Anti-PD-L1 antibodies

Abstract

The present invention relates to antigen-binding molecules, in particular antibodies, fragments and variants thereof, that bind to programmed death-ligand 1 (PD-L1) in a pH-dependent manner and compete with PD-L1 binding to the inhibitory receptor programmed death 1 polypeptide (PD-1) and the costimulatory molecule CD80, and the use of such antigen-binding molecules in the treatment and / or prevention of diseases such as cancer.

Description

[Technical field]

[0001] The present invention relates to antigen-binding molecules, in particular antibodies, fragments and variants thereof, that bind to Programmed Death Ligand 1 (PD-L1) in a pH-dependent manner and compete with PD-L1 binding to the inhibitory receptor Programmed Death 1 Polypeptide (PD-1) and the costimulatory molecule CD80, and the use of said antigen-binding molecules in the treatment and / or prevention of diseases such as cancer. [Background technology]

[0002] Costimulation by two different signals to T cells is an important mechanism to coordinate and tightly regulate immune response activation of resting T lymphocytes by antigen-presenting cells (Non-Patent Document 1). The initial antigen-specific signal is transmitted through the T cell receptor (TCR) after recognition of foreign peptide antigens presented by MHC. The second signal is sent to T cells by costimulatory molecules expressed on antigen-presenting cells (APCs) to induce clonal proliferation, cytokine secretion and effector functions of T cells (Non-Patent Document 2). In the absence of costimulation, T cells may become refractory to antigen stimulation and may not mount an effective immune response, leading to exhaustion or tolerance to foreign antigens.

[0003] T cells receive both positive and negative secondary costimulatory signals, and regulation of such signals is crucial to maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative secondary signals appear to be required for the induction of T cell tolerance, while positive signals promote T cell activation. Although the simple two-signal model still provides a plausible explanation for naive lymphocytes, the host immune response is a dynamic process, and costimulatory signals can also be provided to antigen-exposed T cells.

[0004] The mechanism of costimulation is therapeutically important, as manipulation of costimulatory signals has been shown to provide a means to enhance or halt cell-based immune responses. It has been discovered that T cell dysfunction or anergy occurs concomitantly with induction and sustained expression of the inhibitory receptor programmed death 1 polypeptide (PD-1). Two PD-1 ligands have been described: PD-L1 / B7H1 / CD274 and PD-L2 / B7-DC / CD273. PD-L1 is expressed at low levels on immune cells such as B cells, dendritic cells, macrophages, and T cells, and is upregulated after activation. PD-L1 is also expressed in non-lymphoid organs such as endothelial cells, heart, lung, pancreas, muscle, keratinocytes, and placenta. Expression in non-lymphoid tissues suggests that PD-L1 may regulate the function of autoreactive T and B cells and myeloid cells in peripheral tissues, or may regulate inflammatory responses in target organs. PD-L1 expression is primarily regulated by type 1 and type 2 interferons, which are key regulators of PD-L1 in endothelial and epithelial cells. PD-L1 is expressed in tumor samples and is associated with poor prognosis (Non-Patent Document 3). PD-L2 / B7-DC cell surface expression is primarily restricted to macrophages and dendritic cells. PD-L1 is abundantly found in a variety of human cancers (Non-Patent Document 4), and the interaction between PD-1 and PD-L1 results in a reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and immune evasion by cancerous cells (Non-Patent Document 5, Non-Patent Document 6, Non-Patent Document 7). Immune suppression can be reversed by inhibiting the local interaction of PD-1 and PD-L1 (Non-Patent Document 8). Inhibition of PD-L1 signaling has been shown to enhance T cell immunity for the treatment of cancer (Non-Patent Document 9).

[0005] Immunotherapy with checkpoint inhibitors has now become a standard procedure for an ever-increasing number of cancer types. To date, one anti-CTLA-4 antibody (ipilimumab), two anti-PD-1 antibodies (pembrolizumab and nivolumab) and three anti-PD-L1 antibodies (atezolizumab, avelumab and durvalumab) have been approved and have shown clear benefits through numerous clinical trials. Immune-related adverse events associated with these agents often affect the skin, colon, endocrine glands, lungs and liver. These immune-related adverse events (AEs) occur across different tumor types, with events of all grades occurring in approximately 90% of patients treated with anti-CTLA-4 ipilimumab and approximately 70% of patients treated with any anti-PD-1 or anti-PD-L1 antibody (Non-Patent Document 10). For combination checkpoint inhibitor treatment, the proportion of immune-related AEs of grades 3-4 is significantly higher (Non-Patent Document 11).

[0006] Warburg was the first to report that the energy metabolism of cancer cells is mainly limited to glycolysis even in the presence of oxygen (Non-Patent Document 12). Increased glycolysis allows the diversion of glycolytic intermediates to various biosynthetic pathways, including those that generate nucleosides and amino acids, thereby promoting the biosynthesis of macromolecules and organelles required to support the assembly of new cells and active cell proliferation in neoplastic diseases (Non-Patent Document 13). The result of increased intracellular production of lactate is extracellular tumor acidosis. To maintain a slightly alkaline intracellular pH (pHi) (approximately pH 7.4), tumor cells secrete Na + / H + Exchanger, HCO3 - It upregulates several proton release mechanisms, such as transporters and carbonic anhydrase IX (Non-Patent Document 14). Excess protons are excreted into the extracellular matrix, making the extracellular pH (pHe) of the tumor microenvironment acidic. In certain tumor types, a pH as low as 6-6.5 has been measured (Non-Patent Document 15, Non-Patent Document 16). [Prior art documents] [Non-patent literature]

[0007] [Non-licensed document 1] Mondino and Jenkins, J Leukoc Biol. 1994;55:805-15

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0008] The present inventors have generated an anti-PD-L1 Fab with reduced activity at the physiological pH of 7.4 and maximal activity at the acidic pH of tumors, pH 6-6.5, to reduce peripheral checkpoint inhibitor treatment toxicity as described above while enhancing tumor neutralization. The present invention is particularly useful for reducing pneumonitis, broadly defined as inflammation of the lung parenchyma, which has been described in patients receiving anti-PD-1 or PD-L1 therapy, alone or in combination, and is more commonly seen in patients with lung cancer (Brahmer J et al. N Engl J Med 2015; 373: 123-135, Rivzi NA et al. J Clin Oncol 2015; 33: 15s).

