isolated antibodies, isolated nucleic acid, host cell, method for treating an individual having canc

专利摘要:
the present invention relates to anti-lag3 antibodies, methods of producing these molecules and methods of using them.
公开号:BR112019018779A2
申请号:R112019018779
申请日:2018-04-03
公开日:2020-05-05
发明作者:Zwick Adrian；Klein Christian；Fischer Jens；Codarri Deak Laura；Alexander Aaron Weber Patrick；Dengl Stefan；Seeber Stefan
申请人:Hoffmann La Roche；
IPC主号:

专利说明:

“ISOLATED ANTIBODIES, ISOLATED NUCLEIC ACID, HOSTING CELL, METHOD FOR TREATING AN INDIVIDUAL HAVING CANCER AND TO PRODUCE AN ANTIBODY, PHARMACEUTICAL FORMULATION AND USE OF THE ANTIBODY”
Field of the Invention [001] The present invention relates to anti-LAG3 antibodies, methods of producing these molecules and methods of using them.
Background of the Invention [002] The lymphocyte activation gene 3 (LAG3 or CD223) was initially discovered in an experiment designed to selectively isolate molecules expressed in an IL-2-dependent NK cell line (Triebel F et al., Cancer Lett 235 (2006), 147-153). LAG-3 is a unique transmembrane protein, with structural homology to CD4, with four domains similar to the extracellular immunoglobulin superfamily (D1-D4). The distal IgG domain of the membrane contains a short sequence of amino acids, the so-called extra loop that is not found in other proteins of the IgG superfamily. The intracellular domain contains a unique amino acid sequence (KIEELE) that is necessary for LAG-3 to have a negative effect on T cell function. LAG-3 can be cleaved into the binding peptide (CP) by metalloproteases to generate a soluble form, which is detectable in serum. Like CD4, the LAG3 protein binds to MHC class II molecules, however with greater affinity and at a different site than CD4 (Huard et al. Proc. Natl. Acad. Sci. USA 94 (1997), 5744 -5749). LAG3 is expressed by T cells, B cells, NK cells and plasmacytoid dendritic cells (pDCs) and is up-regulated after T cell activation. It modulates T cell function as well as T cell homeostasis. Conventional tees that are anergic or have impaired functions express LAG3. LAG3 ⁺ T cells are enriched at tumor sites and
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2/126 during chronic viral infections (Sierro et al. Expert Opin. Ther. Targets 15 (2011), 91-101). LAG3 has been shown to play a role in the depletion of CD8 T cells (Blackburn et al. Nature Immunol. 10 (2009), 29-37). Thus, there is a need for antibodies that antagonize LAG3 activity and can be used to generate and restore the immune response to tumors.
[003] Monoclonal antibodies to LAG3 have been described, for example, in WO 2004/078928, in which a composition comprising antibodies that specifically bind to CD223 and a cancer vaccine is claimed. WO 2010/019570 discloses human antibodies that bind to LAG3, for example, antibodies 25F7 and 26H10. US 2011/070238 refers to a cytotoxic anti-LAG3 antibody useful in the treatment or prevention of organ transplant rejection and autoimmune disease. WO 2014/008218 describes LAG3 antibodies with optimized functional properties (i.e., reduced deamidation sites) compared to the 25F7 antibody. In addition, LAG3 antibodies are disclosed in WO 2015/138920 (e.g. BAP050), WO 2014/140180, WO 2015/116539, WO 2016/028672, WO 2016/126858, WO 2016/200782 and WO 2017/015560.
Brief Description of the Invention [004] The invention provides anti-LAG3 antibodies and methods of using them.
[005] The invention provides an isolated antibody that binds to human LAG3, wherein the antibody comprises
A) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of
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3/126
SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or
B) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or
C) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or
D) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or
E) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35; (d) HVR-L1 comprising the amino acid sequence
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4/126 of SEQ ID NO: 36; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
[006] The invention further provides an isolated antibody that binds to human LAG3, wherein the antibody comprises
A) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or
B) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or
C) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21 and (iii) HVR-L3 comprising the
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5/126 amino acid sequence of SEQ ID NO: 22; or
D) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or
E) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 35; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
[007] The invention further provides an isolated antibody that binds to human LAG3, wherein the antibody
i) comprises a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8;
ii) comprises a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16;
iii) comprises a VH sequence of SEQ ID NO: 23 and a VL sequence of SEQ ID NO: 24;
iv) comprises a VH sequence of SEQ ID NO: 31 and a VL sequence of SEQ ID NO: 32; or
v) comprises a VH sequence of SEQ ID NO: 39 and a
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6/126 VL sequence of SEQ ID NO: 40.
[008] The invention further provides isolated antibody that binds to human LAG3, wherein the antibody:
i) competes to bind LAG3 with an anti-LAG3 antibody comprising VH with the amino acid sequence of SEQ ID NO: 7 and VL with the amino acid sequence of SEQ ID NO: 8 and / or ii) binds to a LAG3 human and cinomolgo; and / or iii) inhibits the binding of MHC-II expressed in human A375 tumor cells; and / or iv) increases the release of granzyme B or IL-2 in a mixed lymphocyte reaction (mMLR) assay.
[009] In one embodiment, the anti-LAG3 antibody according to the invention is a monoclonal antibody.
[0010] In one embodiment, the anti-LAG3 antibody according to the invention is a human, humanized or chimeric antibody.
[0011] In one embodiment, the anti-LAG3 antibody according to the invention, which is an antibody component that binds to LAG3.
[0012] In one embodiment, the anti-LAG3 antibody according to the invention, is the Fab fragment.
[0013] The invention provides an isolated nucleic acid that encodes the antibody according to any of the preceding claims.
[0014] The invention provides a host cell comprising that nucleic acid.
[0015] The invention provides a method for producing an antibody that comprises culturing the host cell so that the antibody is produced.
[0016] The invention provides this method for producing a
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7/126 antibody, further comprising recovering the antibody from the host cell.
[0017] The invention provides a pharmaceutical formulation comprising the antibody described herein and a pharmaceutically acceptable carrier.
[0018] The invention provides the antibody described herein for use as a medicament.
[0019] The invention provides the antibody described herein for use in the treatment of cancer.
[0020] The invention provides the use of the antibody described herein in the manufacture of a medicament. In one embodiment the drug is for treating cancer, to treat or slow the progression of an immune-related disease, such as tumor immunity, or to stimulate an immune response or function, such as T-cell activity .
[0021] The invention provides a method for treating an individual with cancer, comprising administering to the individual an effective amount of the antibody described herein.
[0022] The antibodies of the present invention show valuable properties to induce the release of Granzyme B, release of IFN-γ and secretion of IL-2 by human CD4 T cells and therefore can stimulate the immune response through T cells (functions reinforced effectors of tumor antigen-specific T cells) alone or in combination with PD1 inhibitors.
Brief Description of the Figures [0023] Figure 1: Effect of anti-LAG3 antibodies on the release of cytotoxic Granzyme B and secretion of IL-2 by human CD4 T cells co-cultured with mature allogeneic dendritic cells Figure 1 A: Granzyme B secretion; Figure 1B: IL-2 secretion.
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8/126 [0024] Figure 2: Effect of anti-LAG3 antibodies on the release of cytotoxic Granzyme B by human CD4 T cells co-cultured with a B cell lymphoblatoid cell line (ARH77).
[0025] Figure 3: Effect of anti-LAG3 antibodies on Treg suppression of Granzima B and IFN-γ release by human CD4 T cells co-cultured with irradiated allogeneic PBMC. Figure 3A: Granzima B release; Figure 3B: IFN-γ release.
Detailed Description of the Invention [0026] A "human acceptor structure" for the purposes here is a structure comprising the amino acid sequence of a light chain variable domain (VL) structure or a derived heavy chain variable domain (VH) structure of a human immunoglobulin structure or a human consensus structure, as defined below. An acceptor human structure "derived from" a human immunoglobulin structure or a human consensus structure may comprise the same amino acid sequence therein, or may contain changes in the amino acid sequence. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the human VL acceptor structure is identical in sequence to the human immunoglobulin VL structure sequence or human consensus structure sequence.
[0027] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (for example, antibody and antigen). The affinity of a
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9/126 molecule X by its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
[0028] An "affinity matured" antibody refers to an antibody with one or more changes in one or more hypervariable regions (HVRs), compared to an original antibody that does not have such changes, resulting in an improvement in antibody affinity to the antigen.
[0029] The term “LAG3”, as used here, refers to any LAG3 native to any vertebrate source, including mammals such as primates (eg humans) and rodents (eg rats and mice), unless otherwise indicated. contrary. The term encompasses "complete", unprocessed LAG3, as well as any form of LAG3 resulting from processing in the cell. The term also covers naturally occurring variants of LAG3, for example splice variants or allelic variants. In a preferred embodiment, the term "LAG3" refers to human LAG3. The amino acid sequence of an exemplarily processed LAG3 (without signal sequences) shown in SEQ ID NO: 54. The amino acid sequence of an exemplary Extracellular Domain (ECD) LAG is shown in SEQ ID NO: 55.
[0030] The terms "anti-LAG3 antibody" and "an antibody that binds to LAG3" refer to an antibody that is capable of binding to LAG3 with sufficient affinity for the antibody to be useful as a diagnostic agent and / or therapeutic in targeting LAG3. In one embodiment, the extent of binding of an anti-LAG3 antibody to an unrelated non-LAG3 protein is less than about 10% of the antibody's binding to LAG3 as measured, for example, by a radioimmunoassay (RIA). In certain
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10/126 embodiments, an antibody that binds to LAG3 has a dissociation constant (Kd) of <1 μΜ, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM or <0,001 nM (e.g., 10 ^"8 M or less, for example 10" ⁸ M to 10 ^"13 M, for example, 10" ⁹ M to 10 ^"13 M). In certain embodiments, an anti-LAG3 antibody binds to an LAG3 epitope that is conserved among LAG3 of different species. In a preferred embodiment, an "anti-LAG3 antibody", "an antibody that specifically binds to human LAG3" and "an antibody that binds to human LAG3" refers to an antibody that specifically binds to the LAG3 antigen human or its extracellular domain (ECD) with a binding affinity of a Kode value of 1.0 x 10 ' ⁸ mol / L or less, in an embodiment of a Kode value of 1.0 x 10' ⁹ mol / L or less, in an embodiment of a Kode value of 1.0 x 10 ' ⁹ mol / L to 1.0 x 10' ¹³ mol / L. In this context, binding affinity is determined with a standard binding assay, such as the surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden), for example, using the LAG3 extracellular domain.
[0031] The term "antibody" is used here in the broadest sense and encompasses several antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (for example, bispecific antibodies) and antibody fragments as long as exhibit binding activity to the desired antigen.
[0032] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab ', Fab'SH, F (ab') 2; diabodies; linear antibodies; single chain antibody molecules (for example, scFv); and multispecific antibodies formed from antibody fragments.
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11/126 [0033] The term "epitope" denotes the location on an antigen, protein or non-protein, to which an anti-LAG3 antibody binds. Epitopes can be formed either from extensions of contiguous amino acids (linear epitope) or comprising non-contiguous amino acids (conformational epitope), for example, coming in spatial proximity due to the folding of the antigen, that is, by the tertiary folding of a protein antigen . Linear epitopes are typically still linked by an anti-LAG3 antibody after exposure of the protein antigen to denaturing agents, while conformational epitopes are typically destroyed by treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7 or 8 to 10 amino acids in a single spatial conformation.
[0034] Scanning for antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be done using routine methods in the art such as, for example, without limitation scanning for alanine, patches of peptide (see Meth. Mol. Biol. 248 (2004) 443-463), peptide divination analysis, epitope excision, epitope extraction, chemical antigen modification (see Prot. Sci. 9 (2000) 487-496) and cross blocking (see “Antibodies”, Cold Spring Harbor Press, Cold Spring Harb., NY).
[0035] The Antigen Structure-Based Antibody Profile (ASAP), also known as Modified Assisted Profile (MAP), allows a multiplicity of monoclonal antibodies specifically linked to LAG3 to be grouped based on the binding profile of each of the antibodies in the crowd to chemically or enzymatically modified antigen surfaces (see, for example, US 2004/0101920). The antibodies in each group bind to the same epitope, which may be a single epitope, distinctly different or partially overlapping the epitope represented by another group.
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12/126 [0036] Competitive binding can also be used to easily determine whether an antibody binds to the same LAG3 epitope as, or competes for binding with, a reference anti-LAG3 antibody. For example, an “antibody that binds to the same epitope” as a reference anti-LAG3 antibody refers to an antibody that blocks the binding of the reference anti-LAG3 antibody to its antigen in a 50% competition test or more and, conversely, the reference antibody blocks the binding of the antibody to its antigen in a competition assay by 50% or more. Also for example, to determine whether an antibody binds to the same epitope as a reference anti-LAG3 antibody, the reference antibody is allowed to bind to LAG3 under saturation conditions. After removing excess reference anti-LAG3 antibody, the ability of an anti-LAG3 antibody in the matter to bind to LAG3 is assessed. If the anti-LAG3 antibody is able to bind to LAG3 after saturation binding of the reference anti-LAG3 antibody, it can be concluded that the anti-LAG3 antibody in question binds to an epitope other than the reference anti-LAG3 antibody . But, if the anti-LAG3 antibody in question is unable to bind to LAG3 after saturation binding of the reference anti-LAG3 antibody, then the anti-LAG3 antibody in question can bind to the same epitope as the epitope bound by reference LAG3 antibody. To confirm that the antibody in question binds to the same epitope or is only prevented from binding for steric reasons, routine experimentation (for example, peptide mutations and binding analyzes using ELISA, RIA, surface plasmon resonance, flow cytometry or any other qualitative antibody binding assay available in the art). This assay should be performed in two preparation times, that is, with both antibodies being the saturating antibody. If, in both preparation times, only the first antibody (saturant) is able to bind to LAG3, then it can be concluded that
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13/126 the anti-LAG3 antibody in question and the reference anti-LAG3 antibody compete for binding to LAG3.
[0037] In some embodiments, two antibodies are considered to bind to the same or to an overlapping epitope if an excess of 1.5, 10, 20 or 100 fold of one antibody inhibits the binding of the other by at least 50 %, at least 75%, at least 90% or even 99% or more, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 50 (1990) 1495-1502).
[0038] In some embodiments, two antibodies are considered to bind to the same epitope if essentially all of the amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of the other.
[0039] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the rest of the heavy and / or light chain is derived from a source or different species.
[0040] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), for example, IgG-ι, lgG2, IgGa, lgG4, IgAi, and lgA2. In certain embodiments, the antibody is of the lgG4 isotype with the S228P mutation in the hinge region to improve the stability of the lgG4 antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
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14/126 [0041] The term "cytotoxic agent", as used herein, refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (for example, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes of Lu ); chemotherapeutic agents or drugs (for example, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and the various anti-tumor or anti-cancer agents disclosed below.
[0042] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the isotype of the antibody. Examples of antibody effector functions include: binding to C1q and complement-dependent cytotoxicity (CDC); Connection to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (eg, B cell receptor); and activation of B cells.
[0043] An "effective amount" of an agent, for example, a pharmaceutical formulation, refers to an effective amount, in dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
[0044] The term "Fc region" here is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, an Fc region of the chain
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15/126 heavy human IgG extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, C-terminal lysine (Lys447) from the Fc region may or may not be present. In one embodiment, the anti-Lag3 antibody as described herein is of the lgG1 isotype and comprises a constant heavy chain domain of SEQ ID NO: 51 or SEQ ID NO: 52. In one embodiment it comprises adding lysine C- terminal (Lys447). In one embodiment, the anti-Lag3 antibody as described herein from the lgG4 isotype and comprises a constant heavy chain domain of SEQ ID NO: 53. In one embodiment, it further comprises C-terminal lysine (Lys447). Unless otherwise specified here, the numbering of amino acid residues in the Fc region or constant region according to the EU numbering system, also called the EU index, as described in Kabat et al. Protein Sequences of Immunological Interest, 5- Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
[0045] "Framework" or "FR" refers to residues of the variable domain that are not residues of the hypervariable region (HVR). The RF of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Therefore, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.
[0046] The terms "full length antibody", "intact antibody" and "full antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains containing an Fc region as defined here.
[0047] The terms "host cell", "host cell lineage" and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acid has been
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16/126 introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the transformed primary cell and the progeny derived therefrom without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to a parent cell, but they may contain mutations. The mutant progeny that has the same biological function or activity as screened or selected in the originally transformed cell is included here.
