more doses to tumor foci, followed by one or more doses of the anti-LAG3 antibodies of the present invention. In some embodiments, radiation therapy can be applied locally to a tumor site to enhance the local immunogenicity of a patient's tumor (adjuvant radiation) and/or to kill tumor cells (ablative radiation therapy) followed by systemic administration of an anti-LAG3 antibody of the present invention. For example, intracranial irradiation can be used in a patient with a malignant brain tumor (eg, glioblastoma multiforme) in combination with systemic administration of an anti-LAG3 antibody of the present invention. In certain embodiments, the anti-LAG3 antibodies of the present invention may be used in combination with radiation therapy and a chemotherapeutic agent (eg, temozolomide) or a VEGF antagonist (eg, aflibercept).

In certain embodiments, the anti-LAG3 antibodies of the present invention may be administered in combination with one or more antiviral drugs for the treatment of a chronic viral infection caused by LCMV, HIV, HPV, HBV, or HCV. Examples of antiviral drugs include, without limitation, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids.

Additional therapeutically active agents/components may be administered before, after, or simultaneously with administration of the anti-LAG3 antibody of the present invention. For the purposes of the present disclosure, such administration regimens involve the administration of an anti-LAG3 antibody in combination with a second therapeutically active moiety.

Additional(s) therapeutically active(s) component(s) can be administered to the subject prior to the introduction of antibodies to LAG3 of the present invention. For example, the first component may be considered to be administered prior to the administration of the second component if the first component is administered 1 week prior, 72 hours prior, 60 hours prior, 48 hours prior, 36 hours prior, 24 hours prior, 12 h before, 6 h before, 5 h before, 4 h before, 3 h before, 2 h before, 1 h before, 30 min before, 15 min before, 10 min before, 5 min before or less than 1 min before the introduction of the second component. In other embodiments, the additional(s) therapeutically active(s) component(s) can be administered to a subject after administration of an anti-LAG3 antibody of the present invention. For example, the first component can be considered to be administered after the administration of the second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 h after, 3 h after, 4 h after, 5 h after, 6 h after, 12 h after, 24 h after, 36 h after, 48 h after, 60 h after, after 72 h after the introduction of the second component. According to still other embodiments, the additional(s) therapeutically active(s) component(s) can be administered to a subject simultaneously with the administration of an anti-LAG3 antibody of the present invention. Simultaneous administration, for the purposes of the present invention, includes, for example, administering an anti-LAG3 antibody and an additional therapeutically active component to a subject in a single dosage form (for example, as a combined formulation), or in separate dosage forms administered to a subject with a difference of approximately 30 minutes or less apart. When administered in separate dosage forms, each dosage form can be administered by the same route (eg, both the anti-LAG3 antibody and the additional therapeutically active component can be administered intravenously, subcutaneously, etc.); alternatively, each dosage form may be administered by a different route (eg, the anti-LAG3 antibody may be administered intravenously and the additional therapeutically active ingredient may be administered subcutaneously). For the purposes of this disclosure, in any case, the administration of the components in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes, is considered to be simultaneous administration. For the purposes of this disclosure, administration of an anti-LAG3 antibody before, concurrently with, or after (as these terms are defined herein above) administration of an additional therapeutically active moiety is considered to be administration of an anti-LAG3 antibody in combination with an additional therapeutically active moiety).

The present invention includes pharmaceutical compositions in which an anti-LAG3 antibody

- 35 039718 of the present invention is formulated with one or more of the additional therapeutically active components described elsewhere in this document, using a number of dosage combinations.

In exemplary embodiments wherein an anti-LAG3 antibody of the present invention is administered in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody disclosed in US 2015/0203579, incorporated herein by reference in its entirety), including administering combination formulations containing an anti-LAG3 antibody and a PD-1 inhibitor, the individual components may be administered to a subject and/or may be formulated together using a range of dosage combinations. Thus, the present invention includes a combination of (i) an anti-LAG3 antibody of the present invention and (ii) a PD-1 inhibitor (e.g., an anti-PD-1 antibody disclosed in US 2015/0203579, incorporated herein by reference in its entirety ) for joint, separate and/or sequential use in the treatment of cancer or viral infections. For example, each of an anti-LAG3 antibody and a PD-1 inhibitor (e.g., an anti-PD-1 antibody) may be administered to a subject and/or may be present in a combined formulation in an amount selected from the group consisting of 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg and 10.0 mg. Combinations/combination formulations may be administered to a subject according to any of the administration schedules disclosed elsewhere herein, including, for example, administration twice a week, once a week, once every 2 weeks, once every 3 weeks, once per month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, etc. An anti-LAG3 antibody of the present invention may be administered, for example, at a dose of about 0.8 to about 11, about 1 to about 10, about 3 to about 10, about 1, about 3, or about 10 mg/kg , together with a PD-1 inhibitor (eg, an anti-PD-1 antibody disclosed in US 2015/0203579), at a dose of about 3 to 5 or about 3.0 mg/kg. Co-administration can be carried out, for example, every 14 days, 21 days or 28 days.

In exemplary embodiments in which an anti-LAG3 antibody of the present invention is administered in combination with a VEGF antagonist (e.g., a VEGF decoy protein such as aflibercept), including administering combined formulations containing an anti-LAG3 antibody and a VEGF antagonist, the subject may be administered separate the components and/or they can be combined using a variety of dosage combinations. For example, an anti-LAG3 antibody may be administered to a subject and/or may be present in a combination formulation in an amount selected from the group consisting of 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg , 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg , 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg , 8.0 mg, 9.0 mg and 10.0 mg; and a VEGF antagonist (e.g., a VEGF decoy protein such as aflibercept) may be administered to the subject and/or may be present in the combination formulation in an amount selected from the group consisting of 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg and 3.0 mg. Combinations/combination formulations may be administered to a subject according to any of the administration schedules disclosed elsewhere herein, including, for example, administration twice a week, once a week, once every 2 weeks, once every 3 weeks, once per month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, etc.

Introduction schemes

In accordance with certain embodiments of the present invention, multiple doses of an anti-LAG3 antibody (or a pharmaceutical composition containing a combination of an anti-LAG3 antibody and any of the additional therapeutically active agents referred to herein) can be administered to a subject over a period of time. Methods in accordance with this aspect of the present invention provide for sequential administration of multiple doses of an anti-LAG3 antibody of the present invention to a subject. As used herein, sequential administration means that a dose of an anti-LAG3 antibody is administered to a subject at different points in time, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that sequentially administer to a patient a single initial dose of an anti-LAG3 antibody, followed by one or more doses of a second line of anti-LAG3 antibody, and optionally followed by one or more doses of a third line of anti-LAG3 antibody. The anti-LAG3 antibody can be administered at a dose of 0.1 to 100 mg/kg of the subject's body weight.

The terms initial dose, second line doses, and third line doses refer to the time sequence of administration of an anti-LAG3 antibody of the present invention. Thus, the initial dose is the dose that is administered at the beginning of the course of treatment (also called the initial dose); second line doses are doses that are administered after the initial dose; and third line doses are doses that are administered after the second line doses. The initial dose, second and third line doses may contain the same amount of anti-LAG3 antibody, but in general

- 36 039718 may differ from each other, taking into account the frequency of administration. However, in certain embodiments, the amount of anti-LAG3 antibody contained in the initial, second, and third line doses differ from each other (eg, adjusted upwards or downwards as necessary) over the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the course of treatment as loading doses, followed by doses that are administered less frequently (eg, maintenance doses).

In certain embodiments, the amount of anti-LAG3 antibody contained in the initial, second and/or third line doses may be suboptimal or subtherapeutic. As used herein, the terms subtherapeutic or suboptimal refer to a dose of an antibody administered at a level too low to provide a therapeutic effect, or at a level below that needed to treat a disease such as cancer.

According to certain illustrative embodiments of the present invention, each dose of the second and/or third line is administered through 1-26 (for example,

1, 2, 2G, 3, 3G, 4, 4G, 5, 5G, 6, 6G, 7.8, 8G, 9, 9G, 10, 10G, 11, 11G, 12,

12G, 13, 13G, 14, 14G, 15, 15G, 16, 16G, 17, 17G, 18, 18G, 19, 19G, 20, 20G, 21, 21G, 22,

22G, 23, 23G, 24, 24G, 25, 25G, 26, 26G, or more) weeks after the next previous dose. The phrase "next prior dose" as used herein means, in the case of a multiple dose sequence, the dose of anti-LAG3 antibody that is administered to the patient prior to the next dose, in the case of a sequence with no intermediate doses.

Methods in accordance with this aspect of the present invention may involve administering any number of second and/or third doses of an anti-LAG3 antibody to a patient. For example, in certain embodiments, only one dose of the second line is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) doses of the second line. Similarly, in certain embodiments, the patient is administered only one third-in-line dose. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) third dose doses.

In embodiments involving multiple second-line doses, each second-line dose may be administered at the same frequency as the other second-line doses. For example, each dose of the second line can be administered to the patient 1-2 weeks or 1-2 months after the next previous dose. Similarly, in embodiments involving multiple third line doses, each third line dose may be administered at the same frequency as the other third line doses. For example, each third line dose may be administered to a patient 2-12 weeks after the next prior dose. In certain embodiments of the present invention, the frequency with which second and/or third line doses are administered to a patient may vary over the course of treatment. The frequency of administration can also be adjusted by the physician during the course of treatment, depending on the needs of the individual patient participating in the clinical study.

Diagnostic applications of antibodies

Anti-LAG3 antibodies of the present invention can be used to detect and/or measure LAG3 in a sample, for example for diagnostic purposes.

In some embodiments, one or more antibodies of the present invention are contemplated for use in assays for detecting a disease or disorder, such as cancer, an autoimmune disease, or a viral infection. Exemplary diagnostic assays for LAG3 may involve, for example, contacting a sample obtained from a subject (e.g., a patient) with an anti-LAG3 antibody of the present invention, wherein the anti-LAG3 antibody is labeled with a detectable label or reporter molecule or used as a capture ligand. for selective isolation of LAG3 from samples from a subject. Alternatively, an unlabeled anti-LAG3 antibody can be used for diagnostic applications in combination with a secondary antibody that is labeled for detection. The detectable label or reporter molecule may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I; a fluorescent or chemiluminescent substance such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure LAG3 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence-activated cell sorting (FACS).

Samples that can be used in diagnostic assays for LAG3 in accordance with the present invention include any sample of tissue or body fluid that can be obtained from a subject that contains detectable amounts of either LAG3 protein or fragments thereof, under normal or pathological conditions. In general, LAG3 levels in a particular

- 37 039718 a sample obtained from a healthy patient (for example, a patient who does not suffer from a malignant tumor or an autoimmune disease) will be measured first to establish the initial, or standard, level of LAG3. This baseline LAG3 level can then be compared to levels

LAG3 measured in samples obtained from individuals suspected of having a condition associated with a malignant tumor, or symptoms associated with such a condition.

Antibodies specific for LAG3 may not contain additional labels or compounds, or they may contain an N-terminal or C-terminal label or compound. In one embodiment, the label or compound is biotin. In a binding assay, the location of the label (if any) may determine the orientation of the peptide with respect to the surface on which the peptide is bound. For example, if the surface is coated with avidin, the peptide containing the N-terminal biotin will be positioned such that the C-terminal portion of the peptide is away from the surface.

Aspects of the present invention relate to the use of the disclosed antibodies as markers for predicting cancer or viral infection in patients. The antibodies of the present invention can be used in diagnostic assays to predict cancer in a patient and to predict survival rates.

Examples

The following examples are provided for the purpose of providing those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention and are not intended to limit the scope of what the present inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to the values used (eg amounts, temperatures, etc.), however some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is atmospheric or near atmospheric.

Example 1. Obtaining human antibodies to LAG3.

Human anti-LAG3 antibodies were generated using a LAG3 fragment that is approximately amino acid residues 29-450 of GenBank accession number NP_002277.4 (SEQ ID NO: 582) fused to a mouse Fc region. The immunogen, with an adjuvant to stimulate an immune response, was directly administered to a VELOCIMMUNE® mouse (i.e., an engineered mouse containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions) as described in US 8502018 B2, or a Veloclmmune® humanized Universal Light Chain (ULC) mouse as described in WO 2013022782. The humoral immune response was monitored using a LAG3-specific immunoassay. When the desired immune response was achieved, the splenocytes were collected and fused with mouse myeloma cells to preserve the viability of the former and to obtain hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce LAG3-specific antibodies. Using this technique, and the immunogen described above, several chimeric anti-LAG3 antibodies (ie, antibodies having human variable domains and mouse constant domains) have been generated; exemplary antibodies thus obtained from VELOCIMMUNE® technology-derived mice have been designated H1M14985N, H1M14987N, H2M14811N, H2M14885N, H2M14926N, H2M14927N, H2M14931N, H2M18336N, H2M18337N and H3N1481.

Anti-LAG3 antibodies were also isolated directly from antigen positive B cells (from each of the immunized mice) without fusion with myeloma cells, as described in US Patent Application No. 7,582,298, incorporated herein by reference in its entirety. Several fully human anti-LAG3 antibodies (ie, antibodies having human variable domains and human constant domains) have been generated using this method;

exemplary antibodies thus obtained have been designated as

H4H15477R,

N4N15483R, N4N15484R, N4N15491R, N4N17823R, N4N17826R2, N4N17828R2,

H4sH15460P, H4sH15462P, H4sH15463P, H4sH15464P, H4sH15466P, H4sH15467P,

H4sH15470P, H4sH15475P, H4sH15479P, H4sH15480P, H4sH15482P, H4sH15488P,

H4sH15496P2, H4sH15498P2, H4sH15505P2, H4sH15518P2, H4sH15523P2, H4sH15530P2, H4sH15555P2, H4sH15558P2, H4sH15567P2 and H4H17819P.

- 38 039718

Illustrative Antibodies

H4sH15496P2, H4sH15498P2, H4sH15505P2,

H4sH15518P2, H4sH15523P2, H4sH15530P2, H4sH15555P2, H4sH15558P2 and H4sH15567P2 were derived from B cells from ULC-derived Velocimmune® mice.

The biological properties of exemplary antibodies prepared according to the methods in this example are detailed in the examples below.

Example 2 Amino acid and nucleotide sequences for heavy and light chain variable regions.

Table 1 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-LAG3 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are shown in Table 2.

Table 1. Amino acid sequence identifiers

SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H1M14985N 2 four 6 eight ten 12 fourteen 16 H1M14987N eighteen twenty 22 24 26 28 thirty 32 H2M14811N H2M14885N H2M14926N H2M14927N H2M14931N H2M18336N H2M18337N Н4Н15477Р Н4Н15483Р Н4Н15484Р Н4Н15491Р H4N17823R H4H17826R2 H4N17828R2 H4sH15460P H4sH15462P H4sH15463P H4sH15464P H4sH15466P H4sH15467P H4sH15470P H4sH15475P H4sH15479P H4sH15480P H4sH15482P H4sH15488P H4sH15496P2 H4sH15498P2 34 fifty 66 82 98 114 130 146 162 178 194 210 226 242 258 274 290 306 322 338 354 370 386 402 418 434 450 458 36 52 68 84 100 116 132 148 164 180 196 212 228 244 260 276 292 308 324 340 356 372 388 404 420 436 452 460 38 54 70 86 102 118 134 150 166 182 198 214 230 246 262 278 294 310 326 342 358 374 390 406 422 438 454 462 40 56 72 88 104 120 136 152 168 184 200 216 232 248 264 280 296 312 328 344 360 376 392 408 424 440 456 464 42 58 74 90 106 122 138 154 170 186 202 218 234 250 266 282 298 314 330 346 362 378 394 410 426 442 522 522 44 60 76 92 108 124 140 156 172 188 204 220 236 252 268 284 300 316 332 348 364 380 396 412 428 444 524 524 46 62 78 94 110 126 142 158 174 190 206 222 238 254 270 286 302 318 334 350 366 382 398 414 430 446 526 526 48 64 80 96 112 128 144 160 176 192 208 224 240 256 272 288 304 320 336 352 368 384 400 416 432 448 528 528 H4sH15505P2 466 468 470 472 522 524 526 528 H4sH15518P2 474 476 478 480 522 524 526 528 H4sH15523P2 482 484 486 488 522 524 526 528 H4sH15530P2 490 492 494 496 522 524 526 528 H4sH15555P2 498 500 502 504 530 532 534 536 H4sH15558P2 506 508 510 512 530 532 534 536 H4sH15567P2 514 516 518 520 530 532 534 536 H4H14813N 538 540 542 544 546 548 550 552 H4H17819P 554 556 558 560 562 564 566 568