[0009] The antigen-binding molecules of the present invention exhibited significantly higher affinity for PD-L1 at acidic pH (pH 6.0) compared to physiological pH (pH 7.4), which is in contrast to other PD-L1-binding molecules known in the art, which do not exhibit pH-specific binding, and many actually exhibit lower affinity at pH 6.0 compared to pH 7.4.

[0010] Therefore, the antigen-binding molecules of the present invention have much lower peripheral toxicity than PD-L1 antibodies known in the art. This means that tumor cell killing strategies (Fc-mediated or toxic drug conjugates) can be used only against tumor-positive PD-L1 cells, without destroying healthy cells. This reduces the serious side effects and peripheral checkpoint inhibitor treatment toxicity.

[0011] These anti-PD-L1 Fabs can be formatted as monoagents or in bispecific formats with other relevant cancer or immuno-oncology targets. In some embodiments, the antigen-binding molecule is monovalent for PD-L1 binding.

[0012] The PD-L1 antibody or composition may be combined with a treatment regimen that further comprises conventional therapy selected from the group consisting of radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, anti-angiogenesis, and palliative care for cancer therapy. The chemotherapy treatment may be selected from the group consisting of gemcitabine, cyclophosphamide, doxorubicin, paclitaxel, and cisplatin.

[0013] The present inventors have identified an antigen-binding molecule that specifically binds to PD-L1.

[0014] In one aspect, the invention provides an anti-PD-L1 antigen binding molecule comprising a VLCDR1 having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 16, 12, 10, 14, 18 or 2.

[0015] In a further aspect, the present invention provides an antigen binding molecule that specifically binds to PD-L1 and inhibits binding of PD-L1 to an antigen binding molecule of the invention.

[0016] In a further aspect, the present invention provides an antigen binding molecule that specifically binds to an epitope of human PD-L1, wherein the epitope consists of SEQ ID NO:19.

[0017] In a further aspect, the present invention provides an antigen binding molecule that specifically binds to an epitope of cynomolgus PD-L1.

[0018] In a further aspect, the invention provides an anti-PD-L1 antigen binding molecule that is an affinity matured mutant of the antibody 2A09.

[0019] In a still further aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding molecule of the present invention.

[0020] In a further aspect, the present invention provides a kit comprising the antigen-binding molecule of the present invention or the pharmaceutical composition of the present invention, and further comprising an additional therapeutically active agent.

[0021] In a further aspect, the present invention provides the antigen-binding molecule of the present invention or the pharmaceutical composition of the present invention for use in the treatment or prevention of cancer.

[0022] In a still further aspect, the present invention provides a method for treating or preventing a PD-L1 mediated disease or disorder comprising administering to a subject an antigen binding molecule or pharmaceutical composition of the present invention, wherein the PD-L1 mediated disease or disorder is cancer.

[0023] In a further aspect, the invention provides a method for inhibiting binding of human PD-L1 to PD-1 and / or CD80, or binding of a cell expressing PD-L1 to PD-1 and / or CD80, comprising contacting human PD-L1 or a cell expressing PD-L1 with an antigen binding molecule of the invention.

[0024] In a further aspect, the present invention provides a nucleic acid encoding the antigen-binding molecule of the present invention.

[0025] In a further aspect of the present invention, there is provided a vector or plasmid comprising a nucleic acid encoding the antigen-binding molecule of the present invention.

[0026] In a further aspect of the present invention, there is provided a host cell comprising a vector or plasmid containing a nucleic acid encoding an antigen-binding molecule of the present invention.

[0027] In a further aspect, the invention provides a method for producing a cell which expresses an anti-PD-L1 antigen binding molecule comprising transfecting a cell with a plasmid or vector containing a nucleic acid of the invention.

[0028] In a further aspect, the invention provides a method for producing an anti-PD-L1 antigen binding molecule comprising culturing a host cell according to the invention in cell culture medium under conditions that allow intracellular expression of the encoding nucleic acid sequence of the plasmid or vector. [Brief description of the drawings]

[0029] [Figure 1A] Figure 1 shows the results of PD-L1 neutralization ELISA of three lead mAbs, 1A06, 1E08 and 2A09. Anti-HEL mAb was used as a negative control. [Figure 1B] FIG. 1 shows the results of CD80 neutralization ELISA of the three lead mAbs 1A06, 1E08 and 2A09. [Diagram 2] Figure 2 shows the results of BIAcore analysis of anti-PD-L1 mAbs.The Kd (1 / s) of mAbs 2A09, 1A06 and 1E08 binding to PD-L1-Fc at pH 6.0 and pH 7.4. [Diagram 3] FIG. 1 shows binding of 2A09 mAb and Fab to MDA-MB-231 cells in Krebs buffer at pH 7.4, 6.5 and 6.0. [Figure 4] Figure 2 shows affinity matured anti-PD-L1 Fab binding to PD-L1 in KRB buffer at pH 7.4 and pH 6.0 by ELISA. [Diagram 5] Figure 1 shows affinity matured anti-PD-L1 Fab inhibition of PD-L1:PD-1 binding at pH 7.4, 6.5 and 6.0 in KRB buffer by ELISA. OD was normalized to the negative control Fab 2A11 (anti-HEL). [Figure 6] Figure 1 shows affinity matured anti-PD-L1 Fab binding to MDA-MB-231 cells with mAbs at pH 7.4, 6.5 and 6.0 in Krebs buffer. Anti-PD-L1 Fab 2.7A04OPT was used as a positive control. [Figure 7] FIG. 1 shows the heavy and light chain variable regions and CDRs of several antigen-binding molecules of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] As used herein, an "antigen-binding molecule" is a member of a pair of molecules that have binding specificity for each other. The members of the antigen-binding pair may be naturally occurring or wholly or partially synthetically produced. One member of the pair has an area on its surface, which may be a protrusion or a cavity, that specifically binds to, and is therefore complementary to, a particular spatial and polar configuration of the other member of the pair. Thus, the members of the pair have the property of specifically binding to each other. Examples of types of antigen-binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand and enzyme-substrate. The present invention generally relates to antigen-antibody type interactions. The antigen-binding molecule used in the present invention binds to PD-L1, human PD-L1, cynomolgus PD-L1, an epitope on PD-L1, an epitope on hPD-L1 or an epitope on cynomolgus PD-L1 with greater affinity than other molecules, i.e. specifically binds to PD-L1, hPD-L1, cynomolgus PD-L1, an epitope on PD-L1, an epitope on hPD-L1 and / or an epitope on cynomolgus PD-L1. The binding affinity of an antigen-binding molecule to PD-L1, hPD-L1, cynomolgus PD-L1, an epitope on PD-L1, an epitope on hPD-L1 or an epitope on cynomolgus PD-L1 is determined by the dissociation constant (K D The binding affinity of an antigen-binding molecule to PD-L1, hPD-L1, cynomolgus PD-L1, an epitope of PD-L1, an epitope of hPD-L1 or an epitope of cynomolgus PD-L1 can also be measured using the association constant (Ka). The K D Values ​​are the K of non-PD-L1 antigen-binding molecules. D lower than the value.