[0048] A "human antibody" is one that has an amino acid sequence that corresponds to an antibody produced by a human or human cell or derived from a non-human source that uses repertoires of human antibodies or other coding sequences for human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. In certain embodiments, a human antibody is derived from a transgenic non-human mammal, for example, a mouse, a rat or a rabbit. In certain embodiments, a human antibody is derived from a hybridoma cell line.
[0049] A "human consensus framework" is a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH structure sequences. Generally, the selection of human immunoglobulin VL or VH sequences from a subset of variable domain sequences. Generally, the sequence subgroup is a subgroup as in Kabat et al., Sequences of Protein of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 13. In one embodiment, for the VL, the subgroup is kappa I subgroup as in Kabat et al. supra. In one embodiment, for VH, the
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17/126 subgroup is subgroup III as in Kabat et al. supra.
A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, where all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, for example, a non-human antibody, refers to an antibody that has undergone humanization.
[0051] The term "hypervariable region" or "HVR" as used here refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or they form structurally defined loops (“hypervariable loops”) and / or contain residues from contact with the antigen (“antigenic contacts”). Antibodies generally comprise six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Examples of HVRs include:
(a) hypervariable loops that occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));
(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigenic contacts that occur in the residues of
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18/126 amino acids 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93101 (H3) (MacCalIum et al., J Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b) and / or (c), including amino acid residues of HVR 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (HI), 26-35b (HI), 49-65 (H2), 93-102 (H3) and 94-102 (H3).
[0052] In one embodiment, the HVR residues comprise those identified in the Description of the amino acid sequences below.
[0053] Unless otherwise indicated, HVR residues and other residues in the variable domain (for example, RF residues) are numbered here according to Kabat et al. supra.
[0054] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.
[0055] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (for example, cows, sheep, cats, dogs and horses), primates (for example, humans and non-human primates, such as monkeys), rabbits and rodents (for example, mice and rats). In certain embodiments, the individual or subject is a human.
[0056] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, an antibody is purified to a purity greater than 95% or 99% as determined, for example, electrophoretic (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (eg, Ion exchange or reverse phase HPLC). For review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).
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19/126 [0057] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
[0058] "Isolated nucleic acid encoding an antiLAG3 antibody" refers to one or more nucleic acid molecules encoding heavy and light antibody chains (or fragments thereof), including such nucleic acid molecule (s) in a single vector or separate vectors, and such nucleic acid molecule (s) present in one or more locations in a host cell.
[0059] The term "monoclonal antibody", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, mutations that occur naturally or that arise during the production of a monoclonal antibody preparation, these variants being generally present in smaller amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation directed against a single determinant in an antigen. Thus, the "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, antibodies
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Monoclonal 20/126 to be used according to the present invention can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods and methods using transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies are described herein.
[0060] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (for example, a cytotoxic moiety) or radiolabel. The naked antibody can be present in a pharmaceutical formulation.
[0061] "Native antibodies" refer to naturally occurring immunoglobulin molecules with variable structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are linked by disulfide. From the N to the C terminal, each heavy chain has a variable region (VH), also called a heavy variable domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from terminal N to C, each light chain has a variable region (VL), also called a light variable domain or a light chain variable domain, followed by a constant light domain (CL). The light chain of an antibody can be attributed to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.
[0062] The term “package leaflet” is used to refer to the instructions usually included in commercial packaging of therapeutic products, which contain information on the indications, use, dosage, administration, combination therapy, contraindications and / or warnings regarding use of such therapeutic products.
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21/126 [0063] “Percentage (%) of amino acid sequence identity” relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide sequence of reference, after aligning the sequences and inserting gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a number of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign ( DNASTAR) or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the total length of the sequences being compared. For the purposes described here, however, the% amino acid sequence identity values are generated using the ggsearch program from the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was written by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85: 2444-2448; W. R. Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266: 227-258; and Pearson et. al. (1997) Genomics 46: 24-36 and is publicly available at http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml. Alternatively, a public server accessible at http://fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare sequences, using the ggsearch program (global protein: protein) and standard options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure that global, not local, alignment is performed. The percentage
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22/126 amino acid identity is given in the output alignment header.
[0064] The term "pharmaceutical formulation" refers to a preparation that is such that it allows the biological activity of an active ingredient contained therein to be effective and that it does not contain additional components that are unacceptably toxic to a subject to whom the formulation would be administered.
[0065] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is not toxic to an individual. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.
[0066] As used here, "treatment" (and its grammatical variations, such as "treating" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed to prophylaxis or during the course of clinical pathology. The desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, improving or palliating disease and remission or better prognosis. In some embodiments, the antibodies of the invention are used to slow the development of a disease or to slow the progression of a disease.
[0067] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to the antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved structural regions (FR) and three hypervariable regions (HVR). (To see,
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23/126 for example, Kindt et al. Kuby Immunology, 6 ^th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to the antigen to search for a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).
[0068] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to here as "expression vectors".
I. COMPOSITIONS AND METHODS [0069] In one aspect, the invention provides isolated antibodies that bind to LAG3.
[0070] In certain embodiments, antibodies are provided that bind to human LAG3. The antibodies of the invention are useful, for example, for the diagnosis or treatment of cancer, to treat or delay the progression of an immune-related disease, such as tumor immunity, or to stimulate an immune response or function, such as T cell activity; or for use as an immunostimulating / stimulating agent for Granzyme B (GrzB), interferon-gamma (IFN-gamma) and or secretion / release of interleukin 2 (IL-2).
A. Exemplary Anti-LAG3 Antibodies [0071] In certain embodiments, an anti-LAG3 is provided
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24/126 where the antibody:
i) competes for binding to LAG3 with an anti-LAG3 antibody (comprising the VH and VL of aLAG3 (0414)) comprising the VH with the amino acid sequence of SEQ ID NO: 7 and VL with the amino acid sequence of SEQ ID NO : 8 and / or ii) binds to a human and cinomolg LAG3; and / or iii) inhibits the binding of MHC-II expressed in tumor cells
Human A375; and / or iv) increases the release of Granzyme B or IL-2 in a mixed lymphocyte reaction (mMLR) assay (as shown in Example 3).
[0072] In one aspect, the invention provides an antiLAG3 antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
[0073] In one aspect, the invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In another embodiment, the antibody comprises HVRH3 comprising the amino acid sequence of SEQ ID NO: 1 and HVR-L3
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25/126 comprising the amino acid sequence of SEQ ID NO: 6. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3.
[0074] In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1. (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) HVR -L2
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26/126 comprising the amino acid sequence of SEQ ID NO: 5 and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
[0076] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 5; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 6.
[0077] In any of the above embodiments, an anti-LAG3 antibody is human or humanized. In one embodiment, an anti-LAG3 antibody comprises HVRs as in any of the above embodiments, and further comprises a human acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.
[0078] In another aspect, an anti-LAG3 antibody comprises a heavy chain variable domain (VH) sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions with respect to the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 7. In certain embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie
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27/126 is, in FRs). Optionally, the anti-LAG3 antibody comprises the VH sequence in SEQ ID NO: 7, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3.
[0079] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, a VL sequence having at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions with respect to the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 8. In certain embodiments, substitutions, insertions or deletions occur in regions outside of HVRs (i.e., FRs). Optionally, the anti-LAG3 antibody comprises the VL sequence in SEQ ID NO: 8, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
[0080] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a VH as in any of the forms of
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28/126 embodiment provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, including post-translational modifications of those sequences.
[0081] In another aspect, the invention provides an antibody that binds to the same epitope as an anti-LAG3 antibody provided here. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-LAG3 antibody comprising a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8. In certain forms of In this embodiment, an antibody is provided that binds to an epitope of the E3 cluster epitope within LAG3 (see example 2).
[0082] In one aspect, the invention provides an antiLAG3 antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.
[0083] In one aspect, the invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11. In another embodiment, the antibody comprises
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12/29
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10. In another embodiment, the antibody comprises (a) HVR- H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11.
[0084] In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9 (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10 and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from (i) HVR-L1
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30/126 comprising the amino acid sequence of SEQ ID NO: 12, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13 and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 .
[0086] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 10; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 11; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 13; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 14.
[0087] In any of the above embodiments, an anti-LAG3 antibody is human or humanized. In one embodiment, an anti-LAG3 antibody comprises HVRs as in any of the above embodiments and further comprises a human acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.
[0088] In another aspect, an anti-LAG3 antibody comprises a heavy chain variable domain (VH) sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions with respect to the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 15. In certain embodiments,
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31/126 substitutions, insertions or deletions occur in regions outside the HVRs (ie, in the FRs). Optionally, the anti-LAG3 antibody comprises the VH sequence in SEQ ID NO: 15, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11.
[0089] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 16. In certain embodiments, a VL sequence having at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions with respect to the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 16. In certain embodiments, substitutions, insertions or deletions occur in regions outside of HVRs (i.e., FRs). Optionally, the anti-LAG3 antibody comprises the VL sequence in SEQ ID NO: 16, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.
[0090] In another aspect, an anti-LAG3 antibody is provided, in
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32/126 that the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, including post-translational modifications of those sequences.
[0091] In another aspect, the invention provides an antibody that binds to the same epitope as an anti-LAG3 antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-LAG3 antibody comprising a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16. In certain embodiments In this embodiment, an antibody is provided that binds to an epitope of the E3 cluster epitope within LAG3 (see example 2).
[0092] In one aspect, the invention provides an antiLAG3 antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22.
[0093] In one aspect, the invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence
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33/126 of SEQ ID NO: 19. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22. In in a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19.
[0094] In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22.
[0095] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17 (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18 and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) a VL domain comprising at least one, at least one
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34/126 two or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21 and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22.
[0096] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 20; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 21; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 22.
[0097] In any of the above embodiments, an anti-LAG3 antibody is human or humanized. In one embodiment, an anti-LAG3 antibody comprises HVRs as in any of the above embodiments and further comprises a human acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.
[0098] In another aspect, an anti-LAG3 antibody comprises a heavy chain variable domain (VH) sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions with respect to the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced,
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35/126 inserted and / or deleted in SEQ ID NO: 23. In certain embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in the FRs). Optionally, the anti-LAG3 antibody comprises the VH sequence in SEQ ID NO: 23, including post-translational modifications of that sequence. In a particular embodiment, VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19.
[0099] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 24. In certain embodiments, a VL sequence having at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions with respect to the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 24. In certain embodiments, substitutions, insertions or deletions occur in regions outside of HVRs (i.e., FRs). Optionally, the anti-LAG3 antibody comprises the VL sequence in SEQ ID NO: 24, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22.
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36/126 [00100] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 23 and SEQ ID NO: 24, respectively, including post-translational modifications of those sequences.
[00101] In another aspect, the invention provides an antibody that binds to the same epitope as an anti-LAG3 antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-LAG3 antibody comprising a VH sequence of SEQ ID NO: 23 and a VL sequence of SEQ ID NO: 24. In certain embodiments embodiment, an antibody is provided that binds to an epitope of the epitope of cluster E3 within LAG3 (see example 2).
[00102] In one aspect, the invention provides an anti-LAG3 antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.
[00103] In one aspect, the invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-H3 comprising the
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37/126 amino acid sequence of SEQ ID NO: 27. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27 , HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27.
[00104] In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.
[00105] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25 (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26 and (iii) HVR-H3
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38/126 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28, (ii) HVR- L2 comprising the amino acid sequence of SEQ ID NO: 29 and (c) HVR-L3 amino acid sequence of SEQ ID NO: 30.
[00106] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 26; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 27; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 28; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 29; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 30.
[00107] In any of the above embodiments, an anti-LAG3 antibody is human or humanized. In one embodiment, an anti-LAG3 antibody comprises HVRs as in any of the above embodiments and further comprises a human acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.
[00108] In another aspect, an anti-LAG3 antibody comprises a heavy chain variable domain (VH) sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or
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39/126 deletions from the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 31. In certain embodiments, substitutions, insertions or deletions occur in regions outside of HVRs (that is, in FRs). Optionally, the anti-LAG3 antibody comprises the VH sequence in SEQ ID NO: 31, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27.
[00109] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 32. In certain embodiments, a VL sequence having at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions with respect to the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 32. In certain embodiments, substitutions, insertions or deletions occur in regions outside of HVRs (i.e., FRs). Optionally, the antiLAG3 antibody comprises the VL sequence in SEQ ID NO: 32, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1
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40/126 comprising the amino acid sequence of SEQ ID NO: 28; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.
[00110] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 31 and SEQ ID NO: 32, respectively, including post-translational modifications of those sequences.
[00111] In another aspect, the invention provides an antibody that binds to the same epitope as an anti-LAG3 antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-LAG3 antibody comprising a VH sequence of SEQ ID NO: 31 and a VL sequence of SEQ ID NO: 32. In certain embodiments In this embodiment, an antibody is provided that binds to an epitope of the E3 cluster epitope within LAG3 (see example 2).
[00112] In one aspect, the invention provides an anti-LAG3 antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
[00113] In one aspect, the invention provides an antibody comprising at least one, at least two or all three
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41/126 VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34. In another embodiment, the antibody comprises (a ) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35.
[00114] In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
[00115] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least
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42/126 two or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33 (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 35; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36, (ii) HVR- L2 comprising the amino acid sequence of SEQ ID NO: 37 and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
[00116] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 35; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 36; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 37; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 38.
[00117] In any of the above embodiments, an anti-LAG3 antibody is human or humanized. In one embodiment, an anti-LAG3 antibody comprises HVRs as in any of the above embodiments and further comprises a human acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.
[00118] In another aspect, an anti-LAG3 antibody comprises a heavy chain variable domain (VH) sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39. In certain embodiments, a VH sequence having at least
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WA 26
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions in relation to the sequence of reference, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 39. In certain embodiments, substitutions, insertions or deletions occur in regions outside of HVRs (that is, in FRs). Optionally, the anti-LAG3 antibody comprises the VH sequence in SEQ ID NO: 39, including post-translational modifications of that sequence. In a particular embodiment, VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35.
[00119] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40. In certain embodiments, a VL sequence having at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions with respect to the reference sequence, but an anti-LAG3 antibody comprising that sequence retains the ability to bind to LAG3. In certain embodiments, a total of 1 to 10 amino acids have been replaced, inserted and / or deleted in SEQ ID NO: 40. In certain embodiments, substitutions, insertions or deletions occur in regions outside of HVRs (i.e., FRs). Optionally, the antiLAG3 antibody comprises the VL sequence in SEQ ID NO: 40, including modifications
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44/126 post-translation of this sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
[00120] In another aspect, an anti-LAG3 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 39 and SEQ ID NO: 40, respectively, including post-translational modifications of such sequences.
[00121] In another aspect of the invention, an anti-LAG3 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, an anti-LAG3 antibody is an antibody fragment, for example, an Fv, Fab, Fab ', scFv, diabody or F (ab') 2 fragment. In another embodiment, the antibody is an entire antibody, for example, with the substitutions L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, for example, WO 2012/130831 A1).
[00122] In another aspect, an anti-LAG3 antibody according to any of the above embodiments can incorporate any of the characteristics, alone or in combination, as described in Sections 1 to 7 below.
1. Affinity with Antibodies [00123] In certain embodiments, an antibody provided here has a dissociation constant (Kd) of <1 μΜ, <100 nM, <10
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45/126 nM, <1 ηΜ <0.1 ηΜ, <ηΜ 0.01 or <0.001 ηΜ (e.g., 10 'Μ ⁸ or less, for example 10 ^"8 M to 10" ¹³ M, for example, from 10 ' ⁹ M to 10' ¹³ M).
[00124] In one embodiment, the Kd measured by a radiolabeled antigen (RIA) binding assay. In one embodiment, an RIA is performed with the Fab version of an antibody of interest and its antigen. For example, the binding affinity of the Fab solution to the antigen is measured by equilibrium Fab with a minimum concentration of antigen labeled with ( ¹²⁵ l) in the presence of a series of unlabeled antigen titration, then capturing the binding antigen with a plate coated with anti-Fab antibody (see, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish conditions for the assay, the MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 pg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6 ) and subsequently blocked with 2% (w / v) bovine serum light in PBS for two to five hours at room temperature (approximately 23 ° C). In a non-adsorbent plate (Nunc # 269620), 100 pM or 26 pM of [ ¹²⁵ l] are mixed with serial dilutions of a Fab of interest (for example, consistent with the evaluation of the anti-VEGF antibody, Fab-12 , in Presta et al., Cancer Res. 57: 4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation can continue for a longer period (for example, about 65 hours) to ensure that equilibrium is achieved. After that, the mixtures are transferred to the capture plate for incubation at room temperature (for example, for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN20®) in PBS. When the plates have dried, 150 μΙ / well of scintillant (MICROSCINT-20 ™; Packard) is added and the plates are counted in a TOPCOUNT ™ gamma counter (Packard) for ten minutes. At
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46/126 concentrations of each Fab that give less than or equal to 20% of the maximum binding are chosen for use in competitive binding assays.