- 39 039718

Table 2. Nucleic acid sequence identifiers

SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H1M14985N one 3 5 7 9 eleven 13 fifteen H1M14987N 17 19 21 23 25 27 29 31 H2M14811N H2M14885N H2M14926N H2M14927N H2M14931N H2M18336N H2M18337N Н4Н15477Р Н4Н15483Р Н4Н15484Р Н4Н15491Р H4N17823R H4H17826R2 H4N17828R2 H4sH15460P H4sH15462P H4sH15463P 33 49 65 81 97 113 129 145 161 177 193 209 225 241 257 273 289 35 51 67 83 99 115 131 147 163 179 195 211 227 243 259 275 291 37 53 69 85 101 117 133 149 165 181 197 213 229 245 261 277 293 39 55 71 87 103 119 135 151 167 183 199 215 231 247 263 279 295 41 57 73 89 105 121 137 153 169 185 201 217 233 249 265 281 297 43 59 75 91 107 123 139 155 171 187 203 219 235 251 267 283 299 45 61 77 93 109 125 141 157 173 189 205 221 237 253 269 285 301 47 63 79 95 111 127 143 159 175 191 207 223 239 255 271 287 303 H4sH15464P 305 307 309 311 313 315 317 319 H4sH15466P 321 323 325 327 329 331 333 335 H4sH15467P 337 339 341 343 345 347 349 351 H4sH15470P 353 355 357 359 361 363 365 367 H4sH15475P 369 371 373 375 377 379 381 383 H4sH15479P 385 387 389 391 393 395 397 399 H4sH15480P 401 403 405 407 409 411 413 415 H4SH15482P 417 419 421 423 425 427 429 431 H4sH15488P 433 435 437 439 441 443 445 447 H4sH15496P2 449 451 453 455 521 523 525 527 H4sH15498P2 457 459 461 463 521 523 525 527 H4sH15505P2 465 467 469 471 521 523 525 527 H4sH15518P2 473 475 477 479 521 523 525 527 H4sH15523P2 481 483 485 487 521 523 525 527 H4sH15530P2 489 491 493 495 521 523 525 527 H4sH15555P2 497 499 501 503 529 531 533 535 H4sH15558P2 505 507 509 511 529 531 533 535 H4sH15567P2 513 515 517 519 529 531 533 535 H4H14813N 537 539 541 543 545 547 549 551 H4H17819P 553 555 557 559 561 563 565 567

Antibodies are generally named herein according to the following notation: a prefix corresponding to Fc (e.g., H1M, H4sH, H4H, etc.) followed by a numeric identifier (e.g., 14813, 17828, etc.). , as shown in Table 1), followed by the suffix P, P2, or N. Thus, according to this notation, an antibody herein may be named, for example, H4sH14813N, H4H17819P, H4H17828P2, etc. The prefixes H4sH and H4H in antibody designations used herein indicate a particular isotype in the Fc region of the antibody. For example, the H4sH antibody contains a human IgG4 Fc with 2 or more amino acid changes as disclosed in US20140243504 (incorporated herein in its entirety), the H4H antibody contains a human IgG4 Fc with a serine to

- 40 039718 hinge proline (S108P), HIM antibody contains mouse IgG1 Fc, and H2M antibody contains mouse IgG2 Fc (all variable regions are fully human as indicated by the first H in the antibody designation). As one skilled in the art will appreciate, an antibody with a Fc of a particular isotype can be converted into an antibody with an Fc of a different isotype (e.g., an antibody with a mouse IgG1 Fc can be converted into an antibody with a human IgG4 Fc, etc.), but in any case, variable domains (including CDR) - which are designated by the numerical identifiers shown in the table. 1 will remain the same and antigen binding properties are expected to be identical or substantially similar, regardless of the origin of the Fc domain.

In certain embodiments, selected mouse IgG1 Fc antibodies are converted to human IgG4 Fc antibodies. In certain embodiments, the antibody comprises a human IgG4 Fc with 2 or more amino acid changes as disclosed in US20100331527 (incorporated herein in its entirety). In one embodiment, the IgG4 Fc domain contains a serine to proline mutation in the hinge region (S108P) to help stabilize the dimer. In certain embodiments, the Fc region of the antibodies of the present invention comprises the amino acid sequences of SEQ ID NOs: 569, 570, 571, 572, or 573. Table 3 provides amino acid sequence identifiers for the heavy chain and light chain sequences of selected anti-LAG3 antibodies with IgG4 Fc person.

Table 3. Heavy chain and light chain amino acid sequence identifiers

Antibody designation SEQID NO: heavy chain Light chain H4sH15482P 577 578 H4H15482P 579 578 H4sH14813N 580 581

Each heavy chain sequence in Table 3 forms a variable region (V _H OR HCVR containing HCDR1, HCDR2 and HCDR3) and a constant region (containing CH1, CH2 and CH3 domains). Each light chain sequence in Table 3 forms a variable region (VL or LCVR containing LCDR1, LCDR2 and LCDR3) and a constant region (C _L ). SEQ ID NO: 577 generates an HCVR containing amino acids 1-123 and a constant region containing amino acids 124-449. SEQ ID NO: 578 generates an LCVR containing amino acids 1-107 and a constant region containing amino acids 108-214. SEQ ID NO: 579 generates an HCVR containing amino acids 1-123 and a constant region containing amino acids 124-450. SEQ ID NO: 580 generates an HCVR containing amino acids 1-119 and a constant region containing amino acids 120-445. SEQ ID NO: 581 generates an LCVR containing amino acids 1-107 and a constant region containing amino acids 108-214.

Control Constructs Used in the Following Examples For comparison purposes, two control constructs (anti-LAG3 antibodies) were included in the following experiments: Comparator 1, a human anti-LAG3 monoclonal antibody with the VH/VL sequences of the 25F7 antibody according to WO2010/019570; and comparator 2, an anti-LAG3 human monoclonal antibody with v3.5 antibody VH/VL sequences according to US2014/0093511.

Example 3 Antibody binding to LAG3 determined by surface plasmon resonance.

Association and dissociation rate constants (ka and kd, respectively) upon binding, equilibrium dissociation constants, and dissociation half-lives (K _D and t1/ ₂ , respectively) for antigen binding to purified anti- _LAG3 antibodies were determined using an analysis based on the phenomenon of surface plasmon resonance in the mode real time using a biosensor on a BiaCore 4000 or BiaCore T200 instrument. Either a rabbit anti-mouse polyclonal antibody (GE, no. BR-1008-38) or a mouse monoclonal anti-human Fc antibody (GE, no. BR-1008-39) was immobilized on the BiaCore sensor surface to capture approximately 50-85 FU of monoclonal antibodies. to LAG3, expressed respectively with either mouse Fc or human Fc. LAG3 reagents tested for binding to anti-LAG3 antibodies included recombinant human LAG3 expressed with a C-terminal myc-myc-hexahistidine tag (hLAG3-mmh; SEQ ID: 574) and recombinant human LAG3 expressed with a C-terminal tag , which is a mouse IgG2a mFc (hLAG3-mFc; SEQ ID: 575). Different concentrations (50, 25, 12.5 and 6.25 nM) of LAG3 reagents were sprayed onto the surface with captured anti-LAG3 monoclonal antibody at a flow rate of 50 μl/min on a BiaCore T200. Binding of LAG3 reagents to captured monoclonal antibodies was monitored for 4 min, and their dissociation from antibodies was monitored for 8 min, in HBST running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0 05% v/v surfactant P20). Experiments were carried out at 25 and 37°C. The kinetic rate constants of association (k _a ) and dissociation (k _D ) were determined by processing and fitting the data to a 1:1 binding model using Scrubber 2.0c curve fitting software. Dissociation constants (K _D ) FOR equilibrium binding and dissociation half times (t1/ ₂ ) were then calculated from the kinetic rate constants as: K _D (M) = kd/ka and t1/ ₂ (min.) = [ln2/(60* kd)].

Binding kinetics parameters for the binding of various anti-LAG3 monoclonal antibodies to monomeric (mmH labeled) or dimeric (mFc labeled) LAG3 reagents at 25°C and 37°C are summarized in Tables 4-7.

Table 4. Binding kinetic parameters for anti-LAG3 monoclonal antibodies to bind to

human LAG3-mmh at 25°C

25°C ka kd kd tl/2 ID Ab (l/mol*s) (1/s) [M] (min.) H4N15479R 1.09E+06 5.35Е-05 4.93E-11 215.9 H4sH15479P 1.23E+06 5.98E-05 4.87E-11 193.1 H4sH14813N 4.45E+05 7.89Е-05 1.77E-10 146.4 H4H14813N 4D1E+05 8.80E-05 2D4E-10 131.2 H4H15498P2 3.01E+05 7.81E-05 2.60E-10 147.9 H4H17819P 1DZE+06 4.43Е-04 3.91E-10 26 H4H17823P 1.04E+06 4.47Е-04 4.29E-10 25.8 H4sH15498P2 1.57E+05 1.01E-04 6.45E-10 114 H4H15482P 1.99E+05 1.94Е-04 9.72E-10 59.7 H4sH15482P 2D2E+05 2.60E-04 1.23E-09 44.5 H4H15483P 1.88E+05 3.70E-04 1.97Е-09 31.2 H4H17828P2 6D2E+05 1.41Е-03 2.31Е-09 8.2 H4H15462P 9.39E+05 5.40E-03 5.75Е-09 2.1 H4sH15462P 1.07E+06 6.97Е-03 6.51Е-09 1.7 H4H17826P2 4.50E+05 4.57Е-03 1.02E-08 2.5 Isotype control 1 NB NB NB NB Isotype control 2 NB NB NB NB Comparator 1 1.09E+06 8.70E-04 8.02Е-10 13.3 Comparator 2 1.01E+06 5.82E-04 5.77E-10 19.9

*NB indicates that under the experimental conditions, the LAG3 reagent did not bind to the captured anti-LAG3 monoclonal antibody.

Table 4 shows that at 25° C., all anti-LAG3 antibodies of the present invention bind to hLAG3-mmh with K _D values ranging from 49 pM to 10 nM, while comparators bind with K _D values of 0.58 nM and 0.8 nM.

- 42 039718

Table 5. Binding kinetic parameters for binding of anti-LAG3 monoclonal antibodies to _________________hLAG3-mmh at 37°C_________________

37°С to _a ka Kd* tl/2 IDAb (l/mol*s) (1/s) [M] (min.) H4N15479R 1.27E+06 4.08E-05 3.21E-11 283 H4sH15479P 1.24E+06 4.24Е-05 3.41E-11 272.5 Н4Н15498Р2 3.81E+05 2.96E-05 7.83E-11 390.2 H4sH15498P2 3.78E+05 7D8E-05 1.85E-10 163.1 H4sH14813N 5.78E+05 1.54Е-04 2.66E-10 75.1 H4H14813N 5D5E+05 1.52E-04 2.84Е-10 76 H4N17823R 1.76E+06 9.24Е-04 5.24Е-10 12.5 H4N17819R 1.59E+06 8.76Е-04 5.52E-10 13.2 Н4Н15483Р 7.74E+05 1D1E-OZ 1.43Е-09 10.4 H4sH15482P 2.81E+05 5D2E-04 1.82E-09 22.6 H4H17828P2 1D0E+06 2D7E-03 1.89Е-09 5.6 H4H15482P 2.92E+05 5.70E-04 1.96E-09 20.2 H4H17826P2 1.07E+06 2D4E-03 1.99Е-09 5.4 H4H15462P 1.08E+06 6.26E-03 5.80E-09 1.8 H4sH15462P 1.01E+06 7.72E-03 7.66Е-09 1.5 Isotype control 1 NB NB NB NB Isotype control 2 NB NB NB NB A drug 1.23E+06 3.74Е-03 3,OSE-09 3.1 comparisons 1 Comparator 2 1D0E+06 2.31Е-03 2D0E-09 5

*NB indicates that under the experimental conditions, the LAG3 reagent did not bind to the captured anti-LAG3 monoclonal antibody.

Table 5 shows that at 37° C., all anti-LAG3 antibodies of the present invention bind to hLAG3-mmh with KD values ranging from 32 pM to 7.66 nM, while comparators bind with KD values of 2.1 nM and 3.0 nM.

The data in Tables 4 and 5 indicate that all anti-LAG3 antibodies bind to monomeric hLAG3mmh with very similar affinities at both 25° and 37°C.

- 43 039718

Table 6. Binding kinetic parameters for binding of anti-LAG3 monoclonal antibodies to ______________hLAG3 dimer (hLAG3-mFc) at 25°C______________

25°С ka ka kd tl/2 ID Ab (l/mol*s) (1/s) [M] (min.) H4N15479R 2.30E+06 1.00Е-05 4.35E-12 >1155.0 H4sH15479P 3.05E+06 1.00Е-05 3.28E-12 >1155.0 Н4Н15483Р 1.34E+06 1.00Е-05 7.45E-12 >1155.0 H4H14813N 9.58E+05 1.00Е-05 1.04Е-11 >1155.0 H4sH14813N 1.80E+06 1.90E-05 1.06Е-11 606.6 H4H15482P 7.58E+05 1.00Е-05 1.32E-11 >1155.0 H4sH15482P 7.09E+05 1.00Е-05 1.41Е-11 >1155.0 H4H15498P2 3.63E+05 1.00Е-05 2.76E-11 >1155.0 H4sH15498P2 2.84E+05 1.00Е-05 3.52E-11 >1155.0 H4H17819P 1.45E+06 1.80E-04 1.24Е-10 64.2 H4H17823P 1.26E+06 2.18E-04 1.74Е-10 52.9 H4H17828P2 1.88E+06 4.60E-04 2.45Е-10 25.1 H4sH15462P 2.34E+06 1.90E-03 8D4E-10 6.1 H4H17826P2 6.91E+05 5.64Е-04 8D7E-10 20.5 H4H15462P 2.02E+06 2, OZE-OZ 1.01Е-09 5.7 Isotype control 1 NB* NB NB NB isotypic NB NB NB NB control 2 Comparator 1 2.69E+06 4.50E-04 1.67E-10 25.7 Comparator 2 3.09E+06 3.00E-04 9.68E-11 38.6

*NB indicates that under the experimental conditions, the LAG3 reagent did not bind to the captured anti-LAG3 monoclonal antibody.

Table 6 shows that at 25° C., all anti-LAG3 antibodies of the present invention bind to hLAG3 dimeric proteins with KD values ranging from 4.3 pM to 1.0 nM, while comparators bind to KD values of 97 pM and 0.16 nM.

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Table 7. Binding Kinetic Parameters for Anti-LAG3 Monoclonal Antibodies Binding to hLAG3 Dimer (hLAG3-mFc) at 37°C

37°C ka kd kd tl/2 ID Ab (l/mol*s) (1/s) [M] (min.) H4N15479R 2.50E+06 1.00Е-05 3.99E-12 >1155.0 H4sH15479P 2.92E+06 1.00Е-05 3.42E-12 >1155.0 H4sH15498P2 5.15E+05 1.00Е-05 1.94Е-11 >1155.0 H4H15498P2 4.60E+05 1.00Е-05 2D7E-11 >1155.0 H4H14813N 4.27E+06 1D7E-04 2.74Е-11 98.8 H4sH14813N 3.42E+06 1.01E-04 2.95E-11 114.4 H4sH15482P 5D9E+06 1.64Е-04 3.16E-11 70.3 H4H15482P 1.01E+06 9.01Е-05 8.91Е-11 128.1 H4H15483P 1.38E+06 2.98E-04 2.15E-10 38.8 H4H17826P2 1.98E+06 5.47Е-04 2.77E-10 21.1 H4H17819P 1.66E+06 5.27Е-04 3.17E-10 21.9 H4H17828P2 1.73E+06 7D8E-04 4.15E-10 16.1 H4H15462P 2.16E+06 8.95Е-04 4.15E-10 12.9 H4H17823P 1.34E+06 5.85Е-04 4.37E-10 19.7 H4sH15462P 1.80E+06 1.57Е-03 8.76E-10 7.3 Isotype control 1 NB NB NB NB isotypic NB NB NB NB control 2 Comparator 1 3.44E+06 1.56Е-03 4.54Е-10 7.4 Comparator 2 4.36E+06 1.1 SE-OZ 2.58Е-10 10.3

*NB indicates that under the experimental conditions, the LAG3 reagent did not bind to the captured anti-LAG3 monoclonal antibody.

Table 7 shows that at 37° C., all anti-LAG3 antibodies of the present invention bind to hLAG3 dimeric proteins with KD values ranging from 4.0 pM to 0.9 nM, while comparators bind with KD values of 0.26 nM and 0.45 nM.

The data in Tables 6 and 7 indicate that all anti-LAG3 antibodies bind to hLAG3 dimers as reagents with very similar affinities at both 25° and 37°C.

Example 4 Anti-LAG3 Antibody Cross-Competition in the Octet System.

To assess whether two antibodies compete with each other for binding to monomeric human LAG3 (hLAG3.mmh), competition for binding between anti-LAG3 monoclonal antibodies was determined using real-time biolayer interferometric analysis without labeling on an Octet RED384 biosensor (Pall ForteBio Corp.). A cross-competition experiment was performed at 25° C. in 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.05% v/v. Tween-20 surfactant, 0.1 mg/ml BSA (HBS-P Buffer for Octet) with shaking plates at 1000 rpm. All anti-LAG3 and hLAG3 antibody solutions tested were formulated in Octet HBS-P buffer. To assess whether the 2 antibodies were able to compete with each other for binding to hLAG3, first approximately ~0.6-0.85 nM hLAG3.mmh was captured on His antibody-coated tips of Octet biosensors from wells containing 5 μg/ml hLAG3, within 75 s. Octet biosensor tips with captured hLAG3s were saturated by dipping for 5 min in wells containing 50 μg/mL of the first anti-LAG3 monoclonal antibody (referred to herein as mAb-1), followed by dipping in wells containing a second anti-LAG3 monoclonal antibody (referred to as mAb-2 herein). Between each step, Octet biosensor tips were washed in HBS-P buffer for 30 seconds.