[0031] Antigen binding molecules that bind to PD-L1 and / or hPD-L1 include anti-PD-L1 antibodies.The antigen binding molecules used in the present invention are typically antibodies (including fragments thereof).

[0032] As used herein, PD-L1 may refer to human PD-L1 and / or cynomolgus PD-L1.

[0033] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partially or completely synthetically produced. The term also encompasses any polypeptide or protein having a binding domain that is or is homologous to an antibody binding domain. Antibodies can be polyclonal or monoclonal. They may be derived from natural sources or partially or completely synthetically produced. Antibodies are polypeptides that typically contain two identical heavy chains and two identical light chains that are smaller than the heavy chains. In mammals, there are two types of light chains called lambda (λ) and kappa (κ). Each of the heavy chains and each of the light chains are composed of a variable region and a constant region. The heavy chain variable region is referred to as the VH region and the light chain variable region is referred to as the VL region. For kappa light chains, the VL region is sometimes referred to as the VK region. Each of the light and heavy chain variable regions contains three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which are named VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3, respectively. Examples of antibodies are immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotype subclasses, and fragments containing antigen-binding domains such as Fab, F(ab')2, Fv, scFv, dAb, Fd, and diabodies are also contemplated in the present invention.

[0034] The antigen-binding molecule of the present invention is typically an antibody, more typically a monoclonal antibody. In a preferred embodiment, the antibody is a fully human monoclonal antibody in which a human constant region is used. In some embodiments, the monoclonal antibody of the present invention is a humanized antibody.

[0035] Methods for producing monoclonal antibodies are known to those of skill in the art, for example as described in Frenzel et al., "Expression of Recombinant Antibodies", Front Immunol, 2013, 4:217, the contents of which are incorporated herein by reference.

[0036] The monoclonal antibody of the present invention can be humanized by modifying the amino acid sequence of the antibody. Methods for reducing the immunogenicity of the antigen-binding molecule of the present invention include CDR grafting onto a suitable antibody framework scaffold, or variable surface residue remodeling, for example, by site-directed mutagenesis or other commonly used molecular biology techniques (Roguska et al Protein Eng. 9 895-904 (1996)).

[0037] Other applicable methods include the identification of potential T cell epitopes within the molecule and their subsequent removal (deimmunization), for example by site-directed mutagenesis. Humanization of antigen-binding molecules may be desirable when the molecules are used as therapeutic agents. Humanization of the CDR regions or surrounding framework sequences can be performed as desired.

[0038] It is possible to use monoclonal and other antibodies to produce other antibodies or chimeric molecules which retain the specificity of the original antibody, using techniques of recombinant DNA technology. Such techniques may involve introducing DNA encoding the immunoglobulin variable region or complementarity determining regions (CDRs) of an antibody into the constant regions or constant regions plus framework regions of a different immunoglobulin. The hybridoma or other cells which produce the antibodies may undergo genetic mutations or other changes which may or may not alter the binding specificity of the antibody produced.

[0039] In one embodiment, the heavy chain variable region and / or the light chain variable region are at least 85% humanized, at least 90% humanized, at least 95% humanized, at least 96% humanized, at least 97% humanized, at least 98% humanized, or at least 99% humanized. In some embodiments, the antibodies are conservatively humanized, e.g., to retain better antigen binding. Fewer antibody substitutions may be made in such conservatively humanized antibodies compared to humanized antibodies.

[0040] In some embodiments, the antigen-binding molecules of the present invention are deimmunized using methods such as those described in Jones et al., "Deimmunization of monoclonal antibodies", Methods Mol Biol, 2009, 525:405-23, the contents of which are incorporated herein by reference. Deimmunization removes T cell epitopes from the sequence using a combination of immunological and molecular biology techniques.

[0041] Thus, in some embodiments of the invention there is provided a deimmunized anti-PD-L1 antigen binding molecule, or antigen-binding fragment thereof, comprising deimmunized variants of the six CDR regions of an antibody selected from the group consisting of: 2A09, 8G08, 8D06, 8A04, 8B06, and 8D04. In further embodiments of the invention there is provided a deimmunized anti-PD-L1 antigen binding molecule, or antigen-binding fragment thereof, comprising deimmunized variants of the VH and / or VL sequences from an antibody selected from the group consisting of: 2A09, 8G08, 8D06, 8A04, 8B06, and 8D04. In yet further embodiments of the invention there is provided a deimmunized anti-PD-L1 antibody which is a deimmunized variant of an antibody selected from the group consisting of: 2A09, 8G08, 8D06, 8A04, 8B06, and 8D04.

[0042] The antigen-binding molecules and antigen-binding fragments thereof are based on one parent antibody 2A09. In addition to the parent antibodies, the invention particularly relates to affinity matured variants of the parent antibodies. The invention is also based on antibody fragments comprising one or more antigen-binding domains derived from the antibodies of the invention, as well as further variants such as antibodies or antigen-binding fragments thereof having an antigen-binding domain with one or more conservative amino acid substitutions. In one embodiment, the antibody or antigen-binding fragment thereof has an antigen-binding domain with 1-10 conservative amino acid substitutions. In one embodiment, the antibody or antigen-binding fragment thereof has an antigen-binding domain with 1-5 conservative amino acid substitutions. In one embodiment, the antibody or antigen-binding fragment thereof has an antigen-binding domain with one or two conservative amino acid substitutions. All antigen-binding molecules of the invention specifically bind to PD-L1.

[0043] The antigen-binding molecules, particularly antibodies, of the present invention can be of any suitable type, including IgA, IgD, IgE, IgG, IgM and IgY, although IgG may be preferred. An IgG1 backbone may be most preferred. In related embodiments, the constant region of the antibodies of the present invention may be modified for beneficial effects, such as increased stability and reduced Fcγ receptor interaction.