[00125] According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25 ° C with CM5 antigen chips immobilized at ~ 10 response units (RU). In one embodiment, carboxymethylated dextran (CM5, BIACORE, Inc.) biosensor chips are activated with A / -ethyl-A / '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and / V-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg / ml (~ 0.2 μΜ) before injection at a flow rate of 5 μΙ / minute to achieve approximately 10 response units (RU ) of coupled protein. After the injection of the antigen, 1 M of ethanolamine is injected to block the unreacted groups. For kinetic measurements, two serial dilutions of Fab (0.78 nM to 500 nM) are injected into PBS with 0.05% polysorbate 20 (TWEEN-20 ™) surfactant (PBST) at 25 ° C at a flow rate of approximately 25 μΙ / min. Association rates (kon) and dissociation rates (koff) are calculated using a simple Langmuir linkage model (BIACORE® Evaluation Software version 3.2) while simultaneously adjusting the association and dissociation sensograms. The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the on-rate exceeds 106 M-1 s-1 by the above surface plasmon resonance assay, then the attack rate can be determined using a fluorescent cooling technique that measures the increase or decrease in the intensity of fluorescence emission (excitation = 295 nm; emission = 340 nm, 16 nm band pass) at 25 ° C of an anti-antigen antibody 20
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47/126 nM (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured on a spectrometer, such as a flow stop equipped spectrophotometer (Aviv Instruments) or a SLM-AMINCO ™ spectrophotometer from 8000 series (ThermoSpectronic) with agitated cuvette.
2. Antibody Fragments [00126] In certain embodiments, an antibody provided herein is an antibody fragment. The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that retains the ability to specifically bind to an antigen. Antibody fragments include, but are not limited to Fab, Fab ', Fab'-SH, F (ab ') 2, Fv, single chain Fab (scFab); single chain variable fragments (scFv) and single domain antibodies (dAbs). For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).
[00127] In one embodiment, the antibody fragment is a Fab, Fab ', Fab'-SH or F (ab') 2 fragment, in particular a Fab fragment. Papain digestion of intact antibodies produces two identical fragments of antigen binding, called “Fab” fragments containing each of the heavy and light chain variable domains and also the light chain constant domain and the first heavy chain constant (CH1) domain. The term "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a light chain constant domain (CL) and a VH domain and a first constant domain (CH1) of a chain heavy. Fab 'fragments differ from Fab fragments by adding residues at the carboxyl terminus of the CH1 domain of the heavy chain including one or more cysteines from the antibody hinge region. Fab'-SH are Fab 'fragments in which the
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48/126 cysteine residue (s) of the constant domains contain a free thiol group. Treatment with pepsin yields an F (ab ') 2 fragment that has two antigen combining sites (two Fab fragments) and a part of the Fc region. For discussion of Fab and F (ab ') 2 fragments comprising rescue receptor binding epitope residues and having increased in vivo half-life, see US Patent No. 5,869,046.
[00128] In another embodiment, the antibody fragment is a diabody, a three-body or a tetrabody. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al. Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Know. USA 90: 6444-6448 (1993). Tribodies and tetribodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
[00129] In another embodiment, the antibody fragment is a single-stranded Fab fragment. A "single chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain (VH) variable domain, an antibody 1 constant domain (CH1), an antibody light chain variable domain (VL ), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders from the N-terminus to the C-terminus: a) VH-CH1- ligand -VL-CL, b) VL-CL-ligand-VH-CH1, c) VH-CL-ligand-VL-CH1 or d) VL-CH1-ligand-VHCL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single-stranded Fab fragments are stabilized through the natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-stranded Fab molecules can be further stabilized by generating interchain disulfide bonds by inserting cysteine residues
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49/126 (for example, position 44 on the variable heavy chain and position 100 on the variable light chain according to Kabat numbering).
[00130] In another embodiment, the antibody fragment is the single chain variable fragment (scFv). A "single chain variable fragment (scFv)" is a fusion protein from the variable regions of the heavy (Vh) and light (Vl) chains of an antibody, connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is generally rich in glycine for flexibility, as well as serine or threonine for solubility, and can link the N-terminal of Vn with the C-terminal of Vl, or vice versa. This protein retains the specificity of the original antibody, despite the removal of the constant regions and the introduction of the ligand. For a review of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and US Patent Nos. 5,571,894 and 5,587,458.
[00131] In another embodiment, the antibody fragment is a single domain antibody. Single domain antibodies are antibody fragments comprising all or a portion of the variable domain of the heavy chain or all or a portion of the variable domain of the light chain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US Patent No. 6,248,516 B1).
[00132] Antibody fragments can be produced by various techniques, including but not limited to proteolytic digestion of an intact antibody, as well as production by recombinant host cells (e.g., E. coliou phage), as described herein.
3. Chimeric and humanized antibodies [00133] In certain embodiments, an antibody here
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50/126 provided is a chimeric antibody. Certain chimeric antibodies are described, for example, in US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Know. USA, 81: 6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (for example, a variable region derived from a mouse, rat, hamster, rabbit or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "switched class" antibody in which the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include their antigen-binding fragments.
[00134] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while maintaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, for example, CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some RF residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (for example, the antibody from which the HVR residues are derived), for example, to restore or improve specificity or antibody affinity.
[00135] Humanized antibodies and methods of producing them are reviewed, for example, in Almagro and Fransson, Front. Biosci 13: 16191633 (2008), and are described later, for example, in Riechmann et al. Nature 332: 323-329 (1988); Queen et al., Proc. Nat’l Acad. Know. USA 86:
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51/126
10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (describing the specificity-determining region (SDR) graft); Padlan, Mol. Immunol. 28: 489-498 (1991) (describing “covering”); Dall'Acqua et al., Methods 36: 4360 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al. J. Cancer, 83: 252-260 (2000) (describing the “guided selection” approach to shuffling RF).
[00136] Human structural regions that can be used for humanization include, but are not limited to: structural regions selected using the “best fit” mode (see, for example, Sims et al., J. Immunol. 151: 2296 (1993 )); structural regions derived from the human antibody consensus sequence of a particular subgroup of variable regions of the light or heavy chain (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA 89: 4285 (1992); and Presta et al. J. Immunol., 151: 2623 (1993)); mature human structure regions (somatically mutated) or human germline structure regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and structural regions derived from scanning RF libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 2261122618 (1996)).
4. Human Antibodies [00137] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).
[00138] Human antibodies can be prepared
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52/126 administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to the antigen challenge. These animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the animal's chromosomes. In these transgenic mice, the loci of endogenous immunoglobulins were generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, US Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE ™ technology; US Patent No. 5,770,429 describes HuMab® technology; US Patent No. 7,041,870, describing KM MOUSE® technology, and US Patent Application Publication No. 2007/0061900, describing VelociMouse® technology). The human variable regions of intact antibodies generated by such animals can be further modified, for example, by combining with a different human constant region.
[00139] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, p. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al. Proc. Natl. Acad. Know. USA, 103: 3557-3562 (2006). Additional methods include those described, for example, in US Patent No. 7,189,826 (describing the production of human monoclonal IgM antibodies from cell lines of
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53/126 hybridoma) and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3) : 185 -91 (2005).
[00140] Human antibodies can also be generated by isolating sequences from the variable domain of the Fv clone selected from human-derived phage display libraries. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
5. Library Derived Antibodies [00141] Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al. in Nature Reviews 16: 498-508 (2016). For example, a variety of methods are known in the art to generate phage display libraries and screen such libraries for antibodies having the desired binding characteristics. Such methods are reviewed, for example, in Frenzel et al. in mAbs 8: 1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8: 1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36: 276-289 (2016) as well as in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and in Marks and Bradbury in Methods in Molecular Biology 248: 161-175 (Lo, ed. , Human Press, Totowa, NJ, 2003).
[00142] In certain phage display methods, the repertoires of the VH and VL genes are separately cloned by
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54/126 polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. in Annual Review of Immunology 12: 433-455 (1994). Phages typically have antibody fragments, either as single chain Fv fragments (scFv) or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the naive repertoire can be cloned (for example, human) to provide a single source of antibodies for a wide range of non-self and also self-antigens without any immunization, as described by Griffiths et al. in EMBO Journal 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning non-rearranged V gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to perform the in vitro rearrangement, as described by Hoogenboom and Winter in Journal of Journal. Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490, as well as in US Patent Publications Nos. 2005/0079574, 2007/0117126, 2007/0237764 and 2007/0292936.
[00143] Other examples of methods known in the art to screen combinatorial libraries for antibodies with a desired activity or activities include presentation of ribosome and mRNA, as well as methods for presentation and selection of antibodies in bacteria, mammalian cells, insect cells or cells of yeast. The methods for presenting the yeast surface are reviewed, for example, in Scholler et al. in Methods in Molecular Biology 503: 135-56 (2012) and in Cherf
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55/126 et al. in Methods in Molecular biology 1319: 155-175 (2015) and also in Zhao et al. in Methods in Molecular Biology 889: 73-84 (2012). Methods for presenting the ribosome are described, for example, in He et al. in Nucleic Acids Research 25: 5132-5134 (1997) and in Hanes et al. in PNAS 94: 4937-4942 (1997).
[00144] Antibodies or antibody fragments isolated from human antibody libraries are herein considered human antibodies or human antibody fragments.
6. Multispecific Antibodies [00145] In certain embodiments, an antibody provided herein is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites, that is, different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, the multispecific antibody has three or more binding specificities. In certain embodiments, one of the binding specificities is for LAG3 and the other specificity (two or more) is for any other antigen. In certain embodiments, bispecific antibodies can bind to two (or more) different epitopes of LAG3. Multispecific antibodies (for example, bispecific) can also be used to locate cytotoxic agents or cells for cells that express LAG3. Multispecific antibodies can be prepared as whole antibodies or antibody fragments.
[00146] Techniques for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and engineering “Knob-in-hole” (see, for example, US Patent No.
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56/126
5,731,168, and Atwell et al., J. Mol. Biol. 270: 26 (1997)). Multispecific antibodies can also be produced through electrostatic direction effects engineering to produce heterodynamic Fc antibody molecules (see, for example, WO 2009/089004); cross-linking of two or more antibodies or fragments (see, for example, US Patent No. 4676980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992) and WO 2011/034605); using common light chain technology to circumvent the light chain pairing problem (see, for example, WO 98/50431); using "diabody" technology to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152: 5368 (1994)); and preparing specific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).
[00147] Genetically engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies", or DVD-lg are also included here (see, for example, WO 2001/77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO2010 / 145792 and WO 2013/026831. The bispecific antibody or its antigen-binding fragment also includes a "Double-acting FAb" or "DAF" comprising an antigen-binding site that binds LAG3 as well as a different antigen, or two different LAG3 epitopes (see , for example, US 2008/0069820 and WO 2015/095539).
[00148] Multispecific antibodies can also be
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57/126 supplied in an asymmetric form with a crossing domain in one or more binding arms of the same antigen specificity, that is, by exchanging the VH / VL domains (see, for example, WO 2009/080252 and WO 2015/150447 ), the CH1 / CL domains (see, for example, WO 2009/080253) or the complete Fab arms (see, for example, WO 2009/080251, WO 2016/016299, see also Schaefer et al, PNAS, 108 (2011 ) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). In one embodiment, the multispecific antibody comprises a cross-Fab fragment. The term "cross-Fab fragment" or "xFab fragment" or "cross-Fab fragment" refers to a Fab fragment, in which the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the variable region of the light chain (VL) and the constant region of the heavy chain (CH1) and a polypeptide chain composed of the variable region of the heavy chain (VH) and the constant region of the light chain (CL) Asymmetric Fab arms can also be manipulated by introducing loaded or unloaded amino acid mutations at domain interfaces to direct the correct Fab pairing. See, for example, WO 2016/172485.
[00149] Various other molecular formats for multispecific antibodies are known in the art and are included here (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).
7. Antibody variants [00150] In certain embodiments, amino acid sequence variants of the antibodies contemplated herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence that encodes
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58/126 the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody's amino acid sequences. Any combination of deletion, insertion and substitution can be done to arrive at the final construction, as long as the final construction has the desired characteristics, for example, antigen binding.
a) Variants of Substitution, Insertion and Exclusion [00151] In certain embodiments, antibody variants are provided with one or more amino acid substitutions. Sites of interest for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "preferred substitutions". More substantial changes are provided in Table 1 under the heading of “exemplary substitutions”, and as further described below in reference to the classes of amino acid side chains. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
TABLE 1
Original Residue Exemplary Substitutions Preferred Substitutions Wing (A) Go; Read; lie Go Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) To be; Allah To be Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Giy (G) Allah Allah His (H) Asn; Gin; Lys; Arg Arg
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Original Residue Exemplary Substitutions Preferred Substitutions He O Read; Go; Met; Allah; Phe; Norleucine Read Leu (L) Norleucine; lie; Go; Met; Allah; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Read; Phe; lie Read Phe (F) Trp; Read; Go; lie; Allah; Tyr Tyr Pro (P) Allah Allah Being (S) Thr Thr Thr (T) Go; To be To be Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; To be Phe Go (V) lie; Read; Met; Phe; Allah; Norleucine Read
[00152] Amino acids can be grouped according to common side chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence the orientation of the chain: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
[00153] Non-conservative substitutions will involve the exchange of a member of one of these classes for another class.
[00154] One type of substitution variant involves the replacement of one or more residues in the hypervariable region of a parental antibody (for example, a humanized or human antibody). Generally, the resulting variant (s) selected for further study will have modifications (for example, improvements) in certain biological properties (for example, greater affinity, reduced immunogenicity) in relation to the parental antibody and / or has substantially retained certain biological properties of the
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60/126 parental antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on the phage and screened for a particular biological activity (e.g., binding affinity).
[00155] Changes (for example, substitutions) can be made in HVRs, for example, to improve the affinity of the antibody. Such changes can be made in HVR hotspots, that is, codon-encoded residues that mutate at high frequency during the somatic maturation process (see, for example, Chowdhury, Methods Mol. 207: 179-196 ( 2008)), and / or residues that come into contact with the antigen, with the resulting variant VH or VL to be tested for binding affinity. Affinity maturation building and reselecting from secondary libraries has been described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Pressure, Totowa, NJ, (2001).) In some forms of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (for example, error-prone PCR, chain rearrangement or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves approaches aimed at HVR, in which several HVR residues (for example, 4-6 residues at a time) are randomized. The HVR residues involved in binding to the antigen can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are
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61/126 frequently targeted.
[00156] In certain embodiments, substitutions, insertions or deletions can occur within one or more HVRs, provided that such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative changes (for example, conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made on HVRs. Such changes may, for example, be out of residues from contact with the antigen in HVRs. In certain embodiments of the VH and VL variant sequences provided above, each HVR is unchanged or contains no more than one, two or three amino acid substitutions.
[00157] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scan mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a target residue or group of residues (for example, charged residues, such as arg, asp, his, lys and glu) are identified and replaced with a neutral or negatively charged amino acid (for example, alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at the amino acid sites demonstrating functional sensitivity to the initial substitutions. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify points of contact between the antibody and the antigen. Such contact wastes and neighboring wastes can be targeted or disposed of as candidates for substitution. Variants can be traced to determine whether they contain the desired properties.
[00158] Amino acid sequence inserts include amino and / or terminal carboxyl fusions that vary in length
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62/126 of a residue to polypeptides containing one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal inserts include an antibody with an N-terminal methionyl residue. Other variants of insertion of the antibody molecule include fusion to the N or C terminal of the antibody to an enzyme (for example, to ADEPT) or to a polypeptide that increases the serum half-life of the antibody.
b) Glycosylation variants [00159] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence in such a way that one or more glycosylation sites are created or removed.
[00160] When the antibody comprises an Fc region, the oligosaccharide attached to it can be changed. Native antibodies produced by mammalian cells typically comprise a branched, bianternal oligosaccharide, which is generally linked by an N to Asn297 bond of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15: 26-32 (1997). The oligosaccharide can include various carbohydrates, for example, mannose, N-acetyl glucosamine (GlcNAc), galactose and sialic acid, as well as a GlcNAc-linked fucose in the "backbone" of the biantenary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide to an antibody of the invention can be made in order to create antibody variants with certain improved properties.