The real-time binding response was monitored throughout the experiment and at the end of each stage, the binding response was recorded. Linking response

- 45 039718 hLAG3 with mAb-1 and then blocking mAb, corrected for background binding, compared and determined competitive/non-competitive binding pattern of different monoclonal antibodies to Lag.

Table 8 explicitly defines the relationship of antibodies competing in both directions, regardless of the order of binding.

Table 8. Cross-competition of anti-LAG3 antibodies for binding to monomeric hLAG3

First mAb (mAb-Ι) captured using ANS Octet biosensors mAb-2 antibodies shown to compete with mAb-Ι* H4sH15482P H4sH15479P, H4sH14813N, H4H14813N, H4N15479R, N4N15482R, N4N15483R H4sH15479P H4sH15482P, H4sH14813N, H4H14813N, H4N15479R, N4N15482R, N4N15483R H4sH14813N H4sH15482P, H4sH15479P, H4H14813N, H4H15479P, H4H15482P, H4H15483P H4H14813N H4sH15482P, H4sH15479P, H4sH14813N, H4H15479P, H4H15482P, H4H15483P H4H15479P H4sH15482P, H4sH15479P, H4sH14813N, H4H14813N, H4H15482P, H4H15483P H4H15482P H4sH15482P, H4sH15479P, H4sH14813N, H4H14813N, H4H15479P, H4H15483P H4H15483P H4sH15482P, H4sH15479P, H4sH14813N, H4H14813N, H4H15479P, H4H15482P H4sH15498P2 H4H15498P2, H4H17823P H4H17828P2, H4H17819P, H4H15498P2 H4sHl 5498P2, H4H17823P H4H17828P2, H4H17819P, H4H17828P2 H4sH15498P2, H4H17823P H4H15498P2, H4H17819P, H4H17819P H4sH15498P2, H4H17823P H4H15498P2, H4H17828P2, H4H17823P H4sH15498P2, H4H15498P2, H4H17828P2,

H4H17819P H4sH15462P Missing H4H15462P Missing H4H17826P2 Missing

(*Self-competing mAb2 not shown).

Example 5 Antibody binding to LAG3 overexpressing cells determined by flow cytometry.

Stable cell lines HEK293 (HEK293; ATCC, #CRL-1573) were generated that express recombinant hLAG3 (NCBI accession number NP_002277.4) or monkey LAG3 (mfLAG3) [the cynomolgus monkey sequence with NCBI accession number XP_005570011.1 is further modified with the replacement of X at amino acid position 74 with proline according to the sequence of the rhesus monkey (Macaca mulata) with accession number in NCBI XP_001108923.1] (SEQ ID NO: 576) on the cell surface, and they were used to determine the binding specificity of monoclonal antibodies to LAG3 of the present invention by analysis using flow cytometry.

Anti-LAG3 antibody binding was assessed as follows: Stable HEK293 cells expressing either hLAG3 or mfLAG3 were washed with 1x D-PBS (Irvine Scientific, cat. no. 9240), trypsinized (Specialty Media, cat. no. SM-2004-C), and blocked. using HEK293 cell culture medium (DME + 10% FBS + P/S/G + non-essential amino acids). After prices

The cells were resuspended at a concentration of 2.0 x 10 ⁶ cells/ml in staining buffer (D-PBS + 2% FBS). Anti-LAG3 antibodies and isotype controls were serially diluted in staining buffer at a dose ranging from 5 pM to 100 nM, including one buffer without antibody as a control. Serially diluted antibodies were added to the cell suspension and incubated for 15-30 min on ice. Cells were centrifuged and the pellets were washed with staining buffer to remove unbound antibodies. Cells were then incubated for 15-30 min on ice with secondary allophycocyanin-conjugated F(ab')2 human Fc recognition (Jackson ImmunoResearch, no. 109-136-170). Cells were centrifuged and the pellets were washed with staining buffer to remove unbound secondary F(ab')2 and fixed overnight using Cytofix (BD Biosciences, no. 554655) at a 1:1 dilution and staining buffer. The next day, the fixed cells were centrifuged and the pellets were washed with staining buffer, resuspended and filtered. Fluorescence measurements were obtained on a HyperCyt® cytometer and analyzed in ForeCyt™ (IntelliCyt; Albuquerque, NM) to determine mean fluorescence intensity (MFI). EC ₅₀ values were calculated from a four parameter logistic model equation from an 11 point response curve using GraphPad Prism. EC ₅₀ is defined as the concentration of antibody at which 50% of the maximum binding signal is detected. The MFI ratio for each antibody was calculated by dividing the MFI at 100 nM by the MFI at 0 nM antibody concentration.

Table 9 Anti-LAG3 antibody binding to wild-type (wt) HEK293 cells determined by FACS

FACS for HEK293 wt PID# AB EU50 [M] MFI ratio (100 nM/O nM) Isotype control h!gG4 - 1.5 H4sH15462P - 0.9 H4N15462R - 8.2 H4sH15479P - 1.1 H4N15479R - 1.7 H4sH15482 - 1.2 H4H15482P - 4.9 H4sH15498P2 - one H4H15498P2 - one H4sH14813N - 1.5 H4H14813N - 1.3 H4H17828P2 - 1.1 H4H17826P2 - 2.5 H4H17823P - 2.5 H4H15483P - 2.6 H4H17819P - 1.1 Comparator 1 - one Comparator 2 - 1.6

As shown in Table 9, no measurable binding to the original wild-type HEK293 cells was demonstrated in the FACS analysis for comparators and isotopic controls. The calculated MFI ratios ranged from 1.0 to 1.6x. Several anti-LAG3 antibodies showed higher ratios in wild-type cells, indicating non-specific binding, with calculated MFI values ranging from 0.9 to 8.2x. Under these staining conditions, EC ₅₀ values for anti-LAG3 antibodies, as well as comparators, were not determined.

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Table 10 Anti-LAG3 Antibody Binding to HEK293/hLag3 Cells Determined by FACS

FACS for HEK293/hLAG3 PID# AB EU50 [M] MFI ratio (100 nM/O nM) Isotype control h!gG4 - 1.8 H4sH15462P 6.65Ε-10 181.6 H4N15462R ZE-10 62.6 H4sH15479P 9.03Ε-10 198.7 H4N15479R 7.52Ε-10 203.2 H4sH15482 Ι,ΙΙΕ-09 140.8 H4H15482P 1.91Ε-09 103.7 H4sH15498P2 7.92Ε-09 400.6 H4H15498P2 5.76Ε-09 305 H4sH14813N 1D8E-09 197.2 H4H14813N 1.44Ε-09 208.5 H4H17828P2 2.65Ε-09 355.2 H4H17826P2 3.86Ε-09 414.2 H4H17823P 1.25Ε-09 304.9 H4H15483P 2.31Ε-09 206.3 H4H17819P 2.79Ε-09 435.8 Comparator 1 7.52Ε-10 188.2 Comparator 2 6.5Ε-10 150.9

As shown in Table. 10, no measurable binding to HEK293/hLag3 cells was demonstrated for the isotype controls. All 15 anti-LAG3 antibodies of the present invention showed significant binding to HEK293/hLag3 cells with EC ₅₀ values ranging from 300 pM to 7.9 nM. The calculated MFI ratios were in the range of 62-414x. In the binding assay, the EC ₅₀ values for the comparators were 0.65 nM and 0.75 nM, and the MFI ratios were less than 200x for both antibodies.

Table 11 Anti-LAG3 Antibody Binding to HEK293/mfLag3 Cells Determined by FACS

FACS for HEK293/mfLAG3 PID# Ab EU50 [M] MFI ratio (100 nM/O nM) Isotype control hIgG4 - 1.3 H4sH15462P - one H4H15462P - 2.9 H4sH15479P 9.02Ε-10 20.7 H4H15479P 1.35Ε-09 21.5 H4sH15482 1D5E-09 ten H4H15482P 2.2Ε-09 11.9 H4sH15498P2 >10 nM 12.1 H4H15498P2 >10 ηΜ 12.8 H4sH14813N 1.57Ε-09 16.7 H4H14813N 1.42Ε-09 17.1 H4H17826P2 >10 ηΜ 14.3 H4H17823P >10 ηΜ 12.4 H4H15483P >10 ηΜ 8.8 H4H17819P >10 ηΜ 12.1 Comparator 1 >10 ηΜ 6.7 Comparator 2 >10 ηΜ 8.6

As shown in Table. 11, no measurable binding to HEK293/mfLag3 cells was demonstrated for the isotype controls. A total of 13 out of 15 anti-LAG3 antibodies of the present invention showed significant binding to HEK293/mfLAG3 cells with EC50 values ranging from 900 pM to greater than 10 nM. The calculated MFI ratios ranged from 8.8x to 21.5x. Comparator EC50 values were greater than 10 nM and calculated MFI ratios were 6.7x and 8.6x.

Example 6 Antibody Binding to LAG3 Overexpressing Cells Determined by Electrochemiluminescent Immunoassay.

To examine the ability of a panel of anti-LAG3 monoclonal antibodies to bind cell surface expressed LAG3, an in vitro binding assay using human and simian LAG3 expressing cell lines was developed using an electrochemiluminescence detection platform [Meso Scale Diagnostics, Rockville, MD - ( MSD)].

Stable HEK293 cell lines (ATCC, #CRL-1573) were generated by lentiviral transduction to express recombinant hLAG3 (NCBI accession number NP_002277.4) or monkey LAG3 (mfLAG3) [cynomolgus monkey (Macaca fascicularis) sequence accession number XP_005570011. 1 modified to change the X at amino acid position 74 to proline according to the sequence of the rhesus monkey (Macaca mulata) NCBI accession number XP_001108923.1] (SEQ ID NO: 576). The parent HEK293 cell line, which did not show detectable LAG3 expression in fluorescence-activated cell sorting (FACS), was included in the experiment as a background binding control. Unrelated human IgG4 and hIgG4 were included as isotopic control antibodies.

The experiments were carried out according to the following procedure. Cells from the three cell lines described above were washed once in 1x Ca ²⁺ /Mg ²⁺ free PBS buffer followed by a 10 min incubation at 37°C with enzyme-free cell dissociation solution. The detached cells were washed once with 1xPBS with Ca ²⁺ /Mg ²⁺ and counted using a Cellometer™ Auto T4 cell counter (Nexcelom Bioscience). Approximately 10,000 cells per well in cell wash buffer were plated in 96-well carbon electrode plates (MULTI-ARRAY High Binding Plate, MSD) and incubated for 1 hour at 37° C. to allow cell attachment. Non-specific binding sites were blocked with 2% BSA (w/v) in PBS for 1 hour at room temperature. Solutions of antibodies to LAG3 in serial dilutions in the range from 1.7 pM to 100 nM and solutions without antibody were added in duplicate to the cells attached in the plate, and the plates were incubated for 1 h at room temperature. The plates were then washed to remove unbound antibodies using the AquaMax2000 Immunoplate Washer (MDS Analytical Technologies). Plate-bound antibodies were detected with SULFO-TAG™-conjugated anti-human IgG (MSD) for 1 hour at room temperature. After washings, the contents of the plates were developed with Reading Buffer (MSD) according to the manufacturer's recommended procedure, and luminescence signals were recorded using a SECTOR Imager 6000 (MSD). Direct binding signals (in relative light units - RLU) were analyzed depending on the concentration of antibodies, and the data were fitted using a sigmoidal (four-parameter logistic) dose-response model using GraphPad Prism™ software. The effectiveness of each antibody was determined by calculating the EU ₅₀ . The EC ₅₀ for binding of HEK293-hLAG3 and HEK293-mfLAG3 cells is defined as the antibody concentration at which 50% of the maximum binding signal is detected. The ratio of the signal detected when 100 nM antibody binds to HEK293-hLAG3 or HEK293-mfLAG3 cells compared to the original HEK293 cells was recorded as an indication of the specificity of LAG3 binding. For ratios below 2-fold, at the highest antibody concentration (100 nM), the antibody was concluded to be non-specific (NS). The binding results are shown in Table 12.

Table 12 Antibody binding to hLAG3 or mfLAG3 overexpressing cells

ID Ab Cell binding efficiency for HEK293hLAG3 EC50 cells (M) Cell binding efficiency for HEK293mfLAG3 EC50 cells (M) Binding ratio of 100 nM antibodies to HEK293-hLAG3 cells and HEK293 cells Binding ratio of 100 nM antibodies to HEK293-mfLAG3 cells and HEK293 cells H4sH15462P 8.53E-10 NS 6.8 NS H4sH15482P 1.1 SE-08 NS 4.5 NS H4sH15479P 1.34Е-08 NS 17.8 NS H4sH15498P2 2.87Е-09 2.00Е-08 99.8 32.7 H4sH14813N 2.03Е-08 NS 19.8 NS

- 49 039718

H4H14813N 1.99E-08 NS 8.9 NS H4N15462R 6.85Е-09 NS 2.3 NS H4N15482R 2.47Е-08 NS 7.4 NS H4N15479R 5.20E-08 7.39Е-08 20.1 2.1 Н4Н15498Р2 2.95Е-09 9.68Е-09 38.8 13.8 H4N17828R2 1.2E-09 1.31Е-08 8.7 6 H4H17826R2 3.48E-09 7.85Е-09 7 4.7 H4N17823R 1.5E-09 2.78Е-08 5.3 2.8 Н4Н15483Р 1.72E-08 NS 6.6 NS H4N17819R 6.9E-10 4.02E-08 22.7 8.3 isotypic H4H control NS NS NS NS isotypic H4SH control NS NS NS NS Comparator 1 4.03Е-09 NS 7.7 NS Comparator 2 6.77Е-09 NS 8.1 NS

NS = non-specific binding - ratio at 100 nM ab <2.

As shown in Table 12, antibody potency ranged from 690 pM to 52 nM EC50 for binding to HEK293- _hLAG3 cells, with all antibodies specific binding >2-fold higher binding to human LAG3-expressing cells compared to original HEK293 cells when binding 100 nm antibodies. Only seven of the antibodies specifically bind to HEK293-mfLAG3 cells with EC ₅₀ values ranging from 7.9 nM to 74 nM. Comparator binding efficiencies for HEK293hLag3 cells were approximately 4.0 nM and 7.0 nM. No specific binding to HEK293-mfLag3 cells was shown for comparators. Binding of irrelevant controls to HEK293hLAG3 and HEK293-mfLAG3 cells was, as expected, non-specific, with binding of H4H isotype control and H4sH isotype control with ratios ranging from 0.7 to 1.1 fold greater than binding to parental HEK293 cells.

Example 7 Blocking LAG3 Binding to MHC Class II in a Cell Adhesion Assay.

A cell adhesion assay was used to measure the ability of anti-LAG3 monoclonal antibodies to block adhesion of cells expressing either human MHCII or mouse MHCII (RAJI and A20 cells, respectively) to hLAG3-expressing HEK293 cells attached to a microtiter plate (based on the assay described in Eur J Immunol 1995, 25: 2718-21 by Huard, et al.).

Stable HEK293 cell lines (ATCC, #CRL-1573) were generated by lentiviral transduction to express recombinant hLAG3 (NCBI accession number NP_002277.4) or mouse LAG3 (mLAG3) (GenBank accession number NP_032505.1). For the original HEK293 cell line, no detectable expression of human LAG3 was observed using fluorescence-activated cell sorting (FACS) technology, and was used to demonstrate the need for LAG3 for adhesion of MHCII-expressing RAJI and A20 cells.

The experiments were carried out according to the following procedure. Subconfluent hLAG3 expressing HEK293 cells were washed once with 1X PBS, trypsinized and centrifuged at 1200 rpm. within 5 min. Cell pellets were resuspended in 1X PBS and then counted to determine the number of cells. An appropriate number of cells were selected and resuspended in complete medium (DME + 10% FBS + penicillin/streptomycin/glutamine) so that each well contained 1.2 ^L 4 cells per 100 μl. Cells were seeded in sterile black Nunclon™ Delta 96-Well MicroWell™ (Thermo Scientific) clear bottom well plates. Wells around the perimeter of the plate were not included in the analysis to avoid edge effects. 100 μl of 1xPBS was added to the wells around the perimeter instead of cells. The plated HEK293-hLAG3 cells were incubated overnight at 37°C, 5% CO2. After incubation, these cells were treated with anti-LAG3 antibodies and isotype controls.

On the day of treatment, all antibodies were serially (1:3) diluted in RPMI medium (no FBS or additives added). Concentrations at the 9-point dilution ranged from 45 pM to 300 nM, with the last dilution point containing no mAb. For testing H4sH antibodies in an adhesion assay using RAJI cells, concentrations at 9-point dilution ranged from 23 pM to 150 nM, with the last dilution point containing no mAb. Anti-LAG3 antibodies or isotype controls were added to HEK293-hLAG3 cells in a final volume of 50 μl and incubated for 1 h at 37°C, 5% CO2. While plated HEK293-LAG3 cells were incubated with test or control antibodies, non-adherent B cells (RAJI or A20) were labeled with calcs.