[0044] Such advantageous modifications include modifications or substitutions in the Fc region, such as those that enhance effector cell killing functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0045] In one embodiment, the Fc region is modified to enhance ADCC and / or ADCP and / or CDC activity, where enhancement refers to stronger ADCC and / or ADCP and / or CDC activity compared to the antibody prior to modification.

[0046] In one embodiment, the Fc region is modified by protein engineering to enhance ADCC and / or ADCP and / or CDC activity, hi one embodiment, Fc receptor affinity can be increased by amino acid mutations that increase affinity for C1q, thereby enhancing CDC.

[0047] In another embodiment, Fc receptor affinity can be increased by altering the glycosylation profile of the Fc region, hi one embodiment, the Fc region is modified by defucosylation or underfucosylation to enhance ADCC activity.

[0048] Antibody and antigen-binding molecule fragments The antigen-binding molecule of the present invention can be a fragment of an antibody, specifically an antigen-binding fragment of an antibody.Antigen-binding fragments contain one or more antigen-binding regions.It has been shown that fragments of a whole antibody can perform the function of binding to an antigen. Examples of binding fragments include (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains, (ii) an Fd fragment consisting of the VH and CH1 domains, (iii) an Fv fragment consisting of the VL and VH domains of a single antibody, (iv) a dAb fragment consisting of the VH domain (Ward, ES et al., Nature 341:544-546 (1989)), (v) isolated CDR regions, (vi) an F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments, and (vii) a single chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker that enables the two domains to associate to form an antigen-binding site (Bird et al., Science 242:423-426 (1988), Huston et al., PNAS USA 85:5879-5883). (1988)), (viii) bispecific single chain Fv dimers (International Application PCT / US92 / 09965), and (ix) "diabodies," which are multivalent or multispecific fragments constructed by genetic fusion (WO 94 / 13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)). Typically, the fragments are Fab, F(ab')2 or Fv fragments, or scFv molecules. In some embodiments, the fragments may be Fab fragments.

[0049] Bispecific antibodies are antibodies that can simultaneously bind to two target molecules, such as two antigens or two epitopes. Bispecific antibodies are sometimes referred to as dual-binding antibodies. Examples of bispecific antibody formats include, but are not limited to, (mAb)2, Fcab, F(mAb')2, quadroma, scFv (single-chain variable fragment), bsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), BiTE (bispecific T cell-triggering antibody), DART (dual affinity retargeting antibody), charge pairs, tandem antibodies, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, minibody, zybody (zybodies), DNL-F(ab)3 (dock-and-lock trivalent Fab), bssdAb (bispecific single domain antibody) and knobs-in-holes.

[0050] Diabodies are multimers of polypeptides, each of which comprises a first domain comprising a binding region for an immunoglobulin light chain and a second domain comprising a binding region for an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site, which is formed by the association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (WO 94 / 13804).

[0051] When bispecific antibodies are used, they may be conventional bispecific antibodies and may be produced in a variety of ways (Hollinger & Winter, Current Opinion Biotechnol. 4:446-449 (1993)), for example produced chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments described below. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs can be constructed without the Fc region, using only the variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO Journal 10:3655-3659 (1991).

[0052] Bispecific diabodies may also be useful because, in contrast to bispecific whole antibodies, they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificity can be readily selected from libraries using phage display (WO 94 / 13804). If one arm of the diabody is held constant, for example specificity for antigen X, another arm can be varied to generate libraries and select antibodies of appropriate specificity.

[0053] In some embodiments, the antigen-binding molecules of the invention are monovalent to PD-L1, e.g., monovalent antibody fragments. Monovalent antigen-binding molecules (also known as monomeric antigen-binding molecules) are antigen-binding molecules that have only one binding site for an epitope or antigen. For example, Fab, knob-in-hole, one-arm IgG, scFv exhibit monovalent binding, and complete monoclonal antibodies with two Fab regions exhibit bivalent binding. When an antigen-binding molecule is monovalent, the antigen-binding molecule and the antigen bind in a 1:1 ratio. Thus, the present invention provides a monovalent antibody or antibody fragment that specifically binds to PD-L1 in a pH-dependent manner.

[0054] In some embodiments, the antigen-binding molecule is monovalent for PD-L1. This means that the antigen-binding molecule has at most one binding site for PD-L1. Thus, the antigen-binding molecule binds to PD-L1 molecules in a 1:1 ratio. Monovalent antigen-binding molecules are also generally monospecific. In such embodiments, the monovalent antigen-binding molecule specifically binds only to PD-L1. In some embodiments, the antibody-binding molecule may comprise a monovalent binding site that specifically binds to PD-L1 in a 1:1 ratio and one or more additional binding sites that specifically bind to an antigen different from PD-L1. For example, in some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule that comprises a first binding site that is monovalent for PD-L1 (specifically binds to PD-L1 in a 1:1 ratio) and a second binding site that specifically binds to a second, different antigen. In another embodiment, the antigen binding molecule may be a bispecific antigen binding molecule comprising a first binding site that is monovalent for PD-L1 (specifically binds to PD-L1 in a 1:1 ratio) and a second binding site that is monovalent for a second, different antigen (specifically binds to a second, different antigen in a 1:1 ratio). In such an embodiment, the antigen binding molecule binds to its antigen in a ratio of one antigen binding molecule to two antigen molecules, but also in a ratio of one antigen binding molecule to one PD-L1 molecule. Multi- and bispecific configurations are discussed elsewhere, and it is directly contemplated that the multi- and bispecific antigen binding molecules disclosed herein may be monovalent for PD-L1 (i.e., bind to PD-L1 in a ratio of one bispecific antigen binding molecule to one PD-L1 molecule).

[0055] In some embodiments, the antigen binding molecule or antibody of the present invention may be bispecific for PD-L1 and another antigen or epitope. In one embodiment, the other antigen may be selected from the group consisting of T cell costimulatory agonists or T cell costimulatory antagonists. In one embodiment, the other antigen may be selected from the group consisting of CD47, SIRPα, CD25, TIGIT, ICOS, CD70, BTLA, GITR, LAG-3, TIM-3, CTLA-4, CD137, OX40, EGFR, TGF, VEGF, and CD40.

[0056] In one embodiment, a bispecific antigen binding molecule or antibody of the invention specifically binds to PD-L1 and specifically binds to an immune checkpoint inhibitor. In a further embodiment, a bispecific antigen binding molecule or antibody of the invention specifically binds to PD-L1 and specifically binds to an immune checkpoint inhibitor. In a further embodiment, a bispecific antigen binding molecule or antibody of the invention specifically binds to PD-L1 and specifically binds to an immune checkpoint inhibitor, the immune checkpoint inhibitor being selected from the group consisting of PD-1, CTLA-4, TIM-3, CD137, CD40, LAG-3, VISTA, ICOS, BTLA, GITR and TIGIT.