[00161] In one embodiment, antibody variants are provided having a non-fucosylated oligosaccharide, i.e., an oligosaccharide structure that has no fucose linked (directly or indirectly) to an Fc region. Such a non-fucosylated oligosaccharide (also
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63/126 referred to as "afucosylated" oligosaccharide) is particularly an N-linked oligosaccharide that lacks a fucose residue attached to the first GlcNAc in the backbone of the biantenary oligosaccharide structure. In one embodiment, antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or parental antibody. For example, the proportion of non-fucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., not fucosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides without fucose residues, in relation to the sum of all Asn 297-linked oligosaccharides (for example, complex, hybrid and mannose-rich structures) measured by mass spectrometry MALDI-TOF, as described in WO 2006/082515, for example. Asn297 refers to the asparagine residue located approximately at position 297 in the Fc region (EU number of residues in the Fc region); however, Asn297 can also be located about ± 3 amino acids upstream or downstream from position 297, that is, between positions 294 and 300, due to minor sequence variations in antibodies. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcyRIII receptor binding and / or improved effector function, in particular improved ADCC function. See, for example, US 2003/0157108; US 2004/0093621.
[00162] Examples of cell lines capable of producing antibodies with reduced fucosylation include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US 2003/0157108; and WO 2004/056312, especially in Example 11), and knockout cell lines, such as the alpha-1,6Petition gene 870190089671, of 10/09/2019, pg. 194/271
64/126 fucosyltransferase, FUT8, CHO knockout cells (see, for example, YamaneOhnuki et al. Biotech. Bioeng. 87: 614-622 (2004)). Kanda, Y. et al., Biotechnol, Bioeng., 94 (4): 680-688 (2006) and W02003 / 085107), or cells with reduced or abolished activity of a GDP-fucose synthesis or carrier protein (see, e.g., US2004259150, US2005031613, US2004132140,
US2004110282).
[00163] In another embodiment, antibody variants are provided with bisected oligosaccharides, for example, in which a biantenary oligosaccharide linked to the antibody's Fc region is bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.
[00164] Antibody variants with at least one galactose residue are also provided in the oligosaccharide linked to the Fc region. Such antibody variants can have an improved CDC function. Such various antibodies are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.
c) Variants of the Fc region [00165] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating a variant of the Fc region. The Fc region variant may comprise a sequence of the human Fc region (for example, a human Fc region lgG1, lgG2, lgG3 or lgG4) comprising an amino acid modification (for example, a substitution) at one or more amino acid positions.
[00166] In certain embodiments, the invention
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65/126 contemplates an antibody variant that has some but not all effector functions, making it a desirable candidate for applications where the half-life of the antibody in vivo is important but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity tests can be performed to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor binding assays (FcR) can be performed to ensure that the antibody is not bound to FcgamaR (possibly lack of ADCC activity), but maintains the ability to bind to FcRn. Primary cells for ADCC mediation, NK cells, express only FcgamaRIII, while monocytes express FcgamaRI, FcgamaRII and FcgamaRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 by Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays to assess the ADCC activity of a molecule of interest are described in US Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83 : 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Know. USA 82: 14991502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 13511361 (1987)). Alternatively, non-radioactive assay methods can be used (see, for example, the ACTI ™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wl) Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer cells (NK) Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in a model such as that disclosed in Clynes et al. Proc Nat'l Acad Sci. USA 95: 652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to
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66/126 connect to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro etal., J. Immunol. Methods 202: 163 (1996), Cragg, MS et al., Blood 101: 1045- 1052 (2003) and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). Determinations of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 ( 2006); WO 2013/120929 A1).
[00167] Antibodies with reduced effector function include those with replacement of one or more of the residues of the Fc region 238, 265, 269, 270, 297, 327 and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297 and 327, including the so-called Fc mutant "DANA" with substitution of residues 265 and 297 by alanine (US Patent No. 7,332,581).
[00168] Certain antibody variants with improved or decreased binding to FcRs are described. (See, for example, US Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9 (2): 6591 6604 (2001)).
[00169] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and / or 334 of the Fc region (EU residue numbering ).
[00170] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions, which increase FcRn binding, for example, substitutions at positions 252 and / or 254 and / or 256 of the Fc region (EU numbering of
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67/126 residues). In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 252, 254 and 256. In one embodiment, the substitutions are M252Y, S254T and T256E in an Fc region derived from an Fc region of human IgG 1.
[00171] In certain embodiments, an antibody variant comprises an Fc region with amino acid substitutions, which decrease binding to FcyR, for example, substitutions at positions 234 and 235 of the Fc region (EU residue number). In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgG 1 Fc region. (See, for example, WO 2012/130831 A1). In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.
[00172] In some embodiments, changes are made in the Fc region that result in the change (i.e., improved or decreased) of C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in US Patent No. 6,194,551, WO 99/51642 and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[00173] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)), are described in US 2005/0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve the binding of the Fc region to the FcRn. Such Fc variants include those with substitutions in one or more
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68/126 waste from the Fc region: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, substitution of the residue from the Fc 434 region (see, for example, US Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 2351423524).
[00174] Residues from the Fc region critical for the mouse FcRn to mouse Fc interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171- 5180). Residues I253, H310, H433, N434 and H435 (EU numbering according to Kabat) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310 and H435 were considered critical for the interaction of human Fc with murine FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human FcRn-human Fc complex have shown that residues I253, S254, H435 and Y436 are crucial for interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al ., J. Biol. Chem., 276 (2001) 6591-6604). In Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671) several mutants from residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 were reported and examined.
[00175] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions, which reduce the binding of FcRn, for example, substitutions at positions 253 and / or 310, and / or 435 of the region Fc (EU waste numbering). In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310 and 435. In one embodiment the substitutions are I253A, H310A and H435A in an Fc region derived from an Fc region of human IgG1. See, for example,
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Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.
[00176] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions, which reduces binding to FcRn, for example, substitutions at positions 310 and / or 433 and / or 436 of the region Fc (EU waste numbering). In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433 and 436. In one embodiment the substitutions are H310A, H433A and Y436A in an Fc region derived from an Fc region of human IgG1. (See, for example, WO 2014/177460 A1).
[00177] See also Duncan & Winter, Nature 322: 738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94/29351 relating to other examples of variants of the Fc region.
B. Recombinant Methods and Compositions [00178] Antibodies can be produced using recombinant methods and compositions, for example, as described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding an antibody are provided.
[00179] In the case of a native antibody or fragment of native antibody, two nucleic acids are needed, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. Such nucleic acid (s) encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (for example, the light chain (s) and / or heavy (s) of the antibody). These nucleic acids can be in the same expression vector or in different expression vectors.
[00180] In the case of a bispecific antibody with heterodimeric heavy chains, four nucleic acids are needed, one for the
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70/126 first light chain, one for the second light chain comprising the first hetreomonomeric Fc polypeptide, one for the second light chain and one for the second heavy chain comprising the second heteromonomeric polypeptide of the Fc region. The four nucleic acids can be comprised of one or more nucleic acid molecules or expression vectors. Such nucleic acid (s) encode (s) an amino acid sequence comprising the first VL and / or an amino acid sequence comprising the first VH including the first heteromonomeric Fc region and / or an amino acid sequence comprising the second VL and / or an amino acid sequence comprising the second VH including the second heteromonomeric Fc region of the antibody (e.g., the first and / or second light and / or the first and / or second heavy chain of the antibody). These nucleic acids can be in the same expression vector or in different expression vectors, normally these nucleic acids are located in two or three expression vectors, that is, a vector can comprise more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs and bispecific T cells (see, for example, Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonometric heavy chains comprises so-called “knob mutations” (T366W and optionally one of S354C or Y349C) and the other comprises so-called “hole mutations” (T366S, L368A and Y407V and, optionally, Y349C or S354C) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73).
[00181] In one embodiment, isolated nucleic acids encoding an antibody, as used in the methods described herein, are provided.
[00182] In another embodiment, one or more vectors (for example, expression vectors) are provided comprising
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71/126 these nucleic acid (s).
[00183] In a further embodiment, a host cell is provided comprising such nucleic acid (s).
[00184] In such an embodiment, a host cell comprises (for example, it has been transformed with):
in the case of an antibody composed of two identical light chains and two identical heavy chains which are linked by disulfide or a VH and a VL comprising a fragment thereof:
(1) a vector comprising nucleic acids encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody;
in the case of a bispecific antibody with heterodimeric heavy chains:
(1) a first vector comprising a first pair of nucleic acids that encodes amino acid sequences, one comprising the first VL and the other comprising the first VH of the antibody and a second vector comprising a second pair of nucleic acids encoding amino acid sequences , one of them, comprising the second VL and the other comprising the second VH of the antibody, or (2) a first vector comprising a first nucleic acid encoding an amino acid sequence comprising one of the variable domains (preferably a light chain variable domain) , a second vector comprising a pair of nucleic acids that encode
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72/126 amino acid sequences, one comprising a light chain variable domain and the other comprising the first heavy chain variable domain and a third vector comprising a pair of nucleic acids encoding amino acid sequences, one comprising the respective another light chain variable domain as in the second vector and the other comprising the second heavy chain variable domain, or (3) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the first antibody VL, a second vector comprising a nucleic acid encoding an amino acid sequence comprising the first VH of the antibody, a third vector comprising a nucleic acid encoding an amino acid sequence comprising the second VL of the antibody and a fourth vector comprising a nucleic acid encoding an amino acid sequence comprising the second VH of the antibody.
[00185] In one embodiment, the host cell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell or lymphoid cell (for example, Y0, NSO, Sp20 cell). In one embodiment, a method for producing an anti-LAG3 antibody is provided, wherein the method comprises culturing a host cell comprising nucleic acids encoding the antibody, as provided above, under conditions suitable for expression of the antibody and optionally recovering the antibody from the host cell (or host cell culture medium).
[00186] For the recombinant production of an antiLAG3 antibody, nucleic acids encoding an antibody, for example, as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using procedures
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73/126 conventional (for example, using oligonucleotide probes that are able to specifically bind to genes encoding antibody heavy and light chains) or produced by recombinant methods or obtained by chemical synthesis.
[00187] Host cells suitable for cloning or expression of vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not necessary. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523, (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.) , Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can still be purified.
[00188] In addition to prokaryotes, eukaryotic microbes, like filamentous fungi or yeasts, are suitable cloning or expression hosts for antibody-encoding vectors, including strains of fungi and yeast whose glycosylation pathways have been "humanized", resulting in the production of a antibody with a fully human glycosylation pattern. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.
[00189] Host cells suitable for glycosylated antibody expression are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera cells
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74/126 frugiperda.
[00190] Cultures of plant cells can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing PLANTIBODIESTM technology to produce antibodies in transgenic plants).
[00191] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lineage (cells 293 or 293 as described, for example, in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, for example, in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells and FS4 cells. Other mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. Et al., Proc. Nat'l. Acad. Sci. USA 77 (1980) 4216-4220) and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production See, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa , NJ (2004), pp. 255-268.
C. Assays [00192] Anti-LAG3 antibodies provided here can be
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75/126 identified, screened for, or characterized by their physical / chemical properties and / or biological activities by various tests known in the state of the art.
1. Binding assays and other assays [00193] In one aspect, an antibody of the invention is tested for its antigen binding activity, for example, by known methods such as ELISA, Western blot, etc.
[00194] In another aspect, competition assays can be used to identify an antibody that competes with aLAG3 (0414) for binding to LAG3. In certain embodiments, such a competing antibody binds to the same epitope (for example, a linear or conformational epitope) that is linked by aLAG3 (0414). Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
[00195] In an exemplary competition assay, immobilized LAG3 is incubated in a solution comprising a first labeled antibody that binds to LAG3 (for example, aLAG3 (0414)) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to LAG3. The second antibody can be present in a hybridoma supernatant. As a control, the immobilized LAG3 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under permissive conditions for binding the first antibody to LAG3, excess unbound antibody is removed and the amount of marker associated with immobilized LAG3 is measured. If the amount of marker associated with immobilized LAG3 is substantially reduced in the test sample compared to the control sample, then this indicates that the
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76/126 second antibody is competing with the first antibody for binding to LAG3. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
2. Activity assays [00196] In one aspect, assays are provided for the identification of anti-LAG3 antibodies with biological activity. Biological activity may include, for example, the effect of anti-LAG3 antibodies (alone or in combination with anti-PDL1 antibodies) on the release of cytotoxic Granzyme B and secretion of IL-2 by human CD4 T cells from a mixed reaction assay. lymphocytes (mMLR) or the effect of anti-LAG3 antibodies on Treg suppression, by the release of Granzima B and IFN-γ by human CD4 T cells; or inhibiting the binding of LAG-3 to MHC-II expressed in human A375 tumor cells by anti-LAG3 antibodies. Antibodies having such biological activity in vivo and / or in vitro are also provided.
[00197] In certain embodiments, an antibody of the invention is tested for this biological activity. For details, see examples 2 and 3 below.
D. Methods and Compositions for Diagnosis and Detection [00198] In certain embodiments, any of the anti-LAG3 antibodies provided here is useful for detecting the presence of LAG3 in a biological sample. The term "detection", as used herein, encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue, such as tumor tissue.
[00199] In one embodiment, an antiLAG3 antibody is provided for use in a diagnostic or detection method. In another aspect, a method is provided to detect the presence of LAG3 in a biological sample. In certain embodiments, the method comprises
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77/126 contacting the biological sample with an anti-LAG3 antibody as described here under permissive conditions for binding the anti-LAG3 antibody to LAG3, and detecting whether a complex is formed between the anti-LAG3 and LAG3 antibodies. This method can be an in vitro or in vivo method. In one embodiment, an anti-LAG3 antibody is used to select individuals eligible for therapy with an anti-LAG3 antibody, for example, where LAG3 is a biomarker for patient selection.
[00200] Exemplary disorders that can be diagnosed using an antibody of the invention include cancer in different forms, such as chronic lymphocytic leukemia (CLL) breast cancer, etc. (see also the list of cancers below).
[00201] In certain embodiments, labeled anti-LAG3 antibodies are provided. Markers include, but are not limited to, markers or portions that are directly detected (such as fluorescent, chromophoric, electron-dense, chemiluminescent and radioactive markers), as well as portions, such as enzymes or ligands, that are detected indirectly, for example, through an enzymatic reaction or molecular interaction. Exemplary markers include, but are not limited to, ³² P, ¹⁴ C, ¹²⁵ l, ³ H, and ¹³¹ 1 radioisotopes, fluorophores such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansila , umbeliferone, luceriferases e.g. firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glycoamylase, lysozyme, saccharide, saccharide oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin /
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78/126 avidin, spin markers, bacteriophage markers, stable free radicals and the like.
E. Pharmaceutical Formulations [00202] Pharmaceutical formulations of a antiLAG3 antibody as described herein that are prepared by mixing antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Flemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to receptors at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (for example, protein-Zn complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable vehicles here further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble Ph-20 hyaluronidase glycoproteins, such as rHuPH20 (Hylenex®, Baxter International, Inc. ).
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Certain examples of sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publications Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.
[00203] Examples of lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous formulations of antibodies include those described in US Patent No. 6,171,586 and WO 2006/044908, the latest formulations, including a histidine acetate buffer.
[00204] The formulation here may also contain more than one active ingredient as needed for the particular indication to be treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide anti-PD1 or anti PDL1 antibodies, or anti-TIM3 antibodies. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose.
[00205] The active ingredients can be trapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, microcapsules of hydroxymethylcellulose or microcapsules of gelatin and poly- (methylmethacrylate), respectively, in colloidal distribution systems of drugs (eg liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Flemington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).
[00206] Prolonged release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of molded articles, for example
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80/126 example, films or microcapsules.
[00207] The formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.
F. THERAPEUTIC METHODS AND COMPOSITIONS [00208] Any of the anti-LAG3 antibodies provided here can be used in therapeutic methods.
[00209] In one aspect, an anti-LAG3 antibody for use as a medicine is provided. In other respects, an anti-LAG3 antibody or use in the treatment of cancer is provided. In certain embodiments, an anti-LAG3 antibody is provided for use in a treatment method. In certain embodiments, the invention provides an anti-LAG3 antibody for use in a method of treating an individual with cancer comprising administering to the individual an effective amount of the anti-LAG3 antibody. In one embodiment, the antibody for use in treating or delaying the progression of a disease related to the immune system, such as tumor immunity. In one embodiment the antibody is to be used to stimulate an immune response or function, such as T cell activity.