- 50 039718 nom-AM using the following procedure. A suspension of RAJI and A20 cells was collected and centrifuged at 1200 rpm. Within 5 min. Cell pellets were resuspended in 1X PBS and counted. Cells were washed with RPMI and resuspended in RPMI at a concentration of 10 ⁶ cells/ml. Cells were labeled by adding 5 µl of calcein-AM per 1 ml of cell suspension and incubated for 30 min. at 37°C, at 5% CO ² . The labeled cells were washed 1x with RPMI, centrifuged, washed with 1x PBS and resuspended in PBS at a concentration of 5x10 ⁶ cells/ml. Then, 10 µl of BD Pharmingen Fc Block Fc blocking antibody (0.5 mg/ml) was added per 1 ml of calcein-AM labeled cells, which were incubated at RT for 10 min. Calcein-AM-stained, Fc-blocked RAJI or A20 cells were resuspended in RPMI to a final concentration of 3×10 ⁶ cells per ml and then used in adhesion assays.

After treatment of HEK293-LAG3 cells with anti-LAG3 antibody for one hour, 50 μl of labeled RAJI or A20 cells were added to each well at a density of 1.5 x 10 ⁵ cells/well. The labeled cell suspension was incubated with a HEK293 monolayer for 1 h at 37°C, 5% CO ₂ . Non-adherent cells were washed off by gently washing the wells with 4x RPMI, blotting the plates after each wash with paper towels, followed by 2x PBS washes. PBS was added to each well in a final volume of 200 μl and calcein-AM fluorescence was read at an excitation/emission wavelength of 485 nm/535 nm on a VICTOR™ X5 multifunction plate reader. IC ₅₀ values were calculated from a 4-parameter logistic model equation from a 9-point response curve using GraphPad Prism. The IC ₅₀ was defined as the antibody concentration at which 50% blocking of HEK cell adhesion to RAJI or A20 cells was observed.

The ability of anti-LAG3 antibodies to block LAG3 binding to both human and mouse MHCII was assessed using cell adhesion assay. The assay used a format linking fluorescently labeled cells endogenously expressing MHCII [human MHCII (RAJI) or mouse MHCII (A20)] to cells engineered to express human LAG3. The ability of anti-LAG3 antibodies to block this interaction was evaluated and the results are shown in Table 13.

Table 13 Anti-LAG3 IC ₅₀ Values for Blocking RAJI and A20 Cell Adhesion to HEK293-hLAG3 Cells

Adhesion analysis ID# Ab RAJI A20 IC50 [M] IC50 [M] H4sH15462P 2.50E-09 3.60E-09 H4N15462R 3.20E-09 7.10E-09 H4sH15479P 9.60E-09 6, ZOE-09 H4H15479P 3.10E-08 1.10E-08 H4sH15482 3, ZOE-09 3.80E-09 H4H15482P 1.50E-08 1.10E-08 H4sH15498P2 1.60E-08 2.10E-08 H4H15498P2 1.50E-08 3.00E-08 H4sH14813N 6.00E-09 1.00Е-08 H4H14813N 1.70E-08 1.00Е-08 H4H17828P2 1.10E-08 1.60E-08 H4H17826P2 2.70E-08 1.00Е-08 H4H17823P 8.60E-09 9.40E-09 H4H15483P 1.90E-08 1.60E-08 H4H17819P 1.70E-08 1.60E-08 isotypic control - - Comparator 1 3.40E-09 5.10E-09 Comparator 2 7.20E-09 9.60E-09

As shown in Table 13, no measurable blocking of the binding of RAJI or A20 cells to HEK293/hLAG3 cells was demonstrated for the isotype control. All anti-LAG3 antibodies of the present invention blocked 92-98% adhesion of RAJI or A20 cells to HEK293-hLAG3 cells at maximum antibody concentration, with IC ₅₀ values ranging from 2.5 nM to

- 51039718 nM for RAJI cell adhesion and 3.6 nM to 30 nM for A20 cell adhesion to HEK293-hLAG3 cells.

The IC ₅₀ values for the reference drugs were 3.4 nM and 7.2 nM for RAJI cell adhesion and 5.1 nM and

9.6 nM for adhesion of A20 cells to HEK293-Lag3 cells.

Example 8 Blocking of LAG3-induced T cell downregulation in the luciferase reporter gene assay using T cells/APC.

T cell activation is mediated by stimulation of T cell receptors (TCRs) that recognize specific peptides presented by major histocompatibility complex class I or II (MHCI or MHCII) proteins on antigen presenting cells (APCs) (Goldrath et al. 1999, Nature 402 , 255-262). Activated TCRs in turn trigger events in a signaling cascade that can be traced to reporter genes under the control of transcription factors such as activator protein 1 (AP-1), activated T cell nuclear factor (NFAT), or light chain gene enhancer nuclear factor. kappa activated B cells (NFkB). The T cell response is optimized through the involvement of co-receptors expressed on T cells either constitutively or inducibly. One such receptor is LAG3, a negative regulator of T-cell activity. LAG3 interacts with its ligand, MHCII, which is expressed on target cells, including APC or cancer cells, and inhibits T cell activation by suppressing positive signals initiated by the TCR signalosome (Workman et al. 2002, J Immunol 169:5392-5395) .

To characterize the ability of anti-LAG3 antibodies to interfere with LAG3-mediated signaling in T cells, a reporter assay using LAG3-expressing T cells/APC was developed to investigate T cell signaling induced by APC-T cell interaction using a mixed culture, derived from two mammalian cell lines: a Jurkat T cell (clone JRT3.T3.5, as described below) and an engineered APC surrounded by HEK293 (as described below) (FIG. 1).

As regards the first component, a T cell line for reporter bioassay using LAG3 expressing T cells/APC was established as follows: A Jurkat-derived T cell clone, JRT3.T3.5 (ATCC no. transduced using a lentiviral construct with a Cignal AP-1-Luc reporter system (Cignal Lenti AP-1 Luc reporter) (Qiagen Sabiosciences, #CLS-011L) according to the manufacturer's instructions. Lentivirus encodes the firefly luciferase gene under the control of the CMV minimal promoter, tandem repeats of the TPA-inducible transcriptional response element (TRE), and the puromycin resistance gene. After antibiotic selection, puromycin-resistant reporter cell pools were additionally transduced with constructs encoding human CD28 (NP_006130.1), alpha and beta subunits of TCR clone Ob2F3 (alpha AGA92550.1, beta AAA61026.1), human LAG3 chimera (containing extracellular domain Human LAG3 (NP_002277.4) and transmembrane/cytoplasmic domain of human CD300a (amino acids 181-299; accession number NP 009192.2) and human PD-1 (accession number NP_005009.2) Protein expression was confirmed by FACS analysis after each round of transduction A single clone JRT3.T3/APl-Luc/CD28/hLAG3-CD300a/hPD-1 chimera, clone 2D2 was then generated and used in a T cell/APC biological assay The cell line was maintained in RPMI + 10% FBS + penicillin/streptomycin/glutamine (P/S/G) supplemented with 100 μg/ml hygromycin + 500 μg/ml G418 + 1 μg/ml puromycin, referred to herein as selection medium These cells are referred to herein as reporters mi T-cells.

As for the second component, an APC cell line for reporter bioassay using LAG3 expressing T cells/APC was prepared as follows: A stable cell line HEK293 (ATCC No. CRL-1573) expressing human CD20 (number NP_068769.2) was transduced constructs encoding human MHCII subunits, HLADR2A (NP_061984.2) and HLA-DR2B (NP_002115). HLA-DR2 positive cells were isolated by FACS and the resulting cell line, HEK293/CD20/HLA-DR2, was maintained in DME + 10% + P/S/G + non-essential amino acids supplemented with 100 μg/ml hygromycin + 500 μg/ ml of G418, referred to herein as selection medium. These cells are referred to herein as APC cells.

Two approaches have been used to enable interaction between the T cell and APC to initiate TCR-mediated signaling: (1) a bispecific antibody approach; and (2) peptide primed method. In this biological assay, anti-LAG3 antibodies blocked LAG3/MHCII interaction and reactivated T cells by disrupting inhibitory signaling via the cytoplasmic tail of the CD300a molecule, followed by an increase in AP1-Luc signaling. T cell activation was monitored by reading luciferase.

A method based on a bispecific antibody. This method used a bispecific antibody consisting of one Fab arm that binds to CD3 on T cells and a second Fab that binds to CD20 on HEK293 cells (anti-CD3xCD20 bispecific antibody; e.g. disclosed in US20140088295). The presence of the bispecific molecule leads to the formation of an immune synapse and activation of the TCR complex by clustering CD3 molecules on engineered T cells.

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The day before screening, the engineered reporter T cells were cultured in selection medium until a concentration of 5x105 cells/ml was reached and tested in a biological assay the next day. EC ₅₀ values of anti-LAG3 antibodies were determined in the presence of a fixed concentration of an anti-CD3xCD20 bispecific antibody (100 pM). The reagents were added in the following order. Anti-Lag 3 antibodies and isotype controls were serially diluted in RPMI1640 supplemented with 10% FBS and penicillin/streptomycin/glutamine (assay medium). Concentrations at the 10-point dilution ranged from 15 pM to 100 nM, with the end point of the dilution containing no mAb (one buffer as control). Serially diluted antibodies were added to the appropriate wells of 96-well white flat-bottomed plates containing a fixed concentration (100 pM) of the bispecific antibody. Overnight cultured reporter T cells were resuspended at 2×10 ⁶ /ml in assay medium, added to anti-LAG3 and bispecific antibody plates, and incubated for 30 min at 37°C/5% CO ₂ . The final concentration of reporter T cells was 5x10 ^L 4 per well.

APC cells were then washed with 1x D-PBS (Irvine Scientific, cat. no. 9240), trypsinized (Specialty Media, cat. no. SM-2004-C) and blocked with assay medium. After centrifugation, cells were resuspended to a concentration of 4x10 ⁵ /ml in assay medium and added to plates containing reporter T cells incubated with anti-LAG3 and bispecific antibody. The final concentration of APC cells was 1x104 per well. The plates were incubated for 4-6 h at 37°C, 5% CO ₂ . After addition of the ONEGlo™ reagent (Promega, #E6051), API-Luc activity was detected and data collected in relative light units (RLU) on a Victor luminometer. All samples were tested in duplicate.

Peptide priming method using MBP85-99. TCRs recognize a specific MHC/peptide complex and mediate T cell activation. The Ob2F3 clone TCR heterodimer used in this assay was reported to interact with the MHCII protein, HLA-DR2AB, in complex with the MBP85-99 peptide (myelin basic protein, NP_001020272; SEQ ID NO: 583).

The day before screening, engineered reporter T cells were obtained as described above for the bispecific antibody method. APC cells were primed overnight with 0.2 μM MBP85-99 peptide (Celtek HLA-DR2 MBP85-99 in DMSO, ER-15, lot #140411, molecular weight 1797.1 g/mol) in an appropriate cell culture medium containing antibiotic, at 37°C/5% CO ₂ . EC ₅₀ values of anti-LAG3 antibodies were determined in the presence of 1x104 primed APCs. Reagents were added in the following order: Anti-LAG3 antibodies and controls were serially diluted in assay medium. Anti-LAG3 antibody concentrations at 10-point serial dilution ranged from 15 pM to 100 nM, with the last dilution point containing no mAb. Antibodies were added to the appropriate wells of a 96-well white flat-bottomed well plate. Overnight cultured reporter T cells were resuspended to a concentration of 2×10 ⁶ /ml in assay medium, added to plates and incubated for 30 min at 37° C. at 5% CO ₂ . The final concentration of reporter T cells was 5x104 T cells per well. MBP85-99 primed APC cells were then washed with 1x D-PBS (Irvine Scientific, catalog number 9240), trypsinized (Specialty Media, catalog number SM-2004-C) and blocked with assay medium. After centrifugation, the cells were resuspended to reach a concentration of 4x10 ⁵ /ml in the assay medium and added to the plates with anti-LAG3 antibodies and peptide-primed reporter T cells. The final concentration of APC cells was 1x104 per well. Assay plates were incubated for 4-6 hours at 37°/5% CO2. After addition of ONE-Glo™ reagent (Promega, #E6051), API-Luc activity was detected and data collected in relative light units (RLU) on a Victor luminometer. All samples were tested in duplicate.

EC50 values were determined from a 4-parameter logistic model equation from a 10-point response curve using GraphPad Prism. The RLU values for each screened antibody were normalized using the mAb-free last serial dilution as a control of 100%, which theoretically should represent the maximum API-Luc response due to either the bispecific molecule (in the bispecific antibody method) or primed MBP85 peptide -99 ARS. The results are shown in Table 14.

Table 14. Anti-LAG3 antibody-dependent activation of API-Luc signaling in a biological assay using LAG3-expressing T cells/APC.

ECso[Μ] ID# Ab Peptide Priming Method Bispecific antibody method Isotype h!gG4 - -

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H4sH15462P 4.86E-10 1.48Е-10 H4N15462R 3.44Е-10 2.24Е-10 H4sH15479P 1.35E-10 8.72E-11 H4N15479R 2.84Е-10 1.45E-10 H4sH15482P 8.06Е-10 5.18E-10 H4N15482R 2.48Е-09 >100 nM H4sH15498P2 4.61E-09 >100 nM Н4Н15498Р2 5.89Е-09 6.62E-09 H4sH14813N 8D6E-10 6.88E-10 H4H14813N 1.01Е-09 1.07Е-09 Н4Н15483Р 2.54Е-09 3.69Е-09 H4N17819R 2D2E-09 >100 nM H4N17823R 6.88E-10 >100 nM H4H17826R2 8.83Е-09 2.66Е-07 H4N17828R2 4.91Е-09 >100 nM Comparator 1 2.79Е-10 1.57E-10 Comparator 2 3.49Е-10 2.38E-10

As shown in Table 14, all anti-Lag3 mAbs of the present invention that were tested in the T cell/APC bioassay provided relief from LAG3/HLA-DR2 mediated inhibition with EC50 values ranging from 135 pM to 8.83 nM in the peptide primed assay, and with EC50s ranging from 87 pM to over 100 nM in the bispecific antibody assay, while the comparators had EC ₅₀ values of 280 pM and 350 pM. The isotype controls tested did not show a significant reactivation in the biological assay. In an additional experiment, the addition of an anti-PD-1 antibody (REGN 2810; described in Example 12 herein) did not enhance the resumption of T cell activation.

In the second experiment, the HEK293/CD20/HLA-DR2 cell line was transduced with the human PD-L1 genome (amino acids 1-290 of the protein accession number NP_054862.1), which was cloned into a lentiviral vector system (pLVX). As described above, Jurkat-derived T cells expressing hLAG-3 and reporter luciferase under AP-1 control were incubated with PD-L1 positive antigen presenting cells expressing human MHC II complex/peptide. In this assay, anti-LAG-3 antibodies provided reactivation of T cells only in the presence of anti-PD-1 antibody (REGN2810; described in Example 12 herein) and not in the absence of anti-PD-1 antibody. Thus, in this T cell assay, anti-LAG-3 antibodies provided reactivation of T cells by blocking LAG-3/MHC II inhibitory signaling and acted synergistically with anti-PD-1 antibody.

Example 9 Anti-LAG3 antibodies are characterized by binding to CD4+ and CD8+ stimulated cynomolgus monkey T cells.

In this example, the ability of selected anti-LAG3 antibodies to bind to LAG3-expressing CD4+ and/or CD8+ cynomolgus monkey T cells was assessed by FACS.

T cells were first isolated from whole blood of the cynomolgus monkey (Bioreclamation, Westbury, NY). Briefly, whole blood from two cynomolgus monkey donors was diluted 1:1 in PBS and layered on an 85% Ficoll-Paque solution. Following standard procedures, erythrocytes were separated from mononuclear cells and T cells were isolated using a panT cells isolation kit (Miltenyi Biotech).

The isolated T cells were activated using a non-human primate T-cell Activation/Expansion kit (Miltenyi Biotech). The mixture of cells with beads was resuspended at a concentration of 1x10 ⁶ cells/ml in complete medium. After 72 h of incubation, the beads were removed and activated T cells were expanded in complete medium for 96 h.

Then, stimulated cynomolgus monkey T cells from each donor, as described above, were stained with LIVE/DEAD dye (Molecular Probes) to distinguish between live and dead cells by FACS. Cells were then co-incubated with PE-conjugated anti-CD8 and FITC-conjugated anti-CD4 antibodies for 15 min on ice to allow detection.

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CD4- and CD8-positive and negative cell populations.

To evaluate the binding of anti-LAG3 antibodies to previously isolated CD4+ and CD8+ cynomolgus monkey T cells, anti-LAG3 antibodies and isotype controls were serially diluted in blocking buffer and plated in a 96-well V-bottom well plate with final doses ranging from 100 nM up to 50 pM. The serially diluted antibodies were then directly labeled using Zenon AlexaFluor 647 (Molecular Probes) and incubated with a suspension of cynomolgus CD4+/CD8+ T cells for 15-30 minutes. on ice. Cells were centrifuged and the pellets were washed with staining buffer to remove unbound antibodies and fixed for 12 hours with Cytofix (BD Biosciences) at a 1:1 dilution and staining buffer. After the incubation period, the fixation buffer was removed and the pellets were resuspended in staining buffer, filtered, and used for fluorescence measurements.