[0057] In one embodiment, a bispecific antigen binding molecule or antibody of the invention specifically binds to PD-L1 and specifically binds to an immune effector. In a further embodiment, a bispecific antigen binding molecule or antibody of the invention specifically binds to PD-L1 and specifically binds to an immune effector selected from the group consisting of OX40, EGFR, TGF, VEGF, CD40, CD137, GITR, CXCR3, BTLA, CD3, CD70, CD25, CD47, SIRPα, and CD27.

[0058] In one embodiment, the bispecific antigen-binding molecule or antibody of the invention specifically binds to PD-L1 and also acts to recruit and engage other cells, such as T cells. The dual antigen specificity of the bispecific antigen-binding molecule or antibody allows it to simultaneously bind to a PD-L1 specific antigen along with an antigen present on a cytotoxic T cell. This allows the cytotoxic activity of the T cell to be directed to tumor cells by the engaging activity of the bispecific antigen-binding molecule or antibody. In a further embodiment, the bispecific antigen-binding molecule or antibody of the invention specifically binds to PD-L1 and also specifically binds to a T cell recruiter. T cell recruiter molecules include, but are not limited to, CD3 and CD137.

[0059] The antibodies or antigen-binding molecules of the invention may be trispecific. The trispecific antigen-binding molecules or antibodies of the invention comprise a binding site specific for PD-L1 and a binding site for recruiting T cells. In one embodiment, such antigen-binding molecules or antibodies comprise a binding site specific for PD-L1 and a binding site specific for CD3. Furthermore, in these embodiments, the Fc region of the trispecific antigen-binding molecules or antibodies can also bind to Fc receptors on accessory cells. The recruitment of additional accessory cells, such as monocytes, macrophages, NK cells and / or dendritic cells, makes the trispecific antigen-binding molecules or antibodies more potent than other antibodies for the destruction of tumor cells, since the trispecific antibodies allow the linkage of T cells, accessory cells and tumor cells. In one embodiment, the trispecific antigen-binding molecules or antibodies comprise one binding site specific for PD-L1, one binding site specific for CD3 and an intact Fc region capable of interacting with Fc receptors on monocytes, macrophages, NK cells and dendritic cells.

[0060] The antibodies or antigen-binding molecules of the invention may be conjugated to other molecules, such as drugs, prodrugs, or toxic moieties. This serves to target these molecules to tumor cells. The antibodies or antigen-binding molecules of the invention have much lower peripheral toxicity than other PD-L1 antibodies, allowing the use of tumor cell killing strategies only against tumor positive PD-L1 cells, without destroying healthy cells. In one embodiment, the PD-L1 specific antigen-binding molecules of the invention are conjugated to cytotoxins (e.g. maytansinoids, auristatins, dolastatins, cryptophycins, calicheamicins, duocarmycins, pyrrolobenzodiazepines), protein toxins (e.g. Pseudomonas exotoxin, diphtheria toxin, ribosome-inactivating proteins), and radionuclides (e.g. 90 Y, 111 In).

[0061] identity and homology "Identity" as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. There are many methods to measure the identity between two polypeptide or two polynucleotide sequences, but the commonly used methods for determining identity are codified in computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0062] To compare amino acid sequences, programs such as the CLUSTAL program can be used. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as necessary. It is possible to calculate the amino acid identity or similarity (identity + conservation of amino acid type) for the optimal alignment. Programs such as BLASTx align the longest stretches of similar sequences and assign a value to the goodness of fit. Thus, it is possible to obtain comparisons in which several regions of similarity are found, each with a different score. Both types of identity analysis are contemplated in the present invention.

[0063] The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions / total number of positions x 100). Generally, references to % identity in this specification refer to the % identity over the entire length of the molecule, unless otherwise specified or implied by the context.

[0064] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. One example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 incorporate such an algorithm. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment software package, incorporates such an algorithm.Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5, and FASTA, described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8, in which ktup is a control option that sets the sensitivity and speed of the search.

[0065] Typically, the amino acid sequences of the CDRs of the antigen-binding molecules provided in the present invention have at least 70% identity at the amino acid level to the amino acid sequences of the CDRs described below, for example using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403-410 (1990)) provided by HGMP (Human Genome Mapping Project). More typically, the CDR sequences have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity at the amino acid level to the sequences shown below. Typically, each of the CDR sequences of the antigen-binding molecules used in the present invention has this level of identity to the amino acid sequences of the CDRs described below. Alternatively, any one, two, three, four or five of the CDRs of the antigen-binding molecules used in the present invention have this level of identity to the amino acid sequences of the CDRs described below.

[0066] The amino acid sequences of the VH and VL regions of the antigen-binding molecules provided in the present invention have at least 70% identity at the amino acid level to the amino acid sequences of the VH and VL regions described below, for example, using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403-410 (1990)) provided by HGMP (Human Genome Mapping Project). More typically, the VH and VL regions have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity at the amino acid level to the sequences shown below. Typically, each of the VH and VL regions of the antigen-binding molecules used in the present invention has this level of identity to the amino acid sequences of the VH and VL regions described below. Alternatively, any one of the VH and VL regions of an antigen-binding molecule used in the present invention has this level of identity to the amino acid sequences of the VH and VL regions described below.

[0067] Identity, as used herein, may be used interchangeably with "homology" and "similarity." Reference to a specific % identity applies equally to % homology and % similarity. Homology and similarity can be determined using suitable algorithms, such as FASTA, BLAST, and Gapped BLAST. Software for performing these analyses is publicly available.

[0068] Mutants The present invention also extends to variants of the peptide sequences mentioned below. As used herein, the term "variant" relates to proteins having similar amino acid sequences and / or retaining the same function. For example, the term "variant" encompasses proteins or polypeptides comprising one or more amino acid additions, deletions, substitutions, etc. An example of a variant of the present invention is a protein comprising a peptide as defined below, except for the substitution of one or more amino acids with one or more other amino acids. Amino acid substitutions can be made, for example, to reduce or eliminate disorders in the amino acid sequence. Alternatively, amino acid substitutions can be made to improve antigen affinity or to humanize or deimmunize the antibody, as required. Affinity maturation, humanized and deimmunized variants of the specified antibodies, as well as variants comprising amino acid substitutions to reduce or eliminate any disorders in the sequence of the antibody, are provided herein.