[00210] In accordance with other embodiments, the invention provides an anti-LAG3 antibody for use as an immunostimulatory agent / or to stimulate secretion / release of Granzyme B (GrzB), interferon-gamma (IFN-gamma) and / or interleukin 2 (IL-2). In certain embodiments, the invention provides an anti-LAG3 antibody for use in a secretion / release method of Granzyme B (GrzB), interferon-gamma (IFNgama) and or interleukin 2 (IL-2) in an individual, comprising the administration to the individual an effective amount of an anti-LAG3 antibody
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81/126 effective for the secretion / release of Granzyme B (GrzB), interferon-gamma (IFNgama) and or interleukin 2 (IL-2).
[00211] An "individual" according to any of the above embodiments is preferably a human. In another aspect, the invention provides the use of an anti-LAG3 antibody in the manufacture or preparation of a medicament. In one embodiment, the drug is for cancer treatment. In another embodiment, the drug for use in a cancer treatment method comprising administering to an individual having cancer an effective amount of the drug. In another embodiment, the medicament for inducing cell-mediated lysis of cancer cells. In a further embodiment, the medicament for use in a cell-mediated method of inducing cancer cell lysis in an individual suffering from cancer, comprising administering to the individual an effective amount of the medicament to induce apoptosis in a cancer cell / or to inhibit cancer cell proliferation. An "individual" according to any of the above embodiments can be a human.
[00212] The term "cancer" as used herein may be, for example, lung cancer, non-small cell lung cancer (NSCL), bronchiolitis-violet lung cancer, bone cancer, pancreatic cancer, cancer of skin, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of fallopian tubes, carcinoma of the uterus, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, adrenal gland cancer, soft tissue sarcoma, cancer
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82/126 of the urethra, penis cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular cancer, biliary cancer, CNS neoplasms , tumors of the spinal axis, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannomas, ependimonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers or a combination of one or more of the previous cancers. In a preferred embodiment, such cancer is breast cancer, colorectal cancer, melanoma, head and neck cancer, lung cancer or prostate cancer. In a preferred embodiment, such cancer is breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. In another preferred embodiment, such cancer is breast cancer, lung cancer, cervical cancer, ovarian cancer, melanoma cancer, bladder cancer, kidney cancer, kidney cancer, liver cancer, head cancer and neck, colorectal cancer, pancreatic cancer, gastric carcinoma cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myelomas. In a preferred embodiment, these cancers are further characterized by the expression or overexpression of LAG3.
[00213] In another aspect, the invention provides a method for treating cancer. In one embodiment, the method comprises administering to an individual with cancer an effective amount of an anti-LAG3. An "individual" according to any of the above embodiments can be a human.
[00214] In another aspect, the invention provides a method for inducing cell-mediated lysis of cancer cells in an individual suffering from cancer. In one embodiment, the method
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83/126 comprises administering to the individual an effective amount of an anti-LAG3 to induce cell-mediated lysis of cancer cells in the individual suffering from cancer. In one embodiment, an "individual" is a human.
[00215] In another aspect, the invention provides pharmaceutical formulations comprising any of the antiLAG3 antibodies provided herein, for example, for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-LAG3 antibodies provided herein and a pharmaceutically acceptable carrier.
[00216] In another aspect, the invention provides pharmaceutical formulations comprising any of the antiLAG3 antibodies provided herein, for example, for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-LAG3 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-LAG3 antibodies provided herein and at least one additional therapeutic agent, for example, as described below.
[00217] Antibodies of the invention can be used alone or in combination with other agents in a therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is an anti-LAG3 or anti PDL1 or anti TIM3 antibody.
[00218] In addition to the anti-LAG3 antibody, a chemotherapeutic agent can also be administered. In one embodiment, such additional chemotherapeutic agents, which can be administered with anti-LAG3 antibody as described herein and include, but are not limited to, antineoplastic agents including alkylating agents
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84/126 including: nitrogen mustards, such as meclorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU) and semustine (methyl-CCNU); Temodal (TM) (temozolamide), ethylenimines / methylmelamine such as triethylene melamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5azacitidine, 2,2'-difluorodeoxycycline such as 6merca.rho.topurine, 6-thioguamne, azathioprine, T-deoxychoformicin (pentostatin), erythrohydroxinonyladenine (EHNA), fludarabine phosphate and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine and vinorelbine, taxotere, estramustine and stramustine phosphate; pipodophyllotoxins such as etoposide and teniposide; antibiotics such as actinomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mitramycin), mitomycin C and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinum coordination complexes such as oxaliplatin, cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressors such as mitotane (o, p- DDD) and aminoglutetimide; hormones and antagonists including adrenocorticoid steroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutetimide; Gemzar (TM) (gemcitabine), progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and
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85/126 megestrol acetate; estrogen such as the equivalents of diethylstilbestrol and ethinyl estradiol; anti-estrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens, such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal anti-androgens such as flutamide. Therapies targeting the epigenetic mechanism including, but not limited to, histone deacetylase inhibitors, demethylating agents (eg, Vidaza) and release of transcriptional repression therapies (ATRA) can also be combined with antigen-binding proteins. In one embodiment, the chemotherapeutic agent selected from the group consisting of taxanes (such as paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (eg, Abraxane and Opaxio), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar) and other inhibitors of multiquinase, oxaliplatin, cisplatin and carboplatin, etoposide, gemcitabine and vimblastine In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (such as taxol (paclitaxel), docetaxel (Taxotere), modified paclitaxel (for example Abraxane and Opaxio) In one embodiment, the additional chemotherapeutic agent is selected from 5-fluorouracil (5-FU), leucovorin, irinotecan, or oxaliplatin. embodiment the chemotherapeutic agent is 5-fluorouracil, leucovorin and irinotecan (FOLFIRI) In one embodiment, the chemotherapeutic agent is 5-fluorouracil and oxaliplatin (FOLFOX).
[00219] Such combination therapies referred to above encompass combined administration (wherein two or more therapeutic agents are included in the same formulations or in separate formulations) and separate administration, in which case administration of the antibody of the invention may occur before, simultaneously and / or thereafter, administration of the additional therapeutic agent or agents. In a form of
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In this embodiment, administration of the anti-LAG3 antibody and administration of an additional therapeutic agent occurs within about one month, or within about one, two or three weeks, or within about one, two, three , four, five or six days in between. Antibodies of the invention can also be used in combination with radiation therapy.
[00220] An antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. The dosage can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including, but not limited to, single or multiple administrations over various time points, bolus administration and pulse infusion are contemplated herein.
[00221] The antibodies of the invention would be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the place of delivery of the agent, the method of administration, the schedule of administration and other factors known to doctors. The antibody does not need to be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such agents depends on the amount of antibody present in the formulation, the type of disorder or treatment and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about 1 to
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99% of the dosages described herein, or at any dosage and by any route that is empirically / clinically determined to be appropriate.
[00222] For disease prevention or treatment, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, prior therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is properly administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (for example, 0.1 mg / kg to 10 mg / kg) of antibody may be an initial candidate dosage for administration to the patient, for example , by one or more separate administrations, or by continuous infusion. A typical daily dose can vary from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or more, depending on the condition, treatment would generally be continued until a desired suppression of the symptoms of the disease occurs. An exemplary dosage of the antibody would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, every week or every three weeks (for example, in such a way that the patient receives from about two to about twenty, or for example, about six doses of antibody). A higher initial loading dose, followed by one or more smaller doses, can be administered. An exemplary dosage regimen comprises administration. However, other dosing regimens may be useful. O
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88/126 progress of this therapy is easily monitored by conventional techniques and trials.
[00223] It is understood that any of the above formulations or therapeutic methods can be performed using an immunoconjugate of the invention instead of, or in addition to, an antiLAG3 antibody.
G. Articles of manufacture [00224] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package leaflet about or associated with the container. Suitable containers include, for example, bottles, vials, syringes, bags of IV solution, etc. Containers can be formed from a variety of materials, such as glass or plastic. The container contains a composition that is either alone or combined with another composition effective to treat, prevent and / or diagnose the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial) pierceable) by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package leaflet indicates that the composition is used to treat the condition of choice. In addition, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises another cytotoxic agent or otherwise a therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise an information leaflet indicating that the compositions can be used to treat a particular condition.
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Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may also include other materials desirable from a commercial and user point of view, including other buffers, thinners, filters, needles and syringes.
Description of amino acid sequences and NUCLEIC acid sequences
SEQ ID NO: 1 HVR-H1 heavy chain, aLAG3 (0414);
SEQ ID NO: 2 HVR-H2 heavy chain, aLAG3 (0414);
SEQ ID NO: 3 HVR-H3 heavy chain, aLAG3 (0414);
SEQ ID NO: 4 HVL-L1 light chain, aLAG3 (0414);
SEQ ID NO: 5 HVL-L2 light chain, aLAG3 (0414);
SEQ ID NO: 6 HVL-L3 light chain, aLAG3 (0414);
SEQ ID NO: 7 VH heavy chain variable domain, aLAG3 (0414);
SEQ ID NO: 8 light chain variable domain VL, aLAG3 (0414);
SEQ ID NO: 9 HVR-H1 heavy chain, aLAG3 (0403);
SEQ ID NO: 10 HVR-H2 heavy chain, aLAG3 (0403);
SEQ ID NO: 11 HVR-H3 heavy chain, aLAG3 (0403);
SEQ ID NO: 12 HVL-L1 light chain, aLAG3 (0403);
SEQ ID NO: 13 HVL-L2 light chain, aLAG3 (0403);
SEQ ID NO: 14 HVL-L3 light chain, aLAG3 (0403);
SEQ ID NO: 15 VH heavy chain variable domain, aLAG3 (0403);
SEQ ID NO: 16 VL light chain variable domain, aLAG3 (0403);
SEQ ID NO: 17 HVR-H1 heavy chain, aLAG3 (0411);
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SEQ ID NO: 18 HVR-H2 heavy chain, al_AG3 (0411);
SEQ ID NO: 19 HVR-H3 heavy chain, al_AG3 (0411);
SEQ ID NO: 20 HVL-L1 light chain, al_AG3 (0411);
SEQ ID NO: 21 HVL-L2 light chain, al_AG3 (0411);
SEQ ID NO: 22 HVL-L3 light chain, al_AG3 (0411);
SEQ ID NO: 23 VH heavy chain variable domain, al_AG3 (0411);
SEQ ID NO: 24 light chain variable domain VL, aLAG3 (0411);
SEQ ID NO: 25 HVR-H1 heavy chain, aLAG3 (0417);
SEQ ID NO: 26 HVR-H2 heavy chain, aLAG3 (0417);
SEQ ID NO: 27 HVR-H3 heavy chain, aLAG3 (0417);
SEQ ID NO: 28 HVL-L1 light chain, aLAG3 (0417);
SEQ ID NO: 29 HVL-L2 light chain, aLAG3 (0417);
SEQ ID NO: 30 HVL-L3 light chain, aLAG3 (0417);
SEQ ID NO: 31 VH heavy chain variable domain, aLAG3 (0417);
SEQ ID NO: 32 light chain variable domain VL, aLAG3 (0417);
SEQ ID NO: 33 HVR-H1 heavy chain, aLAG3 (0416);
SEQ ID NO: 34 HVR-H2 heavy chain, aLAG3 (0416);
SEQ ID NO: 35 HVR-H3 heavy chain, aLAG3 (0416);
SEQ ID NO: 36 HVL-L1 light chain, aLAG3 (0416);
SEQ ID NO: 37 HVL-L2 light chain, aLAG3 (0416);
SEQ ID NO: 38 HVL-L3 light chain, aLAG3 (0416);
SEQ ID NO: 39 VH heavy chain variable domain, aLAG3 (0416);
SEQ ID NO: 40 light chain variable domain VL, aLAG3 (0416);
SEQ ID NO: 41 VH heavy chain variable domain, BMS986016 (WO 2014/008218 and US 2016/0326248);
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SEQ ID NO: 42 VL light chain variable domain BMS-986016 (WO 2014/008218 and US 2016/0326248);
SEQ ID NO: 43 VH heavy chain variable domain, MDX25F7 (25F7) (US 2011/0150892 and WO 2014/008218);
SEQ ID NO: 44 light chain variable domain VL, MDX25F7 (25F7) (US 2011/0150892 and WO 2014/008218);
SEQ ID NO: 45 VH heavy chain variable domain, humanized BAP050 (LAG525) (US 2015/0259420);
SEQ ID NO: 46 VL light chain variable domain, humanized BAP050 (LAG525) (US 2015/0259420);
SEQ ID NO: 47 VH heavy chain variable domain, MDX 26H10 (26H10) (US 2011/0150892);
SEQ ID NO: 48 VL light chain variable domain, MDX 26H10 (26H10) (US 2011/0150892);
SEQ ID NO: 49 human kappa light chain constant region;
SEQ ID NO: 50 human lambda light chain constant region;
SEQ ID NO: 51 human heavy chain constant region derived from IgG1;
SEQ ID NO: 52 IgG 1 derived human heavy chain constant region with mutations L234A, L235A and P329G;
SEQ ID NO: 53 human heavy chain constant region derived from IgG4;
SEQ ID NO: 54 exemplary human LAG3 sequence (no signal sequence);
SEQ ID NO: 55 human LAG3 extracellular domain (ECD);
SEQ ID NO: 56 primer rbHC.up;
SEQ ID NO: 57 rbHCf.do primer;
SEQ ID NO: 58 primer BcPCR_FHLCJeader.fw;
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SEQ ID NO: 59 initiator BcPCR_huCkappa.rev.
[00225] In the following amino acid sequences of the VL and VH domains including labeled HVRs (bold HVRs, underlined letters) of anti-LAG3 antibodies, aLAG3 (0403) to aLAG3 (0417) are listed:
1) aLAG3 (0403)
SEQ ID NO 15: VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYTMHWVRQAPG KGLEWVSLVSWDGGGTYYTNSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YFCAKAITDTSLYGYDYWGQGILVTVSS
SEQ ID NO 16: VL
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGNA
PKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPLT FGGGTKVEIK
2) aLAG3 (0411)
SEQ ID NO 23: VH
EVHLLESGGGLVQPGGSLRLSCAASGFIVDDYTMNWVRQAPGK GLEWVSVISWDGGATYYADSVKGRFTISRDDFKNTLYLQMNSLRAEDTAVYY CAKGLTDDTLYGSDYWGQGTLVTVSS
SEQ ID NO 24: VL
DIQMTQSPSSLSASVGDRVTITCRASQSIVSYLNWYQQKPGKAP KLLIYASSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPLTF GGGTKVEIK
3) aLAG3 (0414)
SEQ ID NO 7: VH
EVQLLESGGGLVQPGGSLRLSCAASGFIFDDYTMNWVRQAPGK
GLEWVAVISWDGGGTYYTDSVKGRFTISRDDFKNTLYLQMNSLRAEDTAVYY CAKGLTDTTLYGSDYWGQGTLVTVSS
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SEQ ID NO 8: VL
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP
KLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSSPLTF GGGTKVEIK
4) aLAG3 (0416)
SEQ ID NO 39: VH
EVQLVESGGGLVQPGGSLRLACAASGFTFSDYAMSWVRQAPG
KGLEWVSGIDNSGYYTYYTDSVKGRFTISRDDVKNTLYLQMNSLRAEDTAVYL CTKTHSGLIVNDAFDIWGQGTMVTVSS
SEQ ID NO 40: VL
DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP
KLLIYDASSLESGVPSRFSGSGSGTDATLTISSLQPEDFATYYCQQSYSTPLTF GGGTKVEIK
5) aLAG3 (0417)
SEQ ID NO31: VH
EVQLVESGGGLVQPGGSLRLACAASGFTFSDYAMSWVRQAPG
KGLEWVSGIDNSGYYTYYTDSVKGRFTISRDDVKNTLYLQMNSLRAEDTAVYL CTKTHSGLIVNDAFDIWGQGTMVTVSS
SEQ ID NO 32: VL
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP
KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPLTF GGGTKVEIK.