Fluorescence measurements were obtained on a Fortessa (Becton Dickinson) and the data was analyzed in a FlowJo to determine mean fluorescence intensity (MFI). EC50 values were calculated from a four-parameter logistic model equation from an 11-point response curve using GraphPad Prism. The MFI ratio for each antibody was calculated by dividing the MFI at 11.1 nM by the MFI at 0 nM antibody concentration (negative control).

Table 15. EC50 and MFI ratios for binding of selected anti-LAG3 antibodies to CD4+ and CD8+ cynomolgus monkey T cells from multiple donors

FACS-assayed binding against a population of stimulated T cells Donor cynomolgus monkey 1 Donor cynomolgus monkey 2 CD4+ CD8+ CD4+ CD8+ ec ₅₀ [M] Attitude MFI (I nM/ ONM) ec ₅₀ [M] Attitude MFI (I nM/ ONM) EUzo [M] Attitude MFI (I nM/ ONM) EUzo [M] Attitude MFI (11 nM/ONM) H4sH15482P 1.20E-09 1.3 4.60E-10 3 3.80E-10 1.3 4D0E-10 4.7 H4sH14813N 4.20E-10 1.3 8.50E-11 3.5 2.20E-10 1.5 1D0E-10 6.3 Comparator 1 NB 1.1 9.20E-10 2.4 NB 1.2 7.80E-10 4.2 Isotype control hIgG4 NB one NB one NB one NB one

NB: no binding.

As previously shown, anti-LAG3 antibodies H4sH15482P and H4sH14813N were characterized by binding to engineered cynomolgus monkey LAG3-HEK293 cell lines. In this example, H4sH15482P and H4sH14813N showed specific binding to CD4+ and CD8+ LAG3-expressing cynomolgus monkey T cells with an EC ₅₀ ranging from 1.1 nM to 85 pM (Table 15). Thus, the selected anti-LAG3 antibodies described in this example cross-react with LAG3 expressed on activated cynomolgus CD4+ and CD8+ T cells. In contrast, Comparator 1 only showed binding to cynomolgus CD8+ T cells.

Example 10 In vivo efficacy of anti-LAG3 antibodies against Colon 26 tumors.

The in vivo efficacy of mouse anti-LAG3 antibodies alone and in combination with anti-mouse PD-1 antibodies was investigated in the Colon 26 preclinical syngeneic tumor model.

BALB/c mice were purchased from Taconic Biosciences. Colon 26, a mouse adenocarcinoma cell line derived from the BALB/c mouse strain, was obtained from the American Type Culture Collection (ATCC). On day 0, fifty BALB/c mice were implanted subcutaneously with 1 x 10 ⁶ Colon 26 cells (a mouse adenocarcinoma line derived from the BALB/c strain). On day 8, forty mice with a mean tumor volume of 50 mm ³ were selected and randomized into 4 treatment groups (N=10/groups). On days 14, 17, 21, 24, and 28, mice were dosed with antibodies as follows: group 1, isotype control rat IgG2a antibodies (clone 2A3, BioXCell, catalog number BE0089) and isotype control mIgG1 antibodies (clone MOPC-21, BioXCell, catalog number BE0083); group 2, anti-mouse PD-1 antibodies (rat IgG2a for anti-mouse PD-1 antibody, clone RPMI-14, BioXCell, catalog number BE0089) and isotype control mIgG1 antibodies; group 3, anti-mouse LAG3 (rat IgG1 for anti-mouse LAG3, clone C9B7W, BioXCell, catalog number BE0174) and isotype control rat IgG2a; group 4, antibodies to mouse PD-1 and to mouse LAG3. All antibodies were administered by intraperitoneal injection at a concentration of 10 mg/kg. Tumor volumes were monitored by caliper measurement twice a week throughout the experiment (42 days).

Significant suppression of tumor growth was observed in mice treated with anti-mouse PD-1 antibody (FIGS. 2A-B), with 5 out of 10 mice having tumor resolution by day 45 at the end of the experiment. Suppression of tumor growth was also observed in a subgroup of mice treated with anti-mouse LAG3 antibody, with 3 out of 10 mice having tumor resolution by day 45. By striking contrast, the combined anti-LAG3 and anti-PD-1 treatment was superior to either drug as monotherapy, with tumor disappearance in 8 out of 10 mice at the end of the experiment, indicating a strong additive effect of anti-LAG3 and anti-PD-1 treatment. No tumor-free mice were observed in the control group at the end of the experiment. By day 35, there was a statistically significant decrease in tumor volume scores in mice treated with the combined therapeutic drug (p<0.05, FIG. 2B), but not with either drug alone. Despite effective elimination of the tumor, no symptoms of body weight loss were observed (not shown). Thus, in the developed Colon 26 tumor model, the regimen with a combination of antibodies to PD-1 and to LAG3 was significantly more effective than with the corresponding drugs as monotherapy.

In vivo activity of anti-mouse PD-1 and anti-mouse LAG3 antibodies was also studied in preclinical 4T1, RENCA and A20 syngeneic tumor models. Combined treatment with both antibodies provided at least an additive antitumor effect compared to treatment with either antibody alone (data not shown).

Example 11 In vivo efficacy of anti-mouse LAG3 antibodies against MC38 tumors.

The dual blocking effect of PD-1 and LAG3 was studied on established MC38 tumors in C57BL/6 mice using blocking antibodies to mouse PD-1 and to mouse LAG3.

C57BL/6 mice were purchased from Taconic Biosciences. MC38, a mouse adenocarcinoma cell line derived from the C57BL/6 mouse strain, was obtained from the depository of the National Institutes of Health. Anti-LAG3 and anti-PD-1 antibodies, as well as controls, were prepared as described in Example 10.

On day 0, fifty C57BL/6 mice were implanted subcutaneously in the flank with 3x105 MC38 cells. On day 8, forty mice with a mean tumor volume of 45 mm ³ were selected and randomized into 4 treatment groups (N=10/groups). On days 8, 12, 15, 19, and 23, mice were dosed with antibodies as follows: Group 1, isotype control rat IgG2a and mIgG1 antibodies; group 2, anti-mouse PD-1 and mIgG1 antibodies; group 3, anti-mouse LAG3 and rat IgG2a; group 4, anti-mouse PD-1 antibodies and anti-mouse LAG3 antibodies. All antibodies were administered at a concentration of 10 mg/kg by intraperitoneal injection. Tumor volume was monitored by caliper measurement twice a week throughout the experiment (28 days).

Anti-PD-1 antibody therapy provided significant suppression of tumor growth in a subgroup of mice (p<0.0001; day 23) (FIGS. 3A-B), with tumor disappearance at the end of the experiment in 2 out of 10 mice. In the case of monotherapy with an antibody to LAG3, no significant antitumor effect was shown (at the end of the experiment, the tumor disappeared in 0/10 mice), while the combination of antibodies to LAG3 and to PD-1 was superior to therapy with an antibody to PD-1 alone, which indicates about the presence of a synergistic effect.

To test the immunomodulatory properties of anti-PD-1 and anti-LAG3 antibodies in combination, T-cell markers were studied in the spleens and draining lymph nodes (DLN) of treated mice (FIG. 4). Spleens and draining lymph nodes of tumor-bearing mice were removed during preparation and placed in 15 ml of RPMI1640 Glutamax I medium (Invitrogen). Total RNA was isolated and Taqman RT-PCR analysis was performed using a primer/probe mixture specific for mouse CD8P [probe: AGCAGCTCTGCCCTCAT (SEQ ID NO: 584), forward primer: GCTCTGGCTGGTCTTCAGTATG (SEQ ID NO: 585), reverse primer: TTGCCGTATGGTTGGTTTGAAC (SEQ ID NO: 586)] and mouse IFNy (Mm01168134_m1, Applied Biosystems). Samples were normalized to expression levels of the transcript encoding mouse cyclophilin B. The TaqMan assay demonstrated proliferation of CD8+ T cells in both DLN and spleens of mice in the combination treatment group, as well as increased production of the effector molecule IFNy T cells in animals treated with both the combination and anti-PD-1 antibody alone, suggesting that blocking PD-1 and LAG3 results in an increase in T cell proliferation and effector function.

Example 12 In vivo efficacy of anti-human LAG3 antibodies against MC38 tumors.

In this experiment, the efficacy of an exemplary anti-human LAG3 antibody of the present invention against MC38 tumors was studied. anti-human LAG3 mAbs do not bind to mouse LAG3. Therefore, mice humanized to express the human LAG3 protein were used to study the antitumor properties of such antibodies in mice in vivo. For the experiments of this document, two-gene humanized mice were generated using VelociGene® technology that express the extracellular portion of human PD-1 and human LAG3 and the trans-membrane and intracellular portions of murine versions of these proteins (Valenzuela et al 2003; Nat Biotechnol 21 652-659).

Bigene humanized LAG3/PD-1 mice were engineered using VelociGene® technology to replace mouse Pdcd1 and Lag3 gene regions encoding extracellular domains with corresponding regions of the human PD-1 and human LAG3 genes (U.S. Patent Application Serial No. 62/258181 filed November 20, 2015). The expression of humanized PD-1 and LAG3 was confirmed by studying the expression of PD-1 and LAG3 proteins on humanized mouse T cells after stimulation with anti-CD3/CD28 antibody. The interaction of human PD-1 and LAG3 proteins with the corresponding mouse ligands was tested by testing the binding of human LAG3 to mouse MHC II in a cell adhesion assay, and the binding of human PD-1 to mouse PD-L1 was tested in an SPR-Biacore binding assay.

The exemplary anti-LAG3 antibody used for this study is a fully human antibody that specifically binds to human LAG3 and contains HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 SEQ ID NO: 420-422-424-428-430-432 and HCVR/ LCVR SEQ ID NO: 418/426 (also known as H4sH15482P, and as mAb1 hereinafter).

Antibodies to human PD-1 are disclosed in US Patent Application Publication US20150203579, incorporated herein by reference in its entirety. The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810). More specifically, H4H7798N contains the heavy chain sequence of SEQ ID NO: 330 and the light chain of SEQ ID NO: 331 (disclosed in US20150203579). REGN2810 (anti-hPD-1 antibody) binds with high affinity to human PD-1 and blocks the interaction of PD-1 with PD-L1 and PD-L2.

Mouse colon adenocarcinoma (MC38.Ova) cells were engineered to express hen's egg albumin to increase tumor immunogenicity.

On day 0, LAG3 ^hum/hum PD-1 ^hum/hum mice were implanted subcutaneously with 1.5x106 MC38.Ova cells and randomized into three treatment groups (N=7/group). On days 3, 6, 10, 14, and 17, mice were injected intraperitoneally with mAb1 (anti-hLAG3 antibody) (25 mg/kg), REGN2810 (anti-hPD-1 antibody) (10 mg/kg), or isotype control antibody (25 mg /kg). Tumor volumes were monitored by caliper measurement twice a week throughout the experiment (24 days).

On day 0, bigene humanized LAG3 ^hum/hum PD-1 ^hum/hum mice were injected with MC38.Ova cells, and on days 3, 6, 10, 14, and 17 mAb1 (antibodies to hLAG3) (25 mg/day) was administered intraperitoneally. kg), REGN2810 (antibodies to hPD-1) (10 mg/kg) or isotype control (25 mg/kg). REGN2810 (an anti-hPD-1 antibody) was shown to partially inhibit tumor growth (FIG. 5) with tumor regression observed in 2 out of 7 mice, while there was not a single tumor-free mouse in the isotype control group. Monotherapy with mAb1 (anti-hLAG3 antibody) at 25 mg/kg did not show any significant effect on tumor growth, with tumor disappearance at the end of the experiment in only 1 out of 7 mice. Body weight scores of mice were not affected by mAb1 (anti-hLAG3 antibody), REGN2810 (anti-hPD-1 antibody), or isotype control.

Example 13 In vivo efficacy of anti-human LAG3 and anti-human PD-1 antibody combination against MC38 tumors.

This experiment examined the efficacy of mAb1 (an anti-hLAG3 antibody) in combination with REGN2810 (an anti-hPD-1 antibody) against MC38.Ova tumors in LAG3 ^hum/hum PD-1 ^hum/hum bigene humanized mice. Bigene humanized mice are described in Example 12 herein.

The exemplary anti-LAG3 antibody used for this study is a fully human antibody that specifically binds to human LAG3 and contains HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 SEQ ID NO: 420-422-424-428-430-432 and HCVR/ LCVR SEQ ID NO: 418/426 (also known as H4sH15482P, and as mAb1 hereinafter).

The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810). REGN2810 (an anti-hPD-1 antibody) binds with high affinity to human PD-1 and blocks the interaction of PD-1 cPD-Ll and PD-L2.

On day 0, mice were implanted subcutaneously with 1x106 MC38.Ova cells and randomized to four treatment groups (N=7 in control group and N=12 in each of mAb1 (anti-hLAG3), REGN2810 (anti-hPD-1) and combination of mAb1 (anti-hLAG3 antibody) + REGN2810 (anti-hPD-1 antibody)). On days 3, 7, 10, 14, and 17, mice were injected intraperitoneally with mAb1 (anti-hLAG3) (25 mg/kg), REGN2810 (anti-hPD-1) (10 mg/kg), mAb1 (anti-hLAG3) ) (25 mg/kg) and REGN2810 (anti-hPD-1 antibodies) (10 mg/kg) or isotype control (25 mg/kg). Tumor volume values were monitored by caliper measurement twice a week throughout the experiment (32 days) and tumor free animals were monitored for tumor recurrence for up to 80 days.

Monotherapy with REGN2810 (an anti-hPD-1 antibody) resulted in tumor growth inhibition (Fig. 6A

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B), with tumor regression in 2 out of 12 (17%) animals, while mAbl (anti-hLAG3 antibody) was not significantly effective, confirming the conclusion from the previous experiment. Tumor regression was observed in only 1 of 12 mice treated with mAb1 (an anti-hLAG3 antibody). In the isotype control group, there were no tumor-free mice by day 32. In contrast, the combination of mAb1 (anti-hLAG3 antibodies) and REGN2810 (anti-hPD-1 antibodies) demonstrated strong inhibition of MC38.Ova tumor growth, resulting in 5 out of 12 (42%) tumor-free mice at the end of the experiment. None of the tumor-free mice showed tumor recurrence within 80 days of implantation, suggesting the long-term effects of the combination immunotherapy. Using one-way analysis of variance with Tukey's post hoc test for multiple comparisons, there was a significant difference in tumor volume between the mAb1 (anti-hLAG3 antibody) and REGN2810 (anti-hPD-1 antibody) combined treatment groups and the mAb1 (anti-hLAG3 antibody) monotherapy groups. ) (p<0.05), or isotype control (p<0.01) on day 22 (FIG. 6B). The monotherapy drug REGN2810 (anti-hPD1 antibody) also showed significant suppression of tumor growth (p<0.05). Combined treatment with mAb1 (an anti-hLAG3 antibody) and REGN2810 (an anti-hPD-1 antibody) also resulted in a significant improvement in percent survival of the animals (p<0.0001) as analyzed by a log-rank test (Mantel-Cox) (Fig. 6C). Despite the improved efficacy of combination therapy, no symptoms of body weight loss were observed (data not shown). Thus, the combination of mAb1 (anti-hLAG3 antibody) and REGN2810 (anti-hPD-1 antibody) provided improved efficacy, including tumor growth suppression, improved tumor eradication, and increased survival compared to either REGN2810 (anti-hPD-1 antibody) or mAb1 (anti-hPD-1 antibody). to hLAG3) as monotherapy.

Example 14 Studies to determine the optimal dose of anti-LAG3 and anti-PD-1 antibody combination for MC38 tumors.

This example describes a series of optimal dose experiments measuring the in vivo efficacy of a combination of anti-human PD-1 and anti-human LAG3 antibodies against MC38.Ova tumors in LAG3 ^hum/hum PD-1 ^hum/hum bigene humanized mice. Bigene humanized mice are described in Example 12 herein.

The exemplary anti-LAG3 antibody used for this study is a fully human antibody that specifically binds to human LAG3 and contains HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 SEQ ID NO: 420-422-424-428-430-432 and HCVR/ LCVR SEQ ID NO: 418/426 (also known as H4sH15482P, and as mAb1 hereinafter).

The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810). REGN2810 (anti-hPD-1 antibody) binds with high affinity to human PD-1 and blocks the interaction of PD-1 with PD-L1 and PD-L2.

In the first experiment, REGN2810 (an anti-hPD-1 antibody) was titrated and tested at dose levels of 10 mg/kg and 1 mg/kg, both dose groups being tested both as a single agent and in combination with mAb1 (an anti-hLAG3 antibody) ( 25 mg/kg). The results for both combination treatment groups were compared with those of REGN2810 (1 mg/kg or 10 mg/kg) or mAb1 (25 mg/kg) monotherapy to determine if an additive/synergistic antitumor effect was observed. Treatment with an isotype control antibody (25 mg/kg) was also tested. On day 0, LAG-3 ^hum/hum PD1 ^hum/hum mice were injected with MC38.Ova cells and randomized to six treatment groups (N=6 in the isotype control group and N=12 in the mAb1, REGN2810 or REGN2810+ treatment groups). mAb1). Mice were administered: mAb1 (25 mg/kg); REGN2810 (10 mg/kg); REGN2810 (1 mg/kg); mAb1 (25 mg/kg) + REGN2810 (10 mg/kg); mAb1 (25 mg/kg) + REGN2810 (1 mg/kg); or isotype control (25 mg/kg) by intraperitoneal injection on days 3, 7, 10, 14 and 17. Tumor volumes were monitored for 36 days and tumor-free animals were monitored for a period of 80 days.