[0069] replacement Those skilled in the art recognize that various amino acids have similar properties, and one or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of the substance.

[0070] For this reason, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferably used to replace one another (because they have relatively short side chains), and valine, leucine and isoleucine are preferably used to replace one another (because they have more hydrophobic aliphatic side chains). Other amino acids that can often be substituted for one another include phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains), lysine, arginine and histidine (amino acids with basic side chains), aspartate and glutamate (amino acids with acidic side chains), asparagine and glutamine (amino acids with amide side chains), and cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0071] Substitutions of this type are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

[0072] Naturally occurring amino acids may be designated using three-letter and one-letter codes as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gln), cysteine ​​(C or Cys), methionine (M or Met), serine (S or Ser), and threonine (T or Thr). When the residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where the residue may be glutamic acid or glutamine, the symbols Glx or Z may be used. Unless the context indicates otherwise, reference to aspartic acid includes aspartate and reference to glutamic acid includes glutamate.

[0073] Amino acid deletions or insertions can also be made to the amino acid sequences of the fusion proteins mentioned below. Thus, for example, amino acids can be deleted that do not substantially affect, or at least do not eliminate, the activity of the polypeptide. Such deletions can be advantageous, since they can reduce the overall length and molecular weight of the polypeptide while retaining activity. This can reduce the amount of polypeptide required for a particular purpose, for example reducing dosage levels.

[0074] For this reason, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferably used to replace one another (because they have relatively short side chains), and valine, leucine and isoleucine are preferably used to replace one another (because they have more hydrophobic aliphatic side chains). Other amino acids that can often be substituted for one another include phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains), lysine, arginine and histidine (amino acids with basic side chains), aspartate and glutamate (amino acids with acidic side chains), asparagine and glutamine (amino acids with amide side chains), and cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0075] In some embodiments, the following amino acids may be exchanged for conservative amino acid substitutions:

[0076] TIFF0007653408000001.tif31170

[0077] Thus, reference to a "conservative" amino acid substitution refers to an amino acid substitution in which one or more of the amino acids in the sequence of an antibody (e.g., a CDR or a VH or VL sequence) are replaced with another amino acid of the same class as indicated above. Conservative amino acid substitutions may be preferred in the CDR regions to minimize adverse effects on antibody function. However, conservative amino acid substitutions may also occur in framework regions.

[0078] Amino acid changes to the sequences below can be made using any suitable technique, such as site-directed mutagenesis or solid phase synthesis.

[0079] It will be understood that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids, although naturally occurring amino acids may be preferred. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

[0080] In one embodiment of the present invention, there is provided an antigen-binding molecule or antigen-binding fragment thereof, which comprises 1 to 10, preferably 1 to 5, more preferably 1 or 2 amino acid substitutions in the antibody-binding domain or antigen-binding domain.For example, in one embodiment of the present invention, there is provided an anti-PD-L1 antibody or antigen-binding fragment thereof, which comprises six CDR regions of an antibody selected from the group consisting of 2A09, 8G08, 8D06, 8A04, 8B06, and 8D04, and optionally, the antigen-binding molecule has 1 to 10 amino acid substitutions throughout its CDR regions, preferably 1 to 5 amino acid substitutions throughout its CDR regions. In a further embodiment of the invention, there is provided an anti-PD-L1 antigen-binding molecule, or antigen-binding fragment thereof, wherein the anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises the VH and VL sequences of an antibody selected from the group consisting of 2A09, 8G08, 8D06, 8A04, 8B06, and 8D04, and optionally the antigen-binding molecule has from 1 to 10 amino acid substitutions across its VH and VL sequences, preferably from 1 to 5 amino acid substitutions across its VH and VL sequences. In a still further embodiment of the invention, there is provided an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is an antibody selected from the group consisting of 2A09, 8G08, 8D06, 8A04, 8B06, and 8D04, and the antibody has from 1 to 10 amino acid substitutions, preferably from 1 to 5 amino acid substitutions. The substitutions are, of course, with respect to the original CDR or variable chain sequence of the starting antibody.

[0081] In some embodiments, the one or more amino acid substitutions are in the CDR region(s). In other embodiments, the one or more amino acid substitutions are in the framework regions, i.e., the variable heavy and light chains, but not in the CDR region(s). In other embodiments, the one or more amino acid substitutions may be at any position within the variable heavy and / or variable light regions. In some embodiments, the amino acid substitutions do not adversely affect the binding specificity and / or affinity of the antibody. Thus, the variant antibody may have the same or superior functional profile as the antibody from which it is derived.

[0082] For example, in some embodiments there is provided an anti-PD-L1 antibody selected from the group consisting of: 2A09, 8G08, 8D06, 8A04, 8B06, and 8D04, which has between 1 and 10 amino acid substitutions throughout its framework regions, preferably between 1 and 5 amino acid substitutions throughout its framework regions (i.e., the substitutions are found in the framework regions relative to the reference antibody, and the CDR sequences are not altered).

[0083] Affinity maturation mutants Other variants within the scope of the invention include antigen-binding molecules of the invention that have been modified to increase their affinity for PD-L1 or an epitope of PD-L1. In one embodiment, the antigen-binding molecules of the invention are affinity matured antibodies. In one embodiment, the antigen-binding molecules of the invention are fully human affinity matured antibodies.

[0084] Any known method can be used to increase the affinity of the antigen binding molecules of the invention to generate affinity matured antibodies or fully human affinity matured antibodies with increased affinity for PD-L1 or an epitope of PD-L1.

[0085] The invention provides affinity matured variants of the provided antigen-binding agents. The affinity matured variants bind to PD-L1 with greater affinity than the parent antibody. Preferably, the antibody generated binds to PD-L1 with at least 20%, at least 30%, at least 40%, and more preferably at least 50% greater affinity than the parent antibody binds to PD-L1, e.g., as measured by Kd.

[0086] In some embodiments, the invention provides methods of making an antigen binding molecule of the invention comprising providing an antigen binding molecule as described herein (e.g., an anti-PD-L1 binding molecule or antibody, or antigen-binding fragment or variant thereof) and subjecting the antibody to affinity maturation, wherein the generated antibody binds to PD-L1 with greater affinity than the parent antibody. Preferably, the generated antibody binds to PD-L1 with at least 20%, at least 30%, at least 40%, more preferably at least 50% greater affinity than the parent antibody binds to PD-L1, e.g., as measured by Kd. Methods for measuring affinity are known in the art and are described in the Examples below. Affinity matured antibodies generated by such methods can be formulated and used as described herein for other anti-PD-L1 binding molecules.