[00226] The following specific embodiments of the invention are listed:
1. An isolated antibody that binds to human LAG3, where the antibody comprises
A) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of
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SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 5; and (f) HVR-L3 comprising the amino acid sequence of
SEQ ID NO: 6; or
B) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 9; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 10; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 11; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 13; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or
C) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 20; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 21; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or
D) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 26; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 27; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 28; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 29; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or
E) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of
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SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 35; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 36; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 37; and (f) HVR-L3 comprising the amino acid sequence of
SEQ ID NO: 38,
2. An isolated antibody that binds to human LAG3, where the antibody comprises
A) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or
B) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or
C) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) a VL domain comprising (i) HVR-L1 comprising the
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96/126 amino acid sequence of SEQ ID NO: 20; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or
D) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or
E) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 35; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38,
3. An isolated antibody that binds to human LAG3, where the antibody
i) comprises a VH domain with a 95%, 96%, 97% or 98% amino acid sequence identity compared to the SEQ ID NO: 7 sequence and a VL domain with a 95% amino acid sequence identity , 96% 97% or 98% compared to the sequence of SEQ ID NO: 8;
ii) comprises a VH domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to
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97/126 sequence of SEQ ID NO: 15 and a VL domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 16;
iii) comprises a VH domain with a 95%, 96%, 97% or 98% amino acid sequence identity compared to the sequence of SEQ ID NO: 23 and a VL domain with a 95% amino acid sequence identity , 96% 97% or 98% compared to the sequence of SEQ ID NO: 24;
iv) comprises a VH domain with a 95%, 96%, 97% or 98% amino acid sequence identity compared to the sequence of SEQ ID NO: 31 and a VL domain with a 95% amino acid sequence identity , 96% 97% or 98% compared to the sequence of SEQ ID NO: 32; or
v) comprises a VH domain with a 95%, 96%, 97% or 98% amino acid sequence identity compared to the SEQ ID NO: 39 sequence and a VL domain with a 95% amino acid sequence identity , 96% 97% or 98% compared to the sequence of SEQ ID NO: 40,
4. An isolated antibody that binds to human LAG3, where the antibody comprises
A) (a) a VH domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 7 and a VL domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 8; wherein the VH domain comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 and (iii) HVR-H3 comprising a amino acid sequence amino acid sequence selected from
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98/126 of SEQ ID NO: 3; and (b) the VL domain comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or
B) (a) a VH domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 15 and a VL domain with an amino acid sequence identity of 95% 96%, 97% or 98% compared to the sequence of SEQ ID NO: 16; wherein the VH domain comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) the VH domain comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or
C) (a) a VH domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 23 and a VL domain with an amino acid sequence identity of 95%; 96%, 97% or 98% compared to the sequence of SEQ ID NO: 24; wherein the VH comprises a domain (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) the VL domain comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or
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D) (a) a VH domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 31 and a VL domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 32; wherein the VH domain comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26 and (iii) HVR-H3 comprising a amino acid sequence amino acid sequence selected from SEQ ID NO: 27; and (b) the VL domain comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or
E) (a) a VH domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 39 and a VL domain with an amino acid sequence identity of 95%, 96%, 97% or 98% compared to the sequence of SEQ ID NO: 40; wherein the VH domain comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 and (iii) HVR-H3 comprising a amino acid sequence. amino acid sequence selected from SEQ ID NO: 35; and (b) the VL domain comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38,
5. An isolated antibody that binds to human LAG3, where the antibody
i) comprises a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8;
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100/126 ii) comprises a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16;
iii) comprises a VH sequence of SEQ ID NO: 23 and a VL sequence of SEQ ID NO: 24;
iv) comprises a VH sequence of SEQ ID NO: 31 and a VL sequence of SEQ ID NO: 32; or
v) comprises a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ ID NO: 40,
6. An isolated antibody that binds to human LAG3, wherein the antibody comprises a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8,
7. An isolated antibody that binds to human LAG3, wherein the antibody comprises a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16,
8. An isolated antibody that binds to human LAG3, wherein the antibody comprises a VH sequence of SEQ ID NO: 23 and a VL sequence of SEQ ID NO: 24,
9. An isolated antibody that binds to human LAG3, wherein the antibody comprises a VH sequence of SEQ ID NO: 31 and a VL sequence of SEQ ID NO: 32,
10. An isolated antibody that binds to human LAG3, wherein the antibody comprises a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ ID NO: 40,
11. The anti-LAG3 antibody according to any of the previous embodiments, wherein the antibody is independently characterized by one or more of the following properties: the anti-LAG3 antibody,
i) competes for binding to LAG3 with an anti-LAG3 antibody
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101/126 comprising VH with the amino acid sequence of SEQ ID NO: 7 and VL with the amino acid sequence of SEQ ID NO: 8 and / or ii) binds to a human and cinomolgo LAG3; and / or iii) inhibits the binding of MHC-II expressed in human A375 tumor cells; and / or iv) increases the release of Granzyme B or IL-2 in a mixed lymphocyte reaction (mMLR) assay (as shown in Example 3),
12. An isolated antibody that binds to human LAG3, where the antibody:
i) competes to bind LAG3 with an anti-LAG3 antibody comprising VH with the amino acid sequence of SEQ ID NO: 7 and VL with the amino acid sequence of SEQ ID NO: 8 and / or ii) binds to a LAG3 human and cinomolgo; and / or iii) inhibits the binding of MHC-II expressed in human A375 tumor cells; and / or iv) increases the release of Granzyme B or IL-2 in a mixed lymphocyte reaction (mMLR) assay (as shown in Example 3),
13. The antibody of any of the previous embodiments, which is a monoclonal antibody,
14. The antibody according to any of the previous embodiments, which is a human, humanized or chimeric antibody,
15. The antibody according to any of the previous embodiments, which is an antibody fragment that binds to LAG3,
16. The antibody according to any of the previous embodiments, which is a full length IgG1 antibody,
17. The antibody according to any of the previous embodiments, which is a full-length lgG1 antibody with mutations L234A, L235A and P329G (numbered according to the EU index of
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Kabat),
18. Isolated nucleic acid encoding the antibody according to any of the preceding embodiments,
19. A host cell comprising the nucleic acid of embodiment 18,
20. Method for producing an antibody comprising culturing the host cell of embodiment 19 so that the antibody is produced,
21. The embodiment method 20; further comprising recovering the host cell antibody,
22. A pharmaceutical formulation comprising the antibody according to any one of embodiments 1 to 17 and a pharmaceutically acceptable carrier,
23. The antibody according to any of embodiments 1 to 17 for use as a medicament,
24. The antibody according to any of embodiments 1 to 17 for use in the treatment of cancer,
25. Use of the antibody according to any of embodiments 1 to 17, in the manufacture of a drug,
26. The use of embodiment 25 in which the drug is for cancer treatment,
27. Method for treating an individual having cancer, the method comprising administering to the individual an effective amount of the antibody of embodiment 1,
28. Method for treating or slowing the progression of an immune-related disease, such as tumor immunity, the method comprising administering to the individual an effective amount of the antibody of embodiment 1,
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29. Method for stimulating an immune response or function, such as T cell activity, the method comprising administering to the individual an effective amount of the antibody of embodiment 1.
III. EXAMPLES [00227] The following are examples of methods and compositions of the invention. It is understood that several other embodiments can be practiced, given the general description provided above.
[00228] Although the previous invention has been described in some detail by way of illustration and example for the sake of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated in their entirety by reference.
Example 1: Generation of anti-LAG3 antibodies
Rabbit immunization [00229] Roche-owned transgenic rabbits that express a humanized antibody repertoire have been immunized with plasmid DNA that expresses LAG3.
[00230] A set of 3 rabbits was genetically immunized, using a plasmid expression vector encoding full-length human LAG3 (15352_plntronA_fl-hLag3_DNA-IMS), by intradermal application of 400 pg of vector DNA, followed by electroporation (5 square pulses) 750 V / cm, duration 10 ms, interval 1 s). The rabbits received 7 consecutive immunizations on days 0, 14, 28, 49, 70, 98 and 126. Blood (10% of the estimated total blood volume) was collected on days 35, 77, 105 and 133. The serum was prepared , which was used for ELISA titer determination (see below), and peripheral mononuclear cells were isolated, which were used as a source of antigen-specific B cells in the
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104/126 cloning of B cells below.
Determination of serum titers (ELISA) [00231] The recombinant human LAG3 protein was immobilized in a 96-well NUNC Maxisorp plate at 2 pg / mL, 100 μΙ / well, in PBS, followed by: blocking the plate with 2% Crotein C in PBS, 200 μΙ / well; application of serial dilutions of antiserums, in duplicates, in 0.5% Crotein C in PBS, 100 μΙ / well; detection with (1) HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch / Dianova 711036-152; 1/16 000) or (2) HRP-conjugated rabbit anti-human IgG antibody (Pierce / Thermo Scientific 31423; 1 / 5000), or (3) biotinylated goat human anti-kappa antibody (Southern Biotech / Biozol 2063-08, 1/5 000) and streptavidin-HRP; each diluted in 0.5% Crotein C in PBS, 100 μΙ / well. For all stages, the plates were incubated for 1 h at 37 ° C. Between all steps, the plates were washed 3 times with 0.05% Tween 20 in PBS. The signal was developed by adding soluble substrate BM Blue POD (Roche), 100 μΙ / well; and stopped by adding 1 M HCI, 100 μΙ / well. The absorbance was read at 450 nm, against 690 nm as a reference. The titer was defined as dilution of antisera, resulting in half a maximum signal.
Isolation of mononuclear cells from rabbit peripheral blood (PBMC) [00232] Blood samples were collected from immunized transgenic rabbits. Whole blood containing EDTA was diluted with 1x PBS (PAA, Pasching, Austria) before density centrifugation using a lympholytic mammal (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the manufacturer's specifications. PBMCs were washed twice with 1x PBS.
EL-4 B5 medium [00233] RPMI 1640 (Pan Biotech, Aidenbach, Germany)
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105/126 supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2 mM Glutamine, 1% penicillin / streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05 mM b-mercaptoethanol (Gibco, Paisley, Scotland).
Coating of plates with protein antigen [00234] Plates from 6 wells of sterile cell culture were coated with human LAG3 ECD conjugated to a human Fc part (2 pg / mL) in carbonate buffer (0.1 M sodium bicarbonate, 34 mM disodium hydrogen carbonate, pH 9.55) overnight at 4 ° C. The plates were washed in sterile PBS three times before use.
Cell depletion (a) Sterile 6-well plates (grade of cell culture) covered with a confluent monolayer of CHO cells were used to deplete macrophages / monocytes through non-specific adhesion, as well as non-specific binding lymphocytes.
(b) Blank, sterile 6-well plates (cell culture grade) were used to eliminate macrophages and monocytes and other cells through unspecific adhesion.
[00235] Half of the PBMC sample was used for (a) and half for (b).
[00236] Each well was filled with a maximum of 4 ml of medium and up to 6x106 PBMC of the immunized rabbit and allowed to bind for 1 h at 37 ° C in the incubator. The cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen binding (panning) step.
Enrichment of B cells in the LAG3 antigen
Protein Antigen [00237] 6 well coated tissue culture plates
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106/126 with LAG3-ECD-huFc protein were seeded with up to 6 x 10 and 6 PBLs per 4 ml of medium from the depletion steps using the blank 6-well plate and allowed to bind for 1 h at 37 ° C in an incubator. Non-adherent cells were removed by carefully washing the wells 1-2 times with 1x PBS. The remaining adherent cells were separated by trypsin for 10 min at 37 ° C in the incubator. Trypsinization was stopped with EL-4 B5 medium. The cells were kept on ice until immunological fluorescence staining.
Cell surface antigen [00238] 6-well tissue culture plates covered with a monolayer of human LAG3 positive CHO cells were seeded with up to 6x10 ⁶ PBLs per 4 ml of medium from the depletion steps using the 6-well plate covered with CHO and left to stand for 1 h at 37 ° C in the incubator. Non-adherent cells were removed by carefully washing the wells 1-2 times with 1x PBS. The remaining adherent cells were separated by trypsin for 10 min at 37 ° C in the incubator. Trypsinization was stopped with EL-4 B5 medium. The cells were kept on ice until immunological fluorescence staining.
Immunological fluorescence staining and flow cytometry [00239] The anti-IgG FITC antibody (AbD Serotec, Düsseldorf, Germany) and the anti-huCk PE antibody (Dianova, Hamburg, Germany) were used for single-cell screening. For surface staining, cells from depletion and enrichment step were incubated with anti-IgG FITC and anti-huCk PE antibody in PBS and incubated for 45 min in the dark at 4 ° C. After staining, PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Propidium iodide at a concentration of 5 pg / mL (BD Pharmingen, San Diego, CA, USA) was added before FACS analyzes to discriminate between dead and living cells.
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107/126 [00240] A FACSAria Becton Dickinson equipped with a computer and the FACSDiva software (BD Biosciences, USA) were used for the single cell type.
Cultivation of B cells [00241] Cultivation of rabbit B cells was performed by a method described by Seeber et al. (S Seeber et al. PLoS One 9 (2), e86184. 2014 February 4). Briefly, separate rabbit B cells were incubated in 96-well plates with 200 μΙ / well of EL-4 B5 medium containing Pansorbin Cells (1: 100000) (Calbiochem (Merck), Darmstadt, Deutschland), 5% thymocyte supernatant rabbit (MicroCoat, Bernried, Germany) and murine gamma-irradiated murine EL-4 B5 thymoma cells (5 χ 10e5 cells / well) for 7 days at 37 ° C in the incubator. Supernatants from the B cell culture were removed for scanning and the remaining cells were harvested immediately and were frozen at -80 ° C in 100 μΙ of RLT buffer (Qiagen, Hilden, Germany).
Isolation of V-domains from LAG3 antibodies PCR amplification of V-domains [00242] Total RNA was prepared from B cell lysate (resuspended in RLT buffer - Qiagen - Cat. No. 79216) using the NucleoSpin RNA kit 8/96 (Macherey &Nagel; 740709.4, 740698) according to the manufacturer's protocol. The RNA was eluted with 60 μΙ of RNase-free water. 6 μΙ of RNA was used to generate cDNA by reverse transcriptase reaction using SuperMix First-Strand Synthesis Superscript III (Invitrogen 18080-400) and an oligo dT primer according to the manufacturers instructions. All steps were performed on a Hamilton ML Star system. 4 μΙ of cDNA was used to amplify the variable regions of heavy and light chain immunoglobulin (VH and VL) with AccuPrime Supermix (Invitrogen 12344-040) in a final volume of 50 μΙ using the primers rbHC.up and rbHC. for the heavy chain and BcPCR_FHLCJeader.fw and BcPCR_huCkappa.rev for the chain
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108/126 mild (Table 1.1). All forward primers were specific for the signal peptide (respectively VH and VL) while reverse primers were specific for constant regions (respectively VH and VL). The PCR conditions for RbVH were as follows: Hot start at 94 ° C for 5 min; 35 cycles from 20 s to 94, 20 s to 70 ° C, 45 s to 68 ° C and a final extension at 68 ° C for 7 min. The PCR conditions for HuVL were as follows: hot start at 94 ° C for 5 min; 40 cycles of 20 s at 94 ° C, 20 s at 52 ° C, 45 s at 68 ° C and a final extension at 68 ° C for 7 min.
Table 1.1
SEQ ID NO: 56 rbHC.up AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC SEQ ID NO: 57 rbHCf.do ccattggtgagggtgcccgag SEQ ID NO: 58BcPCR FHLCJeader.fw atggacatgagggtccccgc SEQ ID NO: 59BcPCR huCkappa.rev gatttcaactgctcatcagatggc
[00243] 8 μΙ to 50 μΙ of PCR solution were loaded into a 48% 2 E-Gel (Invitrogen G8008-02). Positive PCR reactions were cleared using the NucleoSpin Extract II kit (Macherey &Nagel; 740609250) according to the manufacturer's protocol and eluted in 50 μΙ of elution buffer. All cleaning steps were performed on a Hamilton Starlet System.
Recombinant expression of rabbit monoclonal bivalent antibodies [00244] For recombinant expression of rabbit monoclonal bivalent antibodies, PCR products encoding VH or VL were cloned as cDNA into expression vectors by the outgoing cloning method (RS Haun et al. , Biotechniques (1992) 13, 515-518; MZ Li et al .., Nature Methods (2007) 4, 251-256). The expression vectors contained an expression cassette consisting of a 5 'CMV promoter including intron A and a 3' BGH polyadenylation sequence. In addition to the expression cassette, the plasmids contained an origin of replication derived from pUC18 and a beta-lactamase gene conferring resistance to ampicillin to amplify the
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109/126 plasmid in E. coli. Three variants of the basic plasmid were used: a plasmid containing the rabbit IgG constant region designed to accept the VH regions while containing the human kappa LC constant region to accept the VL regions.
[00245] Linearized expression plasmids encoding the kappa or gamma constant region and the VL / VH inserts were amplified by PCR using overlapping primers.
[00246] The purified PCR products were incubated with T4 DNA polymerase that generated single-stranded protrusions. The reaction was stopped by adding dCTP.