Table 16 shows the mean tumor volume at various time points in the study, and Table 17 shows the number of tumor-free mice at day 36.

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Table 16 Mean Tumor Volumes at Different Time Points

Processing group Tumor volume, mm ³ , mean value (±SEM) Day 7 Day 10 Day 14 Day 17 Day 22 isotypic control 24 (±6) 54 (±11) 104(±25) 269 (±70) 749(±180) shAI (antibody to LAG3) (25 mg/kg) 27(±4) 53 (±20) 83 (±38) 155(±73) 451 (±211) R2810 (10 mg/kg) 27(±4) 29 (±8) 34 (±11) 86 (±26) 313 (±98) R2810 (1 mg/kg) 36 (±8) 92 (±23) 150(±47) 264 (±73) 669 (±183) R2810 (10 mg/kg) + SHAI 25 (±3) 10 (±3) 4 (±4) 8 (±5) 38 (±19) R2810 (1 mg/kg) + shAI 31 (±7) 38(±13) 55 (±35) 106(±58) 378 (±137)

Table 17. Number of tumor-free mice by day 36

Group Mice without tumor (day 36) n/N ^a (%) Isotype control 0/6 (0%) REGN2810 (1 mg/kg) 2/12(16%) REGN2810 (10mg/kg) 4/12(33%) shAI (25 mg/kg) 1/12 (8%) REGN2810 (1mg/kg) + SHAI (25mg/kg) 3/12 (25%) REGN2810 (10mg/kg) + SHAI (25mg/kg) 6/12 (50%)

^a n/N: number of tumor-free mice/total number of mice per group.

REGN2810 monotherapy at a concentration of only 1 mg/kg showed minimal tumor regression over a time period of 36 days, similar to the results for the isotype control treatment group (Fig. 7A-B). By day 36, in the groups treated with REGN2810 (1 mg/kg) and isotype control, respectively, there were 2/12 (~16%) and 0/6 (0%) mice without tumor. In contrast, the mAb1 (25mg/kg), REGN2810 (10mg/kg) and REGN2810 (1mg/kg) + mAb1 (25mg/kg) treatment groups demonstrated similar tumor growth suppression rates over a time period of 22 days (Fig. 7A). On day 36, mAb1 (25mg/kg) treatment resulted in 1/12 (~8%) tumor-free mice, while REGN2810 (10mg/kg) and REGN2810 (1mg/kg) ) + mAb1 (25 mg/kg), respectively, there were 4/12 (~33%) and 3/12 (25%) mice without a tumor. Overall, mAb1 (25mg/kg) + REGN2810 (10mg/kg) showed the strongest inhibition of MC38.Ova tumor growth, with 6 out of 12 (50%) mice having tumor disappearance by day 36. None of the tumor-free mice showed tumor recurrence within 80 days of implantation despite discontinuation of treatment, suggesting the long-term effects of combined immunotherapy. Treatment with mAb1 (25 mg/kg) + REGN2810 (10 mg/kg) also resulted in a significant improvement in percentage animal survival (p = 0.0197) as analyzed by a log-rank test (Mantel-Cox) (Fig. 1C). Also, no symptoms of weight loss were observed.

In the second experiment, mAb1 (an anti-hLAG3 antibody) was titrated and tested at dose levels of 25 mg/kg and 5 mg/kg, with both dose groups tested both as a single agent and in combination with REGN2810 (an anti-hPD-1 antibody) ( 10 mg/kg). The results for both combination treatment groups were compared with those of mAb1 (5 mg/kg or 25 mg/kg) or REGN2810 (10 mg/kg) monotherapy to determine if there was an additive/synergistic antitumor effect. Treatment with an isotype control antibody (25 mg/kg) was also tested. On day 0, LAG-3 ^hum / ^hum PD-1 ^hum / ^h u ^m mice were injected with MC38.Ova cells and randomized to six treatment groups (N=6 in the isotype control group and N=12 in the mAb1, REGN2810 or REGN2810 + mAb1). Mice were administered: mAb1 (5 mg/kg); mAb1 (25 mg/kg); REGN2810 (10 mg/kg); mAb1 (5 mg/kg) +

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REGN2810 (10 mg/kg); mAbl (25 mg/kg) + REGN2810 (10 mg/kg); or isotype control (25 mg/kg) by intraperitoneal injection on days 3, 7, 10, 14 and 17. Tumor volumes were monitored for 31 days.

For mAb1 monotherapy at a concentration of 5 mg/kg, minimal tumor regression was shown, similar to the results for the isotype control treatment group. The combination of mAb1 (25 mg/kg) + REGN2810 (10 mg/kg) showed the strongest tumor suppression (FIG. 8A). A strong combination effect was also demonstrated for the mAb1 (5mg/kg) + REGN2810 (10mg/kg) treatment group. Tumor volume measurements measured on day 23 are shown in FIG. 8B. The combination of anti-LAG-3 antibody and REGN2810 also showed a significant improvement in animal survival (p<0.001, as analyzed by the Mantel-Cox log-rank test) (FIG. 8C). It can be seen from the results that a strong combination effect was observed even at low doses of the anti-LAG3 antibody. Based on the results presented herein, high doses of anti-LAG3 antibodies in combination with anti-PD-1 antibodies may not be required for tumor treatments.

Thus, for the combination of REGN2810 (anti-hPD-1 antibodies) and mAb1 (anti-hLAG3 antibodies) in the MC38.ova tumor model in a bigene humanized LAG3/PD-1 mouse, which allows testing of treatable antibodies that do not bind cross-linked to mouse receptors, improved dose-response efficacy, including tumor growth suppression and increased survival, was demonstrated compared to REGN2810 (anti-hPD-1 antibody) and mAb1 (anti-hLAG3 antibody) as monotherapy. The sustained antitumor efficacy of the combination of REGN2810 (antibodies to hPD-1) and mAb1 (antibodies to hLAG3) in preclinical conditions provides grounds for conducting clinical trials for it as a combined cancer immunotherapy.

Example 15 In vivo efficacy of anti-human LAG3 and anti-human PD-1 antibody combination against established MC38 tumors.

This experiment examined the efficacy of mAb1 (an anti-hLAG3 antibody) in combination with REGN2810 (an anti-hPD-1 antibody) against advanced MC38.Ova tumors in bigene humanized mice LAG3 ^hum/hum PD-1 ^hum/hum Bigene humanized mice described in example 12 in this document.

The exemplary anti-LAG3 antibody used for this study is a fully human antibody that specifically binds to human LAG3 and contains HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 SEQ ID NO: 420422-424-428-430-432 and HCVR/LCVR SEQ ID NO: 418/426 (also known as H4sH15482P, and as mAb1 hereinafter).

The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810). REGN2810 (an anti-hPD-1 antibody) binds with high affinity to human PD-1 and blocks the interaction of PD-1 with PD-1u PD-12.

On day 0, LAG-3 ^hum/hum PD-1 ^{humhum mice} were injected subcutaneously with MC38.Ova cells. On day 10, mice with an average tumor volume of 100 mm ³ were selected and randomized into four treatment groups. On days 10, 14, 17, 22, mice were IP injected with mAb1 (25 mg/kg; N=9), REGN2810 (10 mg/kg; N=10), mAb1 (25 mg/kg) + REGN2810 (10 mg/kg) (N=11) or isotype control antibody (25 mg/kg; N=7). Tumor volume measurements were monitored for 28 days after tumor cell implantation.

Treatment of MC38.ova tumor-bearing humanized mice with a combination of anti-hPD-1 and anti-hLAG-3 antibodies resulted in the activation of intratumoral and peripheral T cells. REGN2810 monotherapy resulted in partial tumor growth inhibition, and mAb1 monotherapy showed no reduction in mean tumor volume compared to isotype control treated mice (FIG. 9). In contrast, the combination of mAb1 (25 mg/kg) and REGN2810 (10 mg/kg) showed strong inhibition of MC38.Ova tumor growth. One-way analysis of variance (ANOVA) with Bonferroni post hoc test for multiple comparison showed a significant difference in mean tumor volume between mAb1 and REGN2810 combination group and mAb1 monotherapy (p<0.01) or isotype control (p<0, 05) (Fig. 10). When comparing combination therapy with REGN2810 monotherapy, the overall mean tumor volume over the entire experiment was lower in the case of combination therapy, however, this difference did not reach statistical significance. The combination of anti-LAG-3 antibody and REGN2810 also provided a significant increase in survival, as well as an increase in the duration of survival of the animals (p<0.01, as analyzed using the Mantel-Cox log-rank test). By day 25, 50% of the mice treated with the combined therapeutic drug survived compared to control and monotherapy (FIG. 11). According to the results, the combination treatment had an additive, dose-dependent antitumor effect compared to the corresponding monotherapeutic drugs.

Example 16 Pharmacokinetics of anti-LAG-3 antibody in cynomolgus monkey.

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The pharmacokinetics (PK) of the anti-LAG3 mAbl antibody were described following administration of a single intravenous (IV) dose of 1.0, 5.0, or 15.0 mg/kg to female cynomolgus monkeys (5 animals per dose group). Blood samples were collected from all animals at various time points to determine the concentration of functional mAb1. Serum functional mAb1 concentrations were determined using enzyme-linked immunosorbent assay (ELISA). Serum anti-mAb1 antibodies were analyzed using a bridged electrochemiluminescence immunoassay. PK parameters were assessed using non-compartmental analysis (NCA). Mean concentration-time profiles are characterized by an initial transient distribution phase followed by a linear beta elimination phase (FIG. 12). No target-mediated clearance phase was observed during the 8 week study. Mean AUC _inf increased in a dose-dependent manner, as demonstrated by similar AUCinf/dose values among the dose levels tested. Consistent with this finding, mean total clearance (CL) values were similar for the 3 dose groups. In addition, the half-lives in the beta phase (t _1/2 ) for the 3 dose groups were comparable, ranging from 10.8 to 11.5 days. Based on the results, up to 50 mg/kg could be taken as the maximum no overt adverse effect dose (NOAEL). These results indicate that mAb1 demonstrated linear kinetics under the conditions of this study.

Example 17 Toxicological evaluation of anti-LAG3 antibody in cynomolgus monkeys.

In vivo toxicological and toxicokinetic profiles of mAb1 were assessed in a 4-week, repeated-dose GLP-compliant toxicology study in male and female cynomolgus monkeys. Monkeys (5 animals of each sex per group) were administered 0, 2, 10 or 50 mg/kg/week of mAb1 by IV infusion. Toxicity scores were based on mortality, morbidity, clinical observations, body weight, food intake, ophthalmic examinations, ECG studies, respiratory rate, pulse oximetry, body temperature and blood pressure, and neurological examinations. Parameters were also evaluated in the framework of laboratory diagnostics (general blood count, coagulation, clinical biochemistry and urinalysis) and immunophenotyping. Macroscopic examinations at autopsy, measurements of organ weights, and histopathological examinations were also performed. A complete autopsy was performed at the end of the dosing period or at the end of the 8-week no-dose period. Selected organs were weighed and tissues were subjected to macroscopic and microscopic examination.

mAb1 was well tolerated at all dose levels evaluated when administered to cynomolgus monkeys by IV infusion. Because mAb1 was well tolerated and no mAb1-related changes were observed in any of the evaluated safety parameters, the No Overt Adverse Effects (NOAEL) dose for this study was considered to be 50 mg/kg, the highest dose administered in this study. research.

Example 18 Hydrogen-deuterium exchange (H/D) epitope mapping for the anti-LAG3 antibody H4sH15482P binding to the extracellular domain of hLAG3.

Experiments were performed to determine the amino acid residues of the human LAG3 extracellular domain (human LAG3 amino acids Leu23-Leu450, UniProt accession number P18627, prepared with a human IgG1 Fc tag at the c-terminus (hLag3.Fc) (R&D Systems, Minneapolis, MN) (SEQ ID NO: 587), with which the anti-LAG3 antibody H4sH15482P interacts. For this purpose, epitope mapping based on hydrogen-deuterium (H/D) exchange with mass spectrometry (HDX-MS) was used. A general description of the HDX method is set forth in Ehring (1999 ) Analytical Biochemistry 267(2):252-259 and Engen and Smith (2001) Anal Chem 73:256A-265A.

Experiment procedure.

HDX-MS experiments were performed using an integrated Waters HDX/MS platform (Waters Corporation, Milford, MA) consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (Auxiliary solvent manager) for digestion and sample loading, a Waters Acquity M-Class (μBinary solvent manager) to create a gradient in the analytical column and a Synapt G2-Si mass spectrometer to measure the mass of pepsin-cleaved peptides.

The labeling solution was prepared in 10 mM PBS buffer in D2O at pD 7.0. For deuterium labeling, 3.8 µl of hLAG3.Fc (5 pmol/µl) or hLAG3.Fc premixed with 1:1 molar ratio of anti-LAG3 antibody H4sH15482P was incubated with 56.2 µl of D2O-based labeling solution for various time intervals (eg, non-deuterated control = 0 s, deuterium labeling: 1 and 20 min).

The deuteration was stopped by transferring 50 µl of the sample to 50 µl of pre-chilled reaction stop buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and the mixture was incubated at 1.0°C for 4 min. The stop sample was then injected into the Waters HDX Manager for real-time pepsin/protease XIII digestion. The digested peptides were captured on an ACQUITY UPLC VEN C18 1.7-µm, 2.1 x 5 mm VanGuard (Waters Corporation) guard column at 0°C and eluted onto an ACQUITY UPLC VEN C18 analytical column 1.7-µm, 1 0x50 mM for a 9 min separation in a 540% B gradient (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The following parameters were adjusted on the mass spectrometer: cone voltage 37 V, scan time 0.5 s, and mass/charge ratio range 50-1700 Thomson (Th).

To identify human LAG3 peptide residues that H4sH15482P interacts with, LC-MSE data from a non-deuterated sample was processed and searched through a database that included human LAG3, pepsin, and random sequences using ProteinLynx Global Server (PLGS) software from Waters . The identified peptides were imported into the DynamX software and filtered according to two criteria: 1) minimum products per amino acid = 0.2 and 2) limit number of repetitions in the file = 3. The DynamX software then automatically determines the deuterium uptake of each of the peptides based on the retention time and the exact mass determinations (<10 ppm) at multiple time points with 3 repetitions for each time point.

Results.

Using a real-time tracking pepsin/protease XIII column, combined with MS ^E data collection, a total of 123 peptides from human LAG3 in the absence or presence of antibody were reproducibly identified, with 78.3% sequence overlap. Four Lag-3 peptides showed significantly reduced deuterium uptake (centroid delta > 0.8 daltons with p-values < 0.05) when bound to H4sH15482P. The registered mass of the peptide corresponds to the average value of the centroids of the MH ⁺ mass from three repetitions. These peptides, corresponding to amino acids 34-77 of hLAG3.Fc, (SEQ ID NO: 587), had lower deuteration rates when bound to H4sH15482P. These identified residues also correspond to human LAG3 residues 28-71 as defined in Uniprot entry P18627 (LAG3_HUMAN, SEQ ID NO: 588) and shown in Table 18.

Table 18. Human LAG3 peptides with altered deuteration rates after binding

H4sH15482P

Amino acid residues SEQ ID NO: 588 T=1 min. deuteration T=20 min. deuteration hLAG3.Fc; MH ⁺ hLAG3.Fc + H4sH15482P; MH ⁺ A hLAG3.Fc; MH ⁺ hLAG3.Fc + H4sH15482P; MH ⁺ A 28-69 4352.30±0.03 4350.14±0.06 -2D5 4352.33±0.20 4351.43±0.12 -0.90 28-71 4672.54±0.03 4670.42±0.01 -2.12 4672.69±0.21 4671.57±0.18 -1.13 31-52 2198.ll±0.10 2196.49±0.01 -1.61 2198.97±0.18 2197.18±0.10 -1.79 32-69 3853.07±0.12 3851.37±0.07 -1.69 3853.23±0.07 3852.35±0.14 -0.88

Example 19 Anti-LAG3 Antibody Clinical Trial in Patients with Advanced Cancer.

This example describes a clinical trial to evaluate the safety, tolerability, and antitumor activity of an anti-LAG3 antibody as a monotherapy and in combination with an anti-PD-1 antibody in patients with advanced cancer.

The exemplary anti-LAG3 antibody used for this study is a fully human antibody that specifically binds to human LAG3 and contains HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 SEQ ID NO: 420-422-424-428-430-432 and HCVR/ LCVR SEQ ID NO: 418/426 (also known as H4sH15482P, and as mAb1 hereinafter).

The anti-PD-1 antibody used for this study is H4H7798N (disclosed in US20150203579; also known as REGN2810). REGN2810 (anti-hPD-1 antibody) binds with high affinity to human PD-1 and blocks the interaction of PD-1 with PD-L1 and PD-L2.