[0087] Affinity maturation can be performed according to any suitable method known to those skilled in the art. For example, in vitro antibody display systems are widely used to generate specific antibodies with high affinity. In these systems, it is possible to combine the phenotype (i.e., antibody fragment) with the genotype (i.e., antibody genes) and directly determine the sequence of the antibody. Several systems have been developed that achieve the display of antibody repertoires and allow the subsequent selection of binders, allowing the selection of even higher affinity variants by increasing the stringency of the selection. Antibody fragments can be expressed in yeast, ribosomes, phage display particles or by direct conjugation to DNA.

[0088] Current antibody affinity maturation methods fall into two mutagenesis categories: stochastic and non-stochastic. Error-prone polymerase chain reaction (PCR), bacterial mutagenization, and saturation mutagenesis are typical examples of stochastic mutagenesis. Non-stochastic methods often use alanine scanning or site-directed mutagenesis to generate a limited collection of specific mutants. In addition, a shuffling approach can be used to obtain shuffled variants of a parent antibody to further improve the affinity of the antibody.

[0089] Thus, in one embodiment of the invention, the method of affinity maturation is selected from the group consisting of stochastic mutagenesis (e.g., error-prone polymerase chain reaction (PCR), mutagenized bacterial strains or saturation mutagenesis), non-stochastic mutagenesis (e.g., alanine scanning or site-directed mutagenesis), shuffling (e.g., DNA shuffling, chain shuffling or CDR shuffling), and the use of the CRISPR-Cas9 system to introduce modifications.

[0090] Affinity maturation methods are described, for example, in Rajpal et al., Proc Natl Acad Sci USA, 2005, 102(24):8466-71; Steinwand et al., MAbs, 2014, 6(1):204-18; and Handbook of Therapeutic Antibodies, Wiley, 2014, Chapter 6, Antibody Affinity (pages 115-140).

[0091] In some embodiments, a method is provided for preparing a pharmaceutical composition, comprising providing an antibody generated according to the methods described above (i.e., generating an antibody by affinity maturation) and co-formulating the antibody with at least one or more pharma- ceutically acceptable excipients. The antibody used to prepare the pharmaceutical composition may be an affinity matured variant of 2A09. The antibody used to prepare the pharmaceutical composition may be an affinity matured variant of 8G08, 8D06, 8A04, 8B06 or 8D04. The pharmaceutical composition generated by such a method may be used in the therapeutic methods of the invention as described herein for other anti-PD-L1 binding molecules. Thus, antigen binding molecules are provided that are affinity matured mutants or variants of the antigen binding molecules of the invention. For example, in one embodiment, affinity matured variants of an antibody selected from the group consisting of 2A09, 8G08, 8D06, 8A04, 8B06 and 8D04 are provided. Generally, affinity matured variants have a higher affinity for PD-L1 than the parent antibody (the antibody from which the mutant is derived). Antigen-binding molecules and antibodies obtainable or obtained by affinity maturation of the antigen-binding molecules or antibodies of the invention are also provided by the present invention.

[0092] Manufacturing obstacles Therapeutic proteins such as antibodies are inherently heterogeneous and complex due to chemical and post-translational modifications (PTMs). Modifications can be caused by a number of factors such as the host cell line, the process used for production, or the conditions during storage or production. Modifications can be related to the chemical stability of the molecule itself, or its ability to aggregate, and the effect this has on the intrinsic physical stability of the antibody. Amino acid motifs or residues in a given antibody sequence that can undergo spontaneous modifications during production or storage are referred to as disorders. Thus, mutations can be made to the antibody sequence to address disorders and reduce the susceptibility of the antibody to modification and degradation.

[0093] Such modifications as a result of obstacles in the antibody sequence may include glycosylation, deamidation, oxidation, and C- and N-terminal mutations. Such modifications may occur during manufacturing. Certain residues and structural or sequence motifs are more susceptible to certain modifications. Examples of such obstacles to modification include N-linked glycosylation of Asn, O-linked glycosylation of Ser / Thr, deamidation of Asn, isomerization / fragmentation of Asp, glycosylation of Lys, oxidation of Met / Trp, free thiol groups, pyroglutamate, C-terminal Lys.

[0094] Those skilled in the art will recognize that computational tools can be used to predict and identify structural and sequence obstacles that may potentially cause modifications. Manufacturing processes can be modified to minimize the occurrence of modifications. Protein engineering can be considered to reduce risk. For example, selective mutation of these obstacles can help identify and reduce the risk of modifications that compromise antibody stability.

[0095] Aspartic acid residues (Asp) can undergo spontaneous modification. Asp-containing motifs, such as Asp-Gly sequences, can undergo spontaneous isomerization to form isoaspartic acid. The formation of isoaspartate can weaken or completely abolish antibody binding. This is even more important when Asp residues are found in the CDRs of antibodies.

[0096] Therefore, the aspartic acid residue (Asp) can be substituted with any naturally occurring amino acid to reduce this barrier to modification. Optionally, the aspartic acid residue (Asp) can be substituted with alanine (Ala), glutamine (Gln) or glutamic acid (Glu) to reduce this barrier to modification. Optimization of production / formulation to reduce isomerization may be investigated. Alternatively, the Asp-Gly motif can be modified to inhibit deamidation by substituting the glycine residue with another naturally occurring amino acid rather than by substituting the Asp residue.

[0097] Methionine residues (Met) may undergo spontaneous modification. The presence of methionine (Met) in CDRs, especially when exposed to solvent, may cause problems if methionine is oxidized, which may prevent binding. Therefore, methionine residues can be substituted with any other naturally occurring amino acid to reduce this barrier to modification. Methionine residues may be preferably substituted with Ala or Leu. Optimization of production / formulation may be investigated to reduce oxidation.

[0098] TIFF0007653408000002.tif41170

[0099] Thus, also provided herein are variant antibodies derived from any of 8A04, 8B06, 8G08, 8D06, 8D04 and 2A09, but which contain one or more amino substitutions to address one or more of the disorders summarized above.

[0100] For example, for any antigen-binding molecule defined by one or more amino acid sequences herein, if one or more Met residues are present, then one or more Met residues may each be independently substituted with an Ala or Leu residue, and if one or more Asp residues are present, then one or more Asp residues may each be independently substituted with an Ala, Gln, or Glu residue.