[00247] In the next step, the plasmid and insert were combined and incubated with recA which induces site-specific recombination. The recombined plasmids were transformed into E. coli. The next day, the cultured colonies were harvested and tested for the correct recombined plasmid by plasmid preparation, restriction analysis and DNA sequencing.
[00248] For antibody expression, isolated HC and LC plasmids were transiently co-transfected into HEK293 cells and supernatants were collected after 1 week.
Example 2: Characterization of anti-LAG3 antibodies
Table 2: Summary of Characterization of different anti-LAG3 antibodies
AntiLag3 Antibodies aLAG3 (0403) aLAG3 (411) aLAG3 (414) aLAG3 (416) aLAG3 (417) MDX-25F7 (25F7) BMS-986016 MDX-26H10(26H10) BAPI 050 humanizedI ILAG525) KD [M] monovalentbivalent tbdtbd tbdtbd 4.63E-10 tbd 2.82E-11tbd tbdtbd tbdtbd tbdtbd tbdtbd tbdtbd
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AntiLag3 Antibodies aLAG3 (0403) aLAG3 (411) aLAG3 (414) aLAG3 (416) aLAG3 (417) MDX-25F7 (25F7) BMS-986016 MDX-26H10(26H10) BAP050 humanizedILLAG5251 kd [1 / s] 5.00AND-06 3.87AND-05 1.95 E-04 2.21 E-04 9.48AND-05 3.86 E-043.99 E-04Epitope Bin E3 E3 E3 E2b E3 E5(D1loop) E5 E4 E2b MHCII /IC50 ELISA [nM] 0.9 0.8 0.9 0.9 0.9 0.8 /0.6 / 0.4 0.9 /0.6 / 1.0 CHO ELISA inflection point[ng / ml] 30.9 41.3 48.1 37.2 27.8 75
HUMAN LAG3 ELISA [00249] Nunc maxisorp plates (Nunc 464718) were coated with 25 μΙ / well of recombinant Human Chimera Fc LAG-3 Protein (R&D Systems, 2319-L3) at a protein concentration of 800 ng / ml and incubated at 4 ° C overnight or for 1 h at room temperature. After washing (3 x 90 μΙ / well with PBST buffer), each well was incubated with 90 μΙ of blocking buffer (PBS + 2% BSA + 0.05% Tween 20) for 1 h at room temperature. After washing (3 x 90 μΙ / well with PBST buffer) 25 μΙ of anti-Lag3 samples were added at a concentration of 1-9 μΙ / mL (1: 3 dilutions in OSEP buffer) and incubated for 1 h at room temperature. After washing (3x90 μΙ / well with PBST buffer) 25 μΙ / well of goat anti-human IgG κ chain antibody conjugated with HRP (Milipore, AP502P) in a 1: 2000 dilution and incubated at room temperature for 1 h . After washing (3 x 90 μΙ / well with PBST buffer), 25 μΙ / well of TMB substrate (Roche, 11835033001) was added and incubated for 2-10 min. The measurement took place in
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111/126 a Tecan Satire 2 instrument at 370 ° C / 492 nm.
LAG3-BINDING ELISA ON THE CELL SURFACE [00250] 25 μΙ / well of Lag3 cells (recombinant CHO cells expressing Lag3, 10,000 cells / well) were seeded in 384-well plates treated with tissue culture (Corning, 3701) and incubated at 37 ° C for one or two days. The next day after removing the medium, 25 μΙ of anti-Lag3 samples (1: 3 dilutions in OSEP buffer, starting at a concentration of 6-40 nM) were added and incubated for 2 h at 4 ° C. After washing (1 x 90 μΙ in PBST) the cells were fixed by adding 30 μΙ / well of glutaraldehyde to a final concentration of 0.05% (Sigma Cat. No: G5882), 10 min at room temperature. After washing (3 x 90 μΙ / well with PBST buffer) 25 μΙ / well of HRP-conjugated goat anti-IgG human κ chain antibody (Milipore, AP502P) was added in a 1: 1000 dilution and incubated at RT for 1 h. After washing (3 x 90 μΙ / well with PBST buffer), 25 μΙ / well of TMB substrate (Roche, 11835033001) was added and incubated for 6 to 10 min. The measurement took place on a Tecan Satire 2 instrument at 370/492 nm.
SPR (Biacore) characterization of anti-LAG3 antibodies [00251] An assay based on surface plasmon resonance (SPR) was used to determine the kinetic parameters of the binding between anti-Lag3 antibodies as monovalent Fab fragments and extracellular domains of human Lag3 ( ECD) labeled with human Fc at 25 ° C.
[00252] Thus, two flow cells from a C1 biosensor chip were prepared in a Biacore T200 immobilizing neutravidine, diluted to 25 pg / ml in acetate buffer at pH 4.5, using the “immobilization assistant”. This yielded immobilization levels of around 1900 RU. Then, the Biotin Anti-IgG-Fc (Human) Conjugate CaptureSelect ™ was turned on
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112/126 to neutravidine, using a 20 pg / ml dilution in running buffer (HBSEP +, GE Healthcare).
[00253] The method itself consisted of four commands per cycle. First command: capture of ~ 46 RU of huLag3-Fc (20s, 10 pl / min). Second command: sample injection for 120s followed by a long dissociation of 1200s at a flow rate of 30 pl / min. Third and fourth command: regeneration by the injection of Glycine-HCI pH 1.5 for 30 seconds.
[00254] A dilution series (3.13 nM - 200 nM, twice dilution in running buffer) of each Fab antibody fragment and additional blank cycles were then measured using the method previously described. The Biacore T200 evaluation software was then used to obtain kinetic values by applying a 1: 1 langmuir adjustment with the adjustment parameter Rmax defined as local ’, since the capture levels were not perfectly reproducible. The results are shown in table 2.
[00255] An assay based on surface plasmon resonance (SPR) was used to determine the apparent affinities of the interaction between Lagag3 ligands in their bivalent format and human Lag3 extracellular domains (ECDs) at 25 ° C.
[00256] Thus, a Biacore biosensor chip was prepared in a Biacore T200, immobilizing a minimum of about 800 RU of specific antibody for P329G point mutation, using standard amine coupling conditions.
[00257] Then, in each cycle, the sample antibody was captured and a concentration of a series of huLag3 ECD concentrations (consisting of four concentrations in total) was applied to the system for 200s, followed by a long dissociation of 1200s. The biosensor chip was then regenerated.
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113/126 [00258] The resulting experimental data was evaluated using the ‘Interaction Map’ feature provided by the Ridgeview Diagnostics TraceDrawer software, to calculate the individual apparent binding contribution of each sample.
[00259] The results are shown in table 2.
Epitope mapping [00260] Epitope storage (binning) was performed using an assay based on surface plasmon resonance (SPR). Therefore, aLag3 ligands were linked to huLag3 in a Biacore T200 instrument. Then, the accessibility of other ligands to the aLag3 - huLag3 binder complex previously formed was evaluated.
[00261] A SA CAP kit (GE Healthcare) was used to perform this test. If not described otherwise, the test was performed according to the SA CAP Kit manual.
[00262] The race included only one type of cycle. After hybridization, a 10 nM dilution of biotinylated huFc-labeled huLag3 was allowed to bind streptavidin on the sensor chip for 20 s at a flow rate of 10 μΙ / min. Then, a first 200 nM sample diluted in running buffer was injected for 180 seconds at a flow rate of 30 μΙ / min, immediately followed by a second sample under the same conditions. The surface was then regenerated.
[00263] The samples were then assigned to different groups of epitopes with similar competition patterns. A first rough categorization was made, based on the relative response of the second injection using a 6.1 RU threshold, which was just above the highest value observed when a binder was injected as the first and second sample. All values and decisions were finally validated by visual inspection of the sensors.
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114/126 [00264] The results are shown in table 2. Three main epitope patterns have been identified (E1, E2 and E3). As aLag30416 and humanized BAP 050 share the same group, but are not completely inhibited, they were assigned to subgroups E2b and E2c.
Binding of anti-Lag3 antibodies from tg rabbits to recombinant cyno Lag3 HEK cells [00265] In addition to the binding analysis using recombinantly HEK cells expressing human Lag3 at the surface, binding to cynomolgus Lag3 positive HEK cells was also evaluated. For this experiment, the frozen HEK293F cells, previously transiently transfected with cyno-LAG-3, were thawed, centrifuged and resupplied in PBS / 2% FBS. 1.5 x 10 ⁵ cells / well were seeded in 96-well plates. Anti-Lag3 antibodies were added to a final normalized concentration of 10 pg / ml. For reference and as controls, autofluorescence antibodies and positive control (Medarex 25F7) as well as isotype control (Sigma hulgGI, cat.no # 15154, data not shown,) were prepared and measured in the experiment. HEK cells were incubated with the indicated antibodies for 45 min on ice, washed twice with 200 μΙ of cold PBS buffer containing 2% FBS, before the secondary antibody (APC-labeled goat anti-human IgG-kappa, Invitrogen, cat.no # MH10515) was added (1:50 diluted in FACS-Puffer / well) and further incubated for 30 min on ice. The cells were again washed twice with 200 μΙ of cold PBS / 2% FBS buffer before the samples were finally resuspended in 150 μΙ of FACS buffer and the binding was measured on the FACS CANTO-II HTS Module.
Results [00266] The table below shows the binding and cross-reactivity of different anti-Lag3 antibodies to HEK293 cells that express
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115/126 cynol_AG3, binding given in% of positive cells or the GeoMean of the signal strength:
LAG3 Antibody % pos GeoMean Reference LAG3 antibody MDX25F7 41.2 3062 aLAG3 (0411) 88.6 11007 aLAG3 (0414) 81.6 9169 aLAG3 (0416) 67.9 4221 aLAG3 (0417) 75.9 7115 aLAG3 (0403) 82.0 7457
Binding of anti-Lag3 antibodies from tg rabbits to (activated) cells
PBMC / T EXPRESSING CYNOMOLGUS LAG3 [00267] After binding to recombinant Lag3 protein and Lag3 expressed recombinantly in mammalian cells, binding to Lag3 expressed in activated Cynomolgus T cells was evaluated / confirmed.
[00268] The binding characteristics of the newly generated antiLag3 antibodies (derived from Roche's transgenic rabbits) to Lag3 expressed on the cell surface of cynomolgus T cells or PBMC were confirmed by FACS analysis. Although Lag3 is not expressed in naive T cells, it is upregulated on activation and / or in depleted T cells. Thus, peripheral cynomolgus blood (PBMC) mononuclear cells were prepared from fresh cynomolgus blood and were then activated by pretreatment with CD3 / CD28 (1 pg / ml) for 2-3 days. Activated cells were subsequently analyzed for LAG-3 expression: Briefly, 1-3x10 ⁵ activated cells were stained for 3060 minutes on ice with the indicated anti-LAG3 antibodies and respective control antibodies at 10 pg / ml final concentration. Bound antiLag3 antibodies were detected via anti-human IgG conjugated to
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116/126 fluorochrome or secondary anti-rabbit IgG antibodies. After staining, the cells were washed twice with PBS / 2% FCS and analyzed on a FACS Fortessa (BD).
Results [00269] The following table summarizes the percentage of Lag3 positive cells within activated cynomolgus PBMCs:
Anti-Lag3 / Ctrl Antibodies % of cyano positive cells (PBLs) after activation by CD3 / CD28 only 2 Ab (hu) 7.62 DP47 (human isotype) 9.19 Reference antibody LAG3(MDX25F7) 22.1 Reference antibody LAG3BMS-986016 18.6 Reference antibody LAG-3(Humanized BAP050(LAG525)) 50.7 only 2 Ab (rb) 5.26 aLAG3 (0403) 44.2 aLAG3 (0411) 46.6 aLAG3 (0414) 43.0 aLAG3 (0416) 38.9 aLAG3 (0417) 35.3
[00270] In activated Cynomolgus T cells, all rabbit anti-Lag3 antibodies demonstrated significant binding to Lag3 ⁺ cells. Hereby, all newly generated antibodies showed an increased percentage of positive cells compared to
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117/126 human anti-Lag3 reference antibodies (for example, as MDX25F7, BMS-986016).
Inhibition of LAG-3 binding to MHC-II expressed in A375 HUMAN tumor cells (BY ELISA) [00271] 25 μΙ / well of A375 cells (10,000 cells / well) were seeded in 384-well plates treated with tissue culture ( Corning, 3701) and incubated at 37 ° C overnight. Anti-Lag3 antibodies were preincubated for 1 h with biotinylated Lag3 (250 ng / ml) in cell culture medium at 1: 3 dilutions starting with 3 pg / ml antibody concentration. After removing the medium from the wells with the seeded cells, 25 μΙ of the pre-incubated Lag3 antibody mixtures were transferred to the wells and incubated for 2 h at 4 ° C. After washing (1 x 90 μΙ in PBST) the cells were fixed by adding 30 μΙ / well of glutaraldehyde to a final concentration of 0.05% (Sigma Cat. No: G5882), 10 min at room temperature. After washing (3 x 90 μΙ / well with PBST buffer) 25 μΙ / well of Poly-HRP40-Streptavidin (Fitzgerald, 65R-S104PHRPx) was added in a dilution of 1: 2000 or 1: 8000 and incubated at room temperature for 1 H. After washing (3 x 90 μΙ / well with PBST buffer), 25 μΙ / well of TMB substrate (Roche, 11835033001) was added and incubated for 2 to 10 minutes. The measurement took place on a Tecan Safire 2 instrument at 370/492 nm.
Inhibition of the binding of LAG-3 to MHC-II expressed in A375 HUMAN tumor cells (BY FACS ANALYSIS)
Assay principle [00272] To study the antagonistic function of anti-Lag3 antibodies, an MHCII: Lag3 competition assay was performed. Human A375 MHCII ⁺ cells were stained with biotinylated Lag3: Fc fusion protein generated in-house, with or without pre-incubation with anti-Lag3 antibodies. It is
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118/126 analysis was studied in a competition experiment with FACS: A375 cells (ATCC, # CRL-1619) were cultured for 2-3 passages in EM Eagle medium supplemented with EBSS (PAN, Catalog No. # P04-00509) , 10% FBS, 2mM L-glutamine, 1x NEAA and 1x sodium pyruvate. All antibodies were diluted in FACS buffer to a final concentration of 20 pg / ml in 25 μΙ (in 96 U-well plates). 25 μΙ of internally generated recombinant biotinylated fusion protein LAG-3: Fc was added to a final concentration of 10 pg / mL, either to the anti-Lag3 antibody or medium or to the controls, and were preincubated for 30 min at room temperature. A375 cells were washed with PBS, and adjusted to 3x10 ⁶ cells / ml in PBS. 100 pL were seeded per well in a 96-well V-bottom plate. The plates were centrifuged and the supernatant was removed. Then, the pre-incubated LAG-3: Fc fusion protein / antibody mixture (50 µl / well) was added to the cells and incubated for 1 h at room temperature. After that, the cells were washed with 200 µl of FACS buffer. For detection of biotinylated Lag3: Fc protein bound to cellular MHCII, a goat anti-biotin antibody conjugated to 3 pl / sample APC (Miltenyi Biotec, Cat. No. 130-090-856) was used and incubated for more 10-15 minutes. After staining, the cells were washed again and then transferred in 150 µl of FACS buffer (PBS / 2% FBS) to a U-bottom plate and analyzed on a FACS Canto-ll using an HTS module.
[00273] Two anti-Lag3 antibodies (clones 25F7 and 26H10; Medarex) served as positive controls and a human IgG1 (Sigma, cat. No. 15154) as an appropriate isotype control. All antibodies were used at a final concentration of 10 pg / ml.
Results [00274] The result of the FACS analysis is shown in the table below, showing the percentage inhibition of Lag3 protein binding to
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MHC-II in cells (calculated as the reduced binding signal with reference to the maximum value in the absence of a blocking antibody):
LAG3 antibody. % inhibition aLAG3 (0403) 34.9 aLAG3 (0414) 67.3 aLAG3 (0411) 45.6 aLAG3 (0416) 68.6 aLAG3 (0417) 59.1 Reference MDX25F7 70.0 Reference MDX26H10 71.7 Isotype control -2.9 No mAb 0.0
[00275] These data support a functional interaction with Lag3 and block the cellular interaction of all antibodies tested.