Objectives of the clinical trial.

The primary objectives of the study are as follows.

In the dose escalation phase: evaluate safety and pharmacokinetics (PK) to determine the phase 2 dose of mAb1 as monotherapy and in combination with REGN2810 in patients with advanced malignancies, including lymphoma. Endpoints include incidence of dose-limiting toxicity (DLT), adverse events (AE) (including those associated with immunity), serious adverse events (SAE), death, and laboratory abnormalities (grade 3 or higher according to the common terminological criteria for adverse events). [CTCAE] events) and pharmacokinetics.

In the dose escalation phase to the maximum tolerated: preliminarily assess the antitumor activity of mAb1 alone and in combination with REGN2810 (separately in separate cohorts), as measured by ORR. Endpoints include overall response rate (ORR) based on Solid Tumor Response Evaluation Criteria (RECIST) 1.1 (Eisenhauer et al 2009, Eur. J. Cancer 45: 228-247) (solid tumors) and Lugano criteria ( Cheson et al 2014, J Clin Oncol 32 3059-3068) (lymphoma).

The secondary goals are: (a) to preliminarily assess the antitumor activity of mAb1 alone and in combination with REGN2810 (individually in selected cohorts), as measured by ORR based on the Immunotherapy Solid Tumor Response Evaluation Criteria (irRECIST) (Wolchok et al 2009, Clin Cancer Res. 15: 7412-7420; Nishino et al 2013, Clin. Cancer Res. 19: 3936-3943), best overall response (BOR), duration of response (DOR), disease control rate, and PFS based on RECIST criteria, irRECIST and Lugano (Cheson et al 2014, J. Clin. Oncol. 32: 3059-3068); (b) characterize the safety profile in each cohort with dose escalation to the maximum tolerated dose as determined in the dose escalation phase; (c) characterize PK mAb1 as a monotherapy drug and mAb1 and REGN2810 when administered in combination; and (d) evaluate immunogenicity as measured by anti-drug antibodies (ADA) for mAb1 and REGN2810.

The exploratory objectives of the study are as follows: (a) estimate the volume of the tumor; (b) to study the pharmacodynamic effects of REGN2810 in serum, plasma, peripheral blood mononuclear cells (PBMCs), and tumor tissue samples (including archival tissue sample) obtained at baseline, during treatment, and during progression from patients treated with mAb1 as a monotherapy or in combination with REGN2810; and (c) evaluate predictive potential and correlation with clinical response for biomarkers of interest, which may include, without limitation: (i) circulating nucleic acids from tumor cells; (ii) distribution of the PBMC subpopulation and expression of molecules that are part of the immunological checkpoint and other biomarkers of interest; (iii) expression of RNA from tumor cells; (iv) number and distribution of tumor infiltrating lymphocytes (CD8+ T cells, CD4+ T cells, regulatory T cells, and other tissue infiltrating subtypes such as B cells, myeloid cells, NK cells, etc.) ; (v) expression levels (messenger RNA and/or protein) of PD-1, PD-L1, LAG3 and possible other checkpoint modulators; (vi) mutations in known oncogenes and potential tumor neoantigens; and (vii) tumor mutation load.

Study design.

The study is a Phase 1 Human First Use (FIH), Open-label, Multicentre, Dose Escalation, Safety, Tolerability, Potency and PK study of mAb1 administered as monotherapy and in combination with REGN2810 in patients with cancer at a late stage. Three dose levels of mAb1 (1.0, 3.0 and 10 mg/kg) were studied as monotherapy and in combination with REGN2810 at 3.0 mg/kg.

At the end of a screening period of up to 28 days, patients underwent up to seventeen cycles of 21 days of treatment (for a total of up to 51 weeks of treatment) followed by a 24-week follow-up period. Each patient received mAb1 (+/- REGN2810) every 21 day cycle.

Treatment continued until the 51-week treatment period was completed or until the onset of disease progression, unacceptable toxicity, withdrawal of consent, or exclusion from the study. Response was assessed every 6 weeks for the first 24 weeks, then every 9 weeks for the next 27 weeks, regardless of study drug dosing delay. After a minimum of 24 weeks of treatment (a minimum of 8 treatment cycles), patients with confirmed complete remission (CR) could choose to discontinue treatment but continue to participate in relation to all relevant study evaluations. Similarly, after consultation with the treating physician, patients who had been treated for at least 24 weeks, with disease stabilization (SD) or partial response (PR) that persisted through 3 subsequent tumor evaluations, could decide to discontinue treatment but continue to participate. regarding the conduct of all relevant assessments within the study. In the case of patients who observed a response and subsequent progression, a tumor biopsy was required during the progression period. In patients who tolerated 4 doses of mAb1 monotherapy and who had at least SD at initial tumor evaluation, but who subsequently developed disease progression (PD), REGN2810 3.0 mg/kg was allowed to be added to the highest dose of mAb1. safely administered up to a certain point (if known) with the aim of resuming the response through the combined blocking of the protein encoded by the lymphocyte activation gene-3 (LAG-3) and programmed cell death protein-1 (PD-1). Safety assessments were performed at each visit for study drug administration.

Dose increase.

Three monotherapy and escalation combination therapy cohorts (1.0, 3.0 and 10 mg/kg mAb1 with or without 3 mg/kg REGN2810) were planned to be included. The first cohort to be included received the monotherapy drug mAb1 at a dose of 1.0 mg/kg. Subsequent inclusion of each additional cohort may be limited by the number of dose-limiting toxicity (DLT) events seen in previous cohorts. Dose reduction cohorts (0.3 mg/kg mAbl with or without 3 mg/kg REGN2810) were included as needed.

Dose escalation rules.

To evaluate all dose levels for both mAb1 and mAb1 + REGN2810 monotherapy cohorts, a modification of the classic 3+3 (4+3) design is used. Although a minimum of 3 patients per dose level is required to be evaluated for DLT, in order to maximize the effectiveness of dose escalation in Phase 1 while maintaining patient safety, include 4 patients per dose level in case the patient stops treatment before conducting an assessment regarding DLT. The duration of the DLT evaluation is 28 days. Dose level tolerance is only achieved when all potentially DLT-evaluable patients complete the 28-day DLT-free evaluation period (0/3 or 0/4). If 3 patients complete the DLT evaluation period without transferring DLT, but a fourth patient is included in the DLT evaluation period, dose level tolerance is achieved only if the fourth patient completes the DLT evaluation period or stops treatment prior to the DLT evaluation. If there is 1 case of DLT in 3 or 4 patients to be DLT-evaluated, then 4 or 3 additional patients (respectively) are included to reach a total of 7 patients. Likewise, 1/6 or 1/7 of the DLT is considered acceptable. Two cases of DLT in 2-7 DLT-evaluable patients will be considered as exceeding the maximum tolerated dose (MTD). At the highest tolerable level, an additional 3-4 patients will be included for additional safety evaluation to reach a total of 6-10 patients eligible for DLT evaluation. 0-1 out of 6-8 or 2 out of 9-10 DLT cases will be considered acceptable. For additional safety evaluation, 3 additional patients may be included at any dose level at the discretion of the sponsor (in consultation with the investigators).

If 1.0 mg/kg mAb1 (dose level 1; DL1) is considered safe (based on results for 3-7 patients), inclusion will begin at 3.0 mg/kg mAb1 (DL2). After enrollment of 4 patients in DL2, enrollment in the first cohort receiving the combination (1.0 mg/kg mAb1 + 3.0 mg/kg REGN2810; DL3) can begin. Dose escalation to 10 mg/kg mAb1 (DL4) may be initiated when 3.0 mg/kg mAb1 (DL2) is considered safe. The second cohort receiving the combination (3.0 mg/kg mAb1 + 3.0 mg/kg REGN2810; DL5) is recruited only after both DL2 and DL3 are considered safe. A third cohort receiving the combination (10 mg/kg mAb1 + 3.0 mg/kg REGN2810; DL6) is recruited only after both DL4 and DL5 are considered safe. If multiple cohorts are available for further inclusion at the same time, the cohort with fewer numbers is preferred (i.e. DL2 is preferred over DL3 or DL3 is preferred over DL4).

Dose-limiting toxicity.

The DLT follow-up period to determine safety with respect to dose escalation or initiation of a new combination therapy is defined as 28 days from cycle 1, day 1, to monitor the safety and tolerability of the first 2 doses of investigational drug(s)(s) ( mAb1 with or without REGN2810 as appropriate). To assess DLT, the patient must have received at least the first 2 doses of the investigational drug(s) (i.e. day 1 and day 22), and be observed for at least 28 days after the first dose, and at least 7 days after the second administration, or transfer DLT (defined below) until the end of the DLT evaluation period. Delays in the second dose of investigational drug(s) beyond 35 days and/or discontinuation of investigational drug(s) are considered DLTs if they are related to the investigational drug(s). The length of the DLT follow-up period is thus increased for patients who have a delayed second dose and for patients who have experienced AE, for which the duration must be assessed to determine if a DLT event has occurred.

In addition to the impossibility of administering (due to investigational drug toxicity) Dose #2 within a defined interval, DLT is generally defined as any of the following investigational drug related toxicities:

Toxicity other than haematological: Grade uveitis >2.

Any toxicity other than hematologic grade >3 (with the exception of non-medically significant laboratory abnormalities such as amylase or lipase without symptoms).

Hematological toxicity:

Grade 4 neutropenia lasting >7 days;

thrombocytopenia 4 degrees;

thrombocytopenia 3 degrees with bleeding;

febrile neutropenia grade >3 or neutropenia grade >3 with confirmed infection.

Both irAEs and non-irAE AEs that meet the DLT definition are considered DLTs.

The maximum tolerated dose.

The MTD is defined as the dose level immediately below the level at which the dose

- 64 039718 is terminated due to the presence of 2 or more DLTs out of 6-7 patients to be evaluated, and will be determined separately for monotherapy and combination therapy. However, in view of the known cases of AEs associated with REGN2810 monotherapy and other anti-PD1/PD-L1 antibodies for cohorts receiving the combination, the severity, frequency, and lack of known combination toxicity data may be taken into account when determining the MTD and deciding whether to add additional patients to the combination. dose level. If the dose increase is not stopped for DLT, the MTD is considered to be undetermined. An additional 3 patients in each of the monotherapy and combination cohorts expected to tolerate the highest dose levels will be included (i.e. 6-10 patients in each of these cohorts). If dose escalation for mAb1 monotherapy or combination therapy with REGN2810 is stopped at 1.0 mg/kg due to the presence of DLT, the 0.3 mg/kg cohort is included. If dose escalation for mAb1 monotherapy or combination therapy is discontinued due to the presence of DLT at dose levels of 3.0 or 10 mg/kg, mAb1 dose is reduced to the previously tested dose level for newly enrolled patients (in monotherapy or combination therapy cohorts). respectively). No patient should be started on a combination therapy with a dose of mAb1 that is not tolerated as a monotherapy.

Recommended dose for phase 2.

The RP2D for escalation cohorts to the maximum tolerated dose will not exceed the MTD or the highest dose tested, and may differ between monotherapy and combination therapy cohorts. The definition of RP2D will be based on safety data and RK.

Patients in monotherapy cohorts who tolerated 4 doses of mAb1 and who had at least SD at initial tumor evaluation but subsequently had PD were allowed to add REGN2810 3.0 mg/kg to the highest dose of mAb1. safely administered up to a certain point (if known) in order to resume the response through the combined blocking of LAG-3 and PD-1.

Dose escalation cohorts up to the maximum tolerated dose.

After mAb1 tolerance has been established, alone and in combination with REGN2810, several escalation cohorts to the maximum tolerated dose using monotherapy or combination therapy for selected indications [non-small cell lung cancer (NSCLC), clear cell renal cell carcinoma (ccRCC), triple-negative breast cancer (TNBC), melanoma, and diffuse large B-cell lymphoma (DLBCL)], are recruited to further evaluate safety and provide preliminary data on antitumor activity in tumors known to express or overexpress LAG3; antitumor activity of mAb1 can be observed when administered alone or in combination with REGN2810. Enrollment in escalation cohorts up to the maximum tolerated dose begins after completion of the escalation. An additional 10 escalation cohorts to the maximum tolerated dose (Table 19), using Simon's 2-step design (Simon 1989, Controlled Clinical Trials 10:1-10), were recruited after confirmation of RP2D. A patient is assigned to a specific treatment cohort based on the patient's tumor type, the presence or absence of prior therapy with anti-PD-1 antibodies/programmed cell death protein ligand 1 (PD-L1) antibodies, the investigator's assessment of the appropriateness of the course of treatment for that patient, and availability of vacancies among patients in the assigned cohort receiving treatment. If during Step 1 of Simon's 2-step design safety concerns arise in a single cohort with dose escalation to the maximum tolerated dose, enrollment may be withheld. After discussion between investigators and Regeneron representatives, inclusion may be restarted at the same or lower dose of mAb1. Security concerns leading to a suspension may include early or late security events. Patients are treated with mAb1 monotherapy (dose determined by dose escalation) or mAb1 and REGN2810 3mg/kg Q3W combination for up to 51 weeks. Inclusion in stage 2 for each cohort with an increase in dose to the maximum tolerated dose occurs only if stage 1 shows a minimum number of tumor responses.

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Table 19. Table of cohorts with increase in dose to the maximum tolerated

Dose escalation to maximum tolerated cohort Type of tumor Not treated antibodies to PD-1/PD-L1 Treated with anti-PD-1/PD-L1 antibodies Treatment one non-small cell lung cancer X Combination of mAb 1 and REGN2810 2 non-small cell lung cancer X Combination of mAb 1 and REGN2810 3 clear cell kidney cancer X Combination of mAb 1 and REGN2810 four clear cell kidney cancer X Combination of mAbl and REGN2810 5 Triple negative breast cancer X Combination of mAb 1 and REGN2810 6 Melanoma X Combination of mAb 1 and REGN2810 7 Melanoma X Combination of mAb 1 and eight 9 Diffuse large cell lymphoma Diffuse large cell lymphoma X X REGN2810 Monotherapy drug mAb 1 Combination of mAb 1 and REGN2810 ten Diffuse large cell lymphoma X Combination of mAb 1 and REGN2810

Study population.

In the dose escalation phase: Patients with advanced cancer who have not received prior anti-LAG3 antibody and/or PD-1/PD-L1 inhibitor drug therapy and who are not candidates for standard therapy, or if for whom the available therapy is not expected to provide clinical benefit, and patients with cancer who are not responding to treatment and who have not experienced a response or have shown tumor progression regardless of the availability of standard therapy.

In escalation to maximum tolerated cohorts: Patients with selected malignancies who have not received prior anti-LAG3 antibody drug therapy and who have not previously received anti-PD-1/PD-L1 antibody therapy but are potential candidates receive anti-PD-1 antibody therapy (cohorts 1, 3, 5, 6, and 9), or have previously received anti-PD-1/PD-L1 antibody therapy and had a confirmed objective response (CR or PR ) or SD for at least 3 months during therapy with anti-PD-1/PD-L1 antibodies, but then there was progression during such therapy, or SD or PR was observed as the best response, followed by a sustained response for 6 months (Cohorts 2, 4, 7, and 10), or are not candidates for standard therapy, or in whom the available therapy is not expected to provide clinical benefit, and is eligible for mAb1 monotherapy (Cohort 8).

Inclusion Criteria.

To be eligible for inclusion in the study, a patient must meet the following criteria.

1. Men and women aged >18 years.

2. Dose escalation cohorts: patients with a histologically or cytologically confirmed diagnosis of malignancy (including lymphoma), with observed tumor progression, for whom there is no alternative standard of care, or there is no alternative standard of care with therapeutic potential (i.e. for who did not respond or who developed tumor progression regardless of the presence of standard therapy), and who were not previously treated with a PD-1/PD-L1 inhibitor. For such patients, the disease is not required to be a tumor measured in size according to RECIST 1.1 or Lugano criteria.

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3. Upscaling cohorts to the maximum tolerated dose: Patients with histologically or cytologically confirmed diagnosis of 1 of the following tumors, which are RECIST 1.1 or Lugano-measured tumor sizes, and meet the following criteria:

i) suffer from untreated anti-PD-1/PD-L1 NSCLC stage IIIB or IV, either without prior therapy for metastatic disease or disease progression/recurrence following a platinum regimen (cohort 1);

ii) suffer from anti-PD-1/PD-L1 antibody-treated stage IIIB or IV NSCLC with no more than 2 prior therapies for metastatic disease (cohort 2);

iii) suffer from late-stage untreated anti-PD-1/PD-L1 ccRCC or metastatic ccRCC with a clear cell component and who received 1-2 prior anti-angiogenic therapeutic drug regimens (cohort 3);

iv) suffering from advanced* anti-PD-1/PD-L1 antibody-treated ccRCC or metastatic ccRCC with a clear cell component and who received 1-2 prior anti-angiogenic therapeutic drug regimens (cohort 4);

v) suffer from untreated anti-PD-1/PD-L1 metastatic TNBC (estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 negative) and who received 5 or fewer previous lines of therapy (cohort 5);

vi) suffering from untreated with antibodies to PD-1/PD-L1, advanced melanoma or metastatic melanoma, and who received no more than 2 previous treatment regimens for metastatic disease (cohort 6);

vii) suffering from advanced* PD-1/PD-L1 antibody-treated melanoma or metastatic melanoma, and who received no more than 2 prior treatment regimens for metastatic disease (cohort 7);

viii) suffer from untreated with anti-PD-1/PD-L1 antibodies, relapsed/refractory DLBCL, and who either progressed after autologous stem cell transplantation or are not candidates for it (cohorts 8 and 9) ix) suffer* from anti-PD-1/PD-L1 antibody-treated relapsed/refractory DLBCL, and who either progressed after autologous stem cell transplant or are not candidates for autologous stem cell transplant (Cohort 10).

Note: * treated with anti-PD-1/PD-L1 antibodies is defined as one who has tolerated or is currently undergoing therapy with anti-PD-1/PD-L1 antibodies (i.e., has not discontinued due to toxicity), and who has As part of the control of the disease, one of two things was observed:

a) CR/PR confirmed after repeat diagnostic imaging, or SD for at least 12 weeks from the first dose until disease progression. Disease progression must have been observed either during anti-PD-1/PD-L1 antibody therapy or within 12 weeks of the last dose;

b) SD or PR as best response followed by sustained response (no more than 70% below baseline) for 6 months during anti-PD-1/PD-L1 antibody therapy.

4. Functional status 0 or 1 according to the scale of the Eastern Joint Cancer Group.

5. Life expectancy of at least 3 months.

6. Adequate organ and bone marrow function according to the following:

(a) hemoglobin >9.0 g/dl;

(b) absolute neutrophil count >1.5 x 109/L;

(c) platelet count >75x109/l;

(d) serum creatinine <1.5x the upper limit of the normal range (ULN) or established glomerular filtration rate >50 ml/min/1.73 m ² ;

(f) total bilirubin <1.5x greater than ULN;

(f) aspartate aminotransferase and alanine aminotransferase (ALT) <3x greater than ULN or <5x greater than ULN, in the presence of liver metastases; and/or (g) alkaline phosphatase <2.5x ULN (or <5.0x ULN if liver or bone metastases are present).

7. Willingness and ability to comply with requirements regarding clinic visits and procedures related to the study.

8. Provide a signed informed consent form.

exclusion criteria. A patient who meets any of the following criteria will be excluded from the study.

1. Currently being treated in another study, or was in an investigational study of an experimental agent and received treatment, or has used an experimental device within 4 weeks of the first dose of investigational therapy, or has been treated with an approved systemic therapy for 3 weeks from the first dose of study therapy, or received any prior systemic therapy within 5 half-lives

- 67 039718 first dose of study therapy (whichever was longer). Only in the case of dose escalation cohorts to the maximum tolerated 2, 4, 7, 10 (treated with anti-PD1/PD-L1 antibodies), prior anti-PD-1/PD-L1 therapy was not allowed within 3 weeks of the first dose investigational therapy, regardless of half-life or drug approval status.

2. Previous treatment with any biologic or small molecule targeting LAG-3.

3. Radiation therapy within 2 weeks prior to randomization and no recovery to baseline after any radiation-induced AE.

4. Only escalation cohorts to the maximum tolerated dose: Other malignancy that is progressive or requiring intensive treatment, except for a non-melanoma skin malignancy that has undergone potentially definitive therapy or in situ cervical carcinoma, or any other tumor that was subjected to presumably effective treatment by radical local tumor control for at least 2 years prior to enrollment.

5. Untreated or active metastases to the central nervous system. Patients with previously treated central nervous system metastases may participate provided they are stable (i.e., no evidence of progression after imaging for at least 6 weeks prior to the first dose of study treatment, and any neurologic symptoms returned to baseline), and there is no evidence of new or spreading CNS metastases, and the patient does not require any systemic corticosteroids to control CNS metastases for 4 weeks prior to the first dose of REGN2810.

6. Encephalitis, meningitis, or uncontrolled fevers one year prior to informed consent.

7. Current or recent (within 5 years) evidence of significant autoimmune disease requiring treatment with systemic immunosuppressants, which may suggest a risk of developing irAE. The following are not exclusion parameters: vitiligo, childhood asthma that has been resolved, hypothyroidism requiring only hormone replacement therapy, type 1 diabetes, or psoriasis that does not require systemic treatment.

8. Corticosteroid therapy (>10 mg prednisone/day or equivalent) for 1 week prior to the first dose of study drug. Patients requiring a short course of steroids are not excluded.

9. Known from history or other evidence of interstitial lung disease or active non-infectious pneumonia (within the last 5 years).

10. Uncontrolled infection caused by the human immunodeficiency virus, infection caused by the hepatitis B or hepatitis C virus; or diagnosed immunodeficiency.

11. Active infection requiring systemic therapy.

12. Receipt of a live vaccine within 30 days prior to the scheduled start of study treatment.

13. Major surgical procedure, surgical biopsy, or significant traumatic injury within 4 weeks prior to screening.

14. Myocardial infarction within 9 months prior to the first dose of study therapy.

15. Previous allogeneic stem cell transplant.

16. Any medical condition that, in the opinion of the Investigator, would result in participation in the study not being in the best interest of the patient.

17. Documented allergic reaction or acute hypersensitivity reaction attributed to antibody-based medicinal products.

18. Known allergy to doxycycline or other tetracycline antibiotics.

19. Known psychiatric or substance abuse disorders that would preclude participation with study requirements.

20. Sexually active men or women of reproductive age who do not wish to use contraceptives during the study. Women who are pregnant, breastfeeding or expecting pregnancy, or men planning to conceive, within the intended duration of the study (screening visit 180 days after the last dose of study drug).

21. Sexually active males or females of reproductive age who are unwilling to use appropriate contraception prior to first treatment, during the study, and for at least 6 months after the last dose of study drug.

Investigated treatment options.

Monotherapy: mAb1 is administered on an outpatient basis by intravenous (IV) infusion over 30 minutes every 21 days for up to 51 weeks at the following monotherapy doses:

DL1: IV infusion of 1.0 mg/kg mAbl over 30 minutes every 21 days for 51 weeks.

DL2: IV infusion of 3.0 mg/kg mAb1 over 30 minutes every 21 days for 51 weeks.

DL4: IV infusion of 10 mg/kg mAb1 over 30 minutes every 21 days for 51 weeks.

DL-1m: IV infusion of 0.3 mg/kg mAb1 over 30 minutes every 21 days for 51 weeks (if needed).

Combined therapy.

In the case of combination therapy, the sequence of administration of the investigational medicinal product includes the administration of mAb1 first, and then REGN2810 on the same day. Study drugs are administered on an outpatient basis by IV infusion over 30 minutes every 21 days for up to 51 weeks. Planned combination regimens to be assigned include:

DL3: IV infusion of 1.0 mg/kg mAb1 and 3.0 mg/kg REGN2810 for 30 minutes every 21 days for 51 weeks;

DL5: IV infusion of 3.0 mg/kg mAb1 and 3.0 mg/kg REGN2810 for 30 minutes every 21 days for 51 weeks;

DL6: IV infusion of 10 mg/kg mAb1 and 3.0 mg/kg REGN2810 for 30 minutes every 21 days for 51 weeks;

DL-1c: IV infusion of 0.3mg/kg mAb1 and 3.0mg/kg REGN2810 over 30 minutes every 21 days for 51 weeks (as required).

Study endpoints.

primary endpoints.

In the dose escalation phase: incidence of DLT, adverse events (AEs; including those related to immunity), serious adverse events (SAEs), deaths, and laboratory abnormalities (grade 3 or higher according to common adverse event terminology criteria [ CTCAE]) and PK. In the dose escalation phase to maximally tolerated: proportion of patients with an objective response (ORR) based on RECIST 1.1 criteria (solid tumors) and Lugano (lymphoma).

secondary endpoints.

ORR based on criteria for assessing the response of solid tumors in immunotherapy (irRECIST); best overall response (BOR), duration of response (DOR), disease control rate, and progression-free survival (PFS) based on RECIST, irRECIST, and Lugano criteria; AE, including those associated with immunity, SAE, deaths and laboratory abnormalities (grade 3 or higher according to CTCAE); PK and ADA.

Procedures and assessments.

The safety and tolerability of mAb1 alone or in combination with REGN2810 was monitored using clinical assessment of AE and repeated clinical assessment measurements, including vital signs (temperature, blood pressure, heart rate and respiratory rate), physical examinations (complete and limited) , 12-lead electrocardiogram (ECG), and laboratory testing including routine CBC, biochemistry, and urinalysis.

Blood samples were collected for the determination of functional mAb1 and functional REGN2810 in serum and ADA (anti-mAb1 or anti-REGN2810 antibodies). Serum and plasma samples were collected for analysis of additional biomarkers. The considered pharmacodynamic, predictive, and prognostic biomarkers associated with mAb1 and REGN2810 treatment exposure, clinical activity, or comorbidity were examined in serum, plasma, peripheral blood mononuclear cells (PBMCs), and tumor tissue. Antitumor activity was assessed using CT and MRI. A genomic DNA sample was collected from patients who agreed to optional participation in the pharmacogenomics substudy.

Results.

The mAb1 administration within the study is expected to be safe and well tolerated by patients with advanced malignancies. Combination with 3mg/kg REGN2810 is expected to provide tumor regression as measured by ORR in patients with solid tumors such as non-small cell lung cancer, melanoma, clear cell kidney cancer, B-cell lymphoma, or breast cancer.

The scope of the present invention is not limited to the specific embodiments described herein. In fact, various modifications of the present invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be covered by the scope of the appended claims.

Claims (32)

CLAIM 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a protein encoded by human lymphocyte activation gene-3 (LAG3), wherein the antibody or antigen-binding fragment contains a heavy chain complementarity determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 420, HCDR2 containing the amino acid sequence of SEQ ID NO: 422 and HCDR3 containing the amino acid sequence of SEQ ID NO: 424, light chain CDR 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 428, LCDR2 containing the amino acid sequence of SEQ ID NO: 430 and LCDR3 containing the amino acid sequence of SEQ ID NO: 432. 2. An isolated antibody or antigen-binding fragment thereof according to claim 1, containing a heavy chain variable region (HCVR) and/or a light chain variable region (LCVR), where the HCVR contains: (i) an amino acid sequence of SEQ ID NO: 418, (ii) an amino acid sequence that shares at least 90% identity with SEQ ID NO: 418, (iii) an amino acid sequence that shares at least 95% identity with SEQ ID NO: 418 ; or (iv) the amino acid sequence of SEQ ID NO: 418 with no more than 5 amino acid substitutions; and LCVR contains: (i) an amino acid sequence of SEQ ID NO: 426, (ii) an amino acid sequence that shares at least 90% identity with SEQ ID NO: 426, (iii) an amino acid sequence that shares at least 95% identity with SEQ ID NO: 426 ; or (iv) the amino acid sequence of SEQ ID NO: 426 with no more than 5 amino acid substitutions. 3. An isolated antibody or antigen-binding fragment thereof that specifically binds to the human LAG3 protein, wherein the antibody or antigen-binding fragment thereof comprises HCVR having the amino acid sequence of SEQ ID NO: 418 and LCVR having the amino acid sequence of SEQ ID NO: 426. 4. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has one or more properties selected from the group consisting of: (a) bind monomeric human LAG3 with a dissociation constant (KD) for equilibrium binding of less than about 1.5 nM as measured in a surface plasmon resonance assay at 25°C; (b) bind monomeric human LAG3 with a KD of less than about 2 nM as measured in a surface plasmon resonance assay at 37°C; (c) bind dimeric human LAG3 with a KD of less than about 20 pM as measured in a surface plasmon resonance assay at 25°C; (d) bind dimeric human LAG3 with a KD of less than about 90 pM as measured in a surface plasmon resonance assay at 37°C; (e) bind to an hLAG3-expressing cell with an EC _{50 of} less than about 2 nM as measured in a flow cytometry assay; (f) bind to an mfLAG3-expressing cell with an EC _{50 of} less than about 2.3 nM as measured in a flow cytometry assay; (g) block hLAG3 binding to human MHC class II with an IC _{50 of} less than about 20 nM as determined by cell adhesion assay; (h) block hLAG3 binding to mouse MHC class II with an IC _{50 of} less than about 15 nM as determined by cell adhesion assay; (i) block hLAG3 binding to MHC class II by more than 90% as determined by cell adhesion assay; (j) provide relief from LAG3 mediated inhibition of T cell activity with an EC50 of less than about 2.5 nM as determined by a luciferase reporter gene assay; and (k) bind to activated CD4+ and CD8+ T cells with an EC _{50 of} less than about 1.2 nM, as determined by fluorescent assay. 5. An isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 418 with no more than 5 amino acid substitutions. 6. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the antibody or antigen-binding fragment thereof contains an LCVR containing an amino acid sequence - 70 039718 the validity of SEQ ID NO: 426 with no more than 5 amino acid substitutions. 7. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO: 418 with no more than 5 amino acid substitutions and an LCVR containing the amino acid sequence of SEQ ID NO: 426 with no more than 5 amino acid substitutions. 8. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, containing an HCVR having the amino acid sequence of SEQ ID NO: 418. 9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, containing an LCVR having the amino acid sequence of SEQ ID NO: 426. 10. An isolated antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence pair HCVR/LCVR SEQ ID NO: 418/426. 11. An isolated antibody according to claim 10, comprising a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 577. 12. An isolated antibody according to claim 10, comprising a heavy chain and a light chain, wherein the light chain contains the amino acid sequence of SEQ ID NO: 578. 13. An isolated antibody that specifically binds to the LAG3 protein, wherein the antibody comprises a heavy chain/light chain amino acid sequence pair of SEQ ID NO: 577/578. 14. A pharmaceutical composition for inhibiting tumor growth in a subject, wherein the tumor or tumor cell contains cells expressing LAG3, the composition comprises an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-13 and a pharmaceutically acceptable carrier or diluent. 15. An isolated polynucleotide molecule comprising a polynucleotide sequence that encodes an HCVR antibody as set forth in any one of claims 1-10. 16. An isolated polynucleotide molecule comprising a polynucleotide sequence that encodes an LCVR antibody as set forth in any one of claims 1-10. 17. An isolated polynucleotide molecule containing a polynucleotide sequence that encodes the heavy chain of an antibody according to claim 11. 18. An isolated polynucleotide molecule containing a polynucleotide sequence that encodes a light chain of an antibody according to claim 12. 19. Expression vector containing polynucleotide molecules according to claims 15 and 16 or 17 and 18. 20. A host cell containing the expression vector of claim 19 for antibody production. 21. A method for inhibiting the growth of a tumor or tumor cell in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the tumor is present in a subject with a disease or disorder selected from the group consisting of from hematopoietic tissue malignancy, brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colon and rectal cancer, mesothelioma, B-cell lymphoma and melanoma. 22. The method of claim 21 wherein the tumor is a primary or recurrent tumor. 23. The method of claim 21 wherein the tumor is an advanced tumor. 24. The method according to any one of claims 21-23, wherein the antibody or antigen-binding fragment thereof is administered as an initial dose, then as one or more doses of the second line, in which each dose of the second line is administered 1-4 weeks after the nearest previous doses. 25. The method of claim 24, wherein the antibody or antigen-binding fragment is administered at a dose of about 1, 3, or 10 mg/kg of the subject's body weight. 26. The method according to any one of claims 21-25, wherein the antibody or antigen-binding fragment thereof is administered to the subject in combination with a second therapeutic agent selected from the group consisting of a PD-1 inhibitor, a CTLA inhibitor, an antibody to a tumor-specific antigen, an antibody to an antigen a virus-infected cell, a PD-L1 inhibitor, a CD20 inhibitor, an anti-CD20 and CD3 bispecific antibody, a dietary supplement such as an antioxidant, a VEGF antagonist, a chemotherapeutic agent, a cytotoxic agent, radiation, an NSAID, a corticosteroid, and any other therapy suitable for attenuating at least one symptom associated with the disease or disorder. 27. The method of claim 26 wherein the second therapeutic agent is a PD1 inhibitor selected from the group consisting of REGN2810, nivolumab, and pembrolizumab. 28. The method of claim 26, wherein the PD-1 inhibitor is administered at a dose of 1, 3, or 10 mg/kg of the subject's body weight. 29. The method of any one of claims 21-28, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. 30. A method of treating a disease or disorder that is treatable by antagon - 71 039718 nistically affecting LAG3, comprising administering to a subject in need a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 13, in which the disease or disorder (a) is selected from the group consisting of a malignant neoplasm of hematopoietic tissue, brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colon and rectal cancer, mesothelioma, B-cell lymphoma and melanoma or (b) is a chronic viral infection caused by a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), papillomavirus human (HPV), lymphocytic choriomeningitis virus (LCMV) and simian immunodeficiency virus (SIV). 31. An isolated antibody or antigen-binding fragment thereof that specifically binds to human LAG3 protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain/light chain amino acid sequence pair of SEQ ID NO: 577/578. 32. A pharmaceutical composition containing an isolated antibody or antigen-binding fragment thereof, which contains a heavy chain/light chain amino acid sequence pair of SEQ ID NO: 577/578 and a pharmaceutically acceptable carrier or diluent.