[0101] Overview of antigen-binding molecules provided A summary of the antigen-binding molecules provided by the present invention are provided below, identifying the SEQ ID NOs assigned in the attached sequence listing. Antigen-binding variants, derivatives and fragments thereof are also provided as part of the present invention.

[0102] TIFF0007653408000003.tif44170

[0103] Variants are also provided that include at least the following variants:

[0104] TIFF0007653408000004.tif86170

[0105] For example, variants of 8G08 provided herein include variants having a VL of SEQ ID NO:35 and a VH of SEQ ID NO:26, or a VL of SEQ ID NO:36 and a VH of SEQ ID NO:27, or a VL of SEQ ID NO:37 and a VH of SEQ ID NO:28. Variants of 8D06 provided herein include variants having a VL of SEQ ID NO:38 and a VH of SEQ ID NO:26, or a VL of SEQ ID NO:39 and a VH of SEQ ID NO:27, or a VL of SEQ ID NO:40 and a VH of SEQ ID NO:28. Variants of 8A04 provided herein include variants having a VL of SEQ ID NO:41 and a VH of SEQ ID NO:26, or a VL of SEQ ID NO:42 and a VH of SEQ ID NO:27, or a VL of SEQ ID NO:43 and a VH of SEQ ID NO:28. Variants of 8B06 provided herein include variants having a VL of SEQ ID NO:47 and a VH of SEQ ID NO:26, or a VL of SEQ ID NO:48 and a VH of SEQ ID NO:27, or a VL of SEQ ID NO:49 and a VH of SEQ ID NO:28. Variants of 8A04 provided herein include variants having a VL of SEQ ID NO:50 and a VH of SEQ ID NO:26, or a VL of SEQ ID NO:51 and a VH of SEQ ID NO:27, or a VL of SEQ ID NO:52 and a VH of SEQ ID NO:28. Variants of 2A09 provided herein include variants having a VL of SEQ ID NO:20 and a VH of SEQ ID NO:26, or a VL of SEQ ID NO:21 and a VH of SEQ ID NO:27, or a VL of SEQ ID NO:22 and a VH of SEQ ID NO:28.

[0106] Other variants of 8G08 provided herein include: VLCDR1 of SEQ ID NO: 10, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 23, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 29 and VHCDR3 of SEQ ID NO: 32, VLCDR1 of SEQ ID NO: 10, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 24, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 30 and VHCDR3 of SEQ ID NO: 33, or Examples of such mutants include those having a VLCDR1 of SEQ ID NO: 10, a VLCDR2 of SEQ ID NO: 3, a VLCDR3 of SEQ ID NO: 25, a VHCDR1 of SEQ ID NO: 6, a VHCDR2 of SEQ ID NO: 31 and a VHCDR3 of SEQ ID NO: 34.

[0107] Other variants of 8D06 provided herein include: VLCDR1 of SEQ ID NO: 12, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 23, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 29 and VHCDR3 of SEQ ID NO: 32, VLCDR1 of SEQ ID NO: 12, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 24, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 30 and VHCDR3 of SEQ ID NO: 33, or Examples of such mutants include those having a VLCDR1 of SEQ ID NO: 12, a VLCDR2 of SEQ ID NO: 3, a VLCDR3 of SEQ ID NO: 25, a VHCDR1 of SEQ ID NO: 6, a VHCDR2 of SEQ ID NO: 31 and a VHCDR3 of SEQ ID NO: 34.

[0108] Other variants of 8A04 provided herein include: VLCDR1 of SEQ ID NO: 44, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 23, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 29 and VHCDR3 of SEQ ID NO: 32, VLCDR1 of SEQ ID NO: 45, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 24, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 30 and VHCDR3 of SEQ ID NO: 33, or Examples of such mutants include those having a VLCDR1 of SEQ ID NO: 46, a VLCDR2 of SEQ ID NO: 3, a VLCDR3 of SEQ ID NO: 25, a VHCDR1 of SEQ ID NO: 6, a VHCDR2 of SEQ ID NO: 31 and a VHCDR3 of SEQ ID NO: 34.

[0109] Other variants of 8B06 provided herein include: VLCDR1 of SEQ ID NO: 16, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 23, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 29 and VHCDR3 of SEQ ID NO: 32, VLCDR1 of SEQ ID NO: 16, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 24, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 30 and VHCDR3 of SEQ ID NO: 33, or Examples of such mutants include those having a VLCDR1 of SEQ ID NO: 16, a VLCDR2 of SEQ ID NO: 3, a VLCDR3 of SEQ ID NO: 25, a VHCDR1 of SEQ ID NO: 6, a VHCDR2 of SEQ ID NO: 31 and a VHCDR3 of SEQ ID NO: 34.

[0110] Other variants of 8D04 provided herein include: VLCDR1 of SEQ ID NO: 16, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 23, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 29 and VHCDR3 of SEQ ID NO: 32, VLCDR1 of SEQ ID NO: 16, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 24, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 30 and VHCDR3 of SEQ ID NO: 33, or Examples of such mutants include those having a VLCDR1 of SEQ ID NO: 16, a VLCDR2 of SEQ ID NO: 3, a VLCDR3 of SEQ ID NO: 25, a VHCDR1 of SEQ ID NO: 6, a VHCDR2 of SEQ ID NO: 31 and a VHCDR3 of SEQ ID NO: 34.

[0111] Other variants of 2A09 provided herein include: VLCDR1 of SEQ ID NO: 2, VLCDR2 of SEQ ID NO: 3, VLCDR3 of SEQ ID NO: 23, VHCDR1 of SEQ ID NO: 6, VHCDR2 of SEQ ID NO: 29 and VHCDR3 of SEQ ID NO: 32, VLCDR1 of SEQ ID NO:2, VLCDR2 of SEQ ID NO:3, VLCDR3 of SEQ ID NO:24, VHCDR1 of SEQ ID NO:6, VHCDR2 of SEQ ID NO:30 and VHCDR3 of SEQ ID NO:33, or Examples of such mutants include those having a VLCDR1 of SEQ ID NO:2, a VLCDR2 of SEQ ID NO:3, a VLCDR3 of SEQ ID NO:25, a VHCDR1 of SEQ ID NO:6, a VHCDR2 of SEQ ID NO:31 and a VHCDR3 of SEQ ID NO:34.

[0112] Various embodiments of the invention will now be discussed in more detail.

[0113] Antigen-binding molecules