Neutralizing potency of new anti-LAG3 antibodies in a LAG-3 Bio / Reporterassay blocking pattern [00276] To test the neutralizing potency of new anti-Lag3 antibodies in restoring a suppressed T cell response in vitro, a commercially reporter system was used available. This system consists of effector cells Lag3 ⁺ NFAT Jurkat (Promega, cat. No. # CS194801), Raji MHC-II ⁺ cells (ATCC, # CLL-86) and a super antigen. In summary, the reporter system is based on three steps: (1) activation of NFAT cells induced by superantigen; (2) inhibition of the activation signal mediated by the inhibitory interaction between MHCII (Raji cells) and effector cells Lag3 ⁺ NFAT Jurkat; (3) recovery of the NFAT activation signal by Lag3 antagonist / neutralizing aVH-Fc fusion constructs.
[00277] For this experiment, the effector T cells Raji and
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Lag-3 ⁺ Jurkat / NFAT-luc2 were grown as described by the supplier. Serial dilutions (40 pg / ml to 50 pg / ml) of various anti-Lag3 and reference antibodies were prepared in assay medium (RPMI 1640 (PAN Biotech, cat. No. # P04-18047), 1% FCS ) in 96-well white flat-bottom culture plates (Costar, cat. No. # 3917). 1x10 ⁵ Lag3 cells ⁺ NFAT-Jurkat / well) were added to the antibody solution. After this step, 2.5 x 10 ⁴ Raji cells / well were added to the Jurakt cell / antibody mixture, as well as 50 ng / ml of final antigen concentration to super antigen SED (Toxin technology, cat. No. DT303). After a six-hour incubation at 37 ° C and 5% CO2, the Bio-GIo substrate (Promega, # G7940) was warmed to room temperature and 75 pl were added per well, incubated for 5-10 min before luminescence overall was measured on a Tecan Infinite reader according to the kit manufacturer's recommendation.
[00278] In the diagrams, the restoration of an MHCII / Lag3-mediated suppression of the NFAT luciferase signal by different anti-Lag3 antibodies in SED stimulation (given as EC50 values) is shown:
Anti-LAG3 EC50 [nM] in Jurkat LAG3 + SED + Raji 1st ^the essay 2nd ^the essay 3rd ^the rehearsal Reference MDX25F7 7.8 / 5.9 8.6 nt Reference BMS-986016 nt 9.6 nt Reference humanized BAP050 (LAG525) nt 22.6 nt Lag3 IgG -Fc nt no effect nt aLAG3 (0411) 1.1 1.0 nt aLAG3 (0414) 1.1 1.0 1.8 aLAG3 (0416) 3.1 2.5 3.5 aLAG3 (0417) 1.0 nt nt
nt. molecules not tested in this experiment
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Example 3: Biological Activity in different tests: Effect of different
ANTI-LAG3 ANTIBODIES (ONLY OR IN COMBINATION WITH ANTIPD1 ANTIBODIES)
Table 3: Summary of the biological activity of different antiLAG3 antibodies (ALONE OR IN COMBINATION WITH ANTI-PP1 ANTIBODIES)
Test type Anti-Lag3 aLAG3 (0403) Anti-Lag3 aLAG3 (0411) Anti-Lag3 aLAG3 (0414) Anti-Lag3 aLAG3 (0416) Anti-Lag3 aLAG3 (0417) Reference1 BMS 986016 Reference 2 ΒΑΡ050 humanized (LAG525) mMLR(GrzB) + - +++ ++ + - ++ mMLR (IL-2) - - + + ++ + ++ CD4 + ARH77 +++ ++++ + Treg Suppression (GrzB) +++ +- + Treg suppression (IFN-g) +++ +++ + PBMCs of melanoma patients +++
Effect of blocking PD-1 and LAG-3 on the cytotoxic release of Granzyme
B SECRETION OF IL-2 BY HUMAN CD4 T CELLS CO-CULTIVATED WITH ALLOGENIC MATURE DENDRITIC CELLS [00279] To screen for antiLAG3 blocking antibodies in combination with anti-PD-1 in an allogeneic environment, we have developed an assay in which recently CD4 T cells Purified cells are co-cultured for 5 days in the presence of allogeneic mature dendritic cells (mDCs) derived from monocytes. Monocytes were isolated from fresh PBMCs one week before by plastic adhesion followed
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122/126 by removing non-adherent cells. Then, we generated immature DCs from monocytes by cultivating them for 5 days in medium containing GM-CSF (50 ng / ml) and IL-4 (100 ng / ml). To induce maturation of the iDCs, we add TNF-alpha, IL-1beta and IL-6 (50 ng / ml each) to the culture medium for an additional 2 days. Then, the maturation of the DCs was evaluated by measuring the surface expression of the Class II Major Histocompatibility Complex (MHCII), CD80, CD83 and CD86 by flow cytometry (LSR Fortessa, BD Biosciences).
[00280] On the day of the minimal mixed lymphocyte reaction (mMLR), CD4 T cells were enriched using a microsphere kit (Miltenyi Biotec) from 108 PBMC obtained from an unrelated donor. Before culturing, CD4 T cells were labeled with 5 μΜ of Carboxy-Fluorescein-Succinimidyl Esther (CFSE). 10 ⁵ CD4 T cells were then plated on a 96-well plate along with mature aloDCs (5: 1) in the presence or absence of aPD1 blocking anti-PD-1 antibody (0376) (= PD1-0103-0312, from PCT Order
PCT / EP2016 / 073248) alone or in combination with chimeric anti-LAG3 antibodies (aLAG3 (0403) to aLAG (0418) ((0403) to (0418)) or reference antibodies (humanized BAP050 (LAG525) and BMS 986016), at a concentration of 10 pg / ml DP47 is a non-binding human IgG with a LALA mutation in the Fc portion to avoid recognition by FcyR and was used as a negative control.
[00281] Five days later, we collected the cell culture supernatants, used later to measure IL-2 levels by ELISA (R&D systems), and left the cells at 37 degrees Celsius for another 5 hours in the presence of Golgi Plug (Brefeldin A) and Golgi Stop (Monensin). The cells were then washed, stained on the surface with anti-human CD4 antibody and the fixable dye Live / Dead Aqua (Invitrogen) before being
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123/126 fixed / permeabilized with Fix / Perm Buffer (BD Bioscience). We performed intracellular staining for Granzima B (BD Bioscience) and IFN-γ (eBioscience). The results are shown in Figures 1A and B.
Effect of PD-1 and LAG-3 blockade on the release of cytotoxic Granzyme B BY HUMAN CD4 T-CELLS CO-CULTIVATED WITH A B-CELL LYMPHBLATOID CELL LINE (ARH77).
[00282] In functional studies, we co-cultured CD4 T cells with the tumor cell line ARH77, a B-cell lymphoblastoid cell line that expresses lower levels of PDL-1 than CDMs, to better characterize the contribution of LAG- antagonism. 3 for block PD1. The rest of the setup and experimental reading remained unchanged from mMLR. Our anti-LAG3 antibodies (aLAG3 (0414) and aLAG3 (0416), chosen based on their ability to co-secrete IL2 and Granzima B in the mMLR) in combination with the anti-PD-1 antibody, showed a significant increase in secretion of Granzyme B by CD4 T cells than the reference anti-LAG3 antibodies ((humanized BAP050 (LAG525) and BMS 986016)) (P <0.05) and anti-PD-1 isolated (P <0.01) , Figure 2.
Effect of blocking PD-1 and LAG-3 on Treg suppression of Granzyme B and IFN-γ release by human CD4 T cells co-cultured with RADIATED ALLOGENIC PBMC.
[00283] In functional studies involving suppression tests of regulatory T cells (Treg), from the same donor PBMCs were split into two samples: one was enriched on CD4 T cells and the other in Tregs defined as CD4 CD25 ^high cells CD127 ^low through of a microsphere kit (Miltenyi Biotec). Once both populations were purified, CD4 T cells were labeled with 5 μΜ of Carboxy-Fluorescein-Succinimidyl Esther (CFSE) while Tregs with 5 μΜ Cell-Trace-Violet (CTV) to be able to distinguish them in FACS later .
Petition 870190089671, of 10/09/2019, p. 254/271
124/126 [00284] Both CD4 (10 ⁵ ) and Tregs (10 ⁵ ) T cells were then co-cultured in a 1: 1 96-well plate together with irradiated PBMCs (10 ⁵ ) from a non-donor related in the presence or absence of our anti-LAG-3 antibodies (aLAG-3 (0414) and aLAG-3 (0416) or reference the anti-LAG3 antibodies (humanized BAP050 (LAG525) and BMS 986016) in combination with our antibody anti-PD-1 at a concentration of 10 pg / ml As a control to estimate the magnitude of the suppression of effector functions of CD4 T cells by Tregs, CD4 T cells (10 ⁵ ) were also co-cultured with irradiated PBMC (10 ⁵ ) in the absence of Tregs.
[00285] Five days later, cell culture supernatants were collected, used later to measure IFNy levels by ELISA (R&D Systems), and the cells were left at 37 degrees Celsius for an additional 5 hours in the presence of Golgi Plug ( Brefeldina A) and Golgi Stop (Monensina). The cells were then washed, stained on the surface with anti-human CD4 antibody and the fixable dye Live / Dead Aqua (Invitrogen) before being fixed / permeabilized with Fix / Perm Buffer (BD Bioscience). We performed intracellular staining for Granzima B (BD Bioscience) and IFN-γ (eBioscience). The results are shown in Figures 3A and B.
[00286] The anti-LAG3 antibodies (aLAG3 (0414) and aLAG3 (0416), in combination with the anti-PD-1 antibody aPD1 (0376) (= PD1-01030312, from PCT application PCT / EP2016 / 073248) elicited escape of Tconv from tight control of regulatory T cells, as demonstrated by the secretion of significantly greater amounts of Granzima B than Tconv in the presence of anti-DP-1 antibodies alone (P <0.05) or in the absence of checkpoint inhibitors (P <0.001) Reference anti-LAG3 antibodies (humanized BAP050 (LAG525) and BMS 986016) in combination with non-PD-1
Petition 870190089671, of 10/09/2019, p. 255/271
125/126 significantly rescued the effector functions of Tconv from the suppression of Treg. Similar results were obtained for IFN-g even if the difference did not reach statistical significance with only 4 donors.
Effect of blocking PD-1 and LAG-3 on the secretion of Granzyme B and IFN-r by CD4 T CELLS OF PBMCS OF MELANOMA PATIENTS AFTER CONVOCATION WITH IMMUNOGENIC POOLS OF ANTIGEN-MELANOMA PEPTIDES [00287] It has been previously described that PBMCs of melanoma patients contain detectable frequencies of tumor antigen-specific T cells. Therefore, for POC purposes, we tested the anti-LAG3 antibody (0414) plus anti-PD-1 versus or anti-PD-1 alone in PBMCs from a patient with restimulated melanoma overnight with peptide pools of antigens associated with immunogenic melanoma.
[00288] 10 ⁵ to 10 ⁶ PBMCs from melanoma patients, were incubated at room temperature in the presence or absence of saturating concentrations (10 pg / ml) of anti-DP-1 antibodies alone (0376), in combination with anti-LAG3 (aLAG3 (0414) = (0414), 10 pg / ml) of antibody. The T cells were then re-stimulated overnight with a pool of immunogenic tumor related antigens such as MAGEA1, MAGEA3, MAGEA4, Melan-A / MART-1, NYESO-1, melanocyte protein Pmel 17 gp100, tyrosinase, protein 2 related to tyrosinase in the presence of protein transport inhibitors, Golgi Plug (Brefeldin A) and Golgi Stop (Monensin).
[00289] The cells were then washed, stained on the surface with anti-human CD4 antibody and the fixable dye Live / Dead Aqua (Invitrogen) before being fixed / permeabilized with Fix / Perm Buffer (BD Bioscience). We performed intracellular staining for Granzima B (BD Bioscience) and IFN-γ (eBioscience).
[00290] The combination of anti-LAG3 and anti-PD-1 antibodies (P <0.01 and P <0.001) significantly (P <0.01 and P <0.0001) increased the
Petition 870190089671, of 10/09/2019, p. 256/271
126/126 effector functions of tumor antigen-specific T cells (ie, secretion of Granzyme B and IFN-γ), while blocking PD-1 alone showed no effect (data not shown).
[00291] Similarly, a person skilled in the art can extend the above mentioned in cell culture / animal studies of example 3 for human treatment methods.

权利要求:
Claims (20)
[1]
Claims
1. ISOLATED ANTIBODY that binds to human Iag3, characterized by the antibody comprising
A) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 5; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or
B) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 9; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 10; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 11; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 13; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or
C) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 20; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 21; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or
D) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 26; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 27; (d) HVR-L1 comprising the amino acid sequence of
Petition 870190089671, of 10/09/2019, p. 258/271
[2]
2/7
SEQ ID NO: 28; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or
E) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 35; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 36; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 37; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
2. ANTIBODY according to claim 1, characterized in that the antibody comprises
A) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 5; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or
B) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 35; (d) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 36; (e) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 37; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
[3]
3. ISOLATED ANTIBODY that binds to human Iag3, characterized by the antibody comprising
Petition 870190089671, of 10/09/2019, p. 259/271
3/7
A) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or
B) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or
C) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or
D) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ
Petition 870190089671, of 10/09/2019, p. 260/271
[4]
4/7
ID NO: 27; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or
E) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 35; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37 ° C and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
4. ANTIBODY according to claim 3, characterized in that the antibody comprises
A) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5 and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or
B) (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 and (iii) HVR -H3 comprising an amino acid sequence selected from SEQ ID NO: 35; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (ii) HVR-L2 comprising the
Petition 870190089671, of 10/09/2019, p. 261/271
[5]
5/7 amino acid sequence of SEQ ID NO: 37 ° C and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.
5. ISOLATED ANTIBODY BINDING TO HUMAN LAG3, characterized by the antibody
i) comprise a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8;
ii) comprising a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16;
iii) comprising a VH sequence of SEQ ID NO: 23 and a VL sequence of SEQ ID NO: 24;
iv) comprising a VH sequence of SEQ ID NO: 31 and a VL sequence of SEQ ID NO: 32; or
v) comprise a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ ID NO: 40.
[6]
6. ANTIBODY according to claim 5, characterized by the antibody
i) comprise a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8; or ii) comprise a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ ID NO: 40.
[7]
7. ISOLATED ANTIBODY BINDING TO HUMAN LAG3, characterized by the antibody:
i) compete for binding to LAG3 with an anti-LAG3 antibody comprising VH with the amino acid sequence of SEQ ID NO: 7 and VL with the amino acid sequence of SEQ ID NO: 8 and / or ii) binding to a LAG3 human and cinomolgo; and / or iii) inhibit MHC-II binding expressed in human A375 tumor cells; and / or
Petition 870190089671, of 10/09/2019, p. 262/271
6/7 iv) increase the release of Granzyme B or IL-2 in a mixed lymphocyte reaction (mMLR) assay.
[8]
ANTIBODY according to any of claims 1 to 7, characterized in that it is a human, humanized or chimeric antibody.
[9]
ANTIBODY according to any one of claims 1 to 8, characterized in that it is a full-length IgG1 antibody with mutations L234A, L235A and P329G (numbered according to the EU index of Kabat).
[10]
10. ISOLATED NUCLEIC ACID, characterized by encoding the antibody, as defined in any one of claims 1 to 9.
[11]
11. HOSTING CELL, characterized by comprising the nucleic acid, as defined in claim 10.
[12]
12. METHOD TO PRODUCE AN ANTIBODY, characterized by comprising the culture of the host cell, as defined in claim 11, so that the antibody is produced.
[13]
13. METHOD according to claim 12; characterized by further comprising the recovery of the host cell antibody.
[14]
14. PHARMACEUTICAL FORMULATION, characterized in that it comprises the antibody, as defined in any one of claims 1 to 9, and a pharmaceutically acceptable carrier.
[15]
ANTIBODY according to any one of claims 1 to 9, characterized in that it is for use in the treatment of cancer.
[16]
ANTIBODY according to any one of claims 1 to 9, characterized in that it is for use as a medicine.
[17]
ANTIBODY according to any one of claims 1 to 9, characterized in that it is for use in the treatment of cancer.
[18]
18. USE OF THE ANTIBODY, as defined in any
Petition 870190089671, of 10/09/2019, p. 263/271
7/7 one of claims 1 to 9, characterized in that it is in the manufacture of a medicament.
[19]
19. USE, according to claim 18, characterized by the medication being for the treatment of cancer.
[20]
20. METHOD FOR TREATING AN INDIVIDUAL HAVING CANCER, characterized by the method comprising administering to the individual an effective amount of the antibody, as defined in claim 1.

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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EP17164917|2017-04-05|

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PCT/EP2018/058385|WO2018185046A1|2017-04-05|2018-04-03|Anti-lag3 antibodies|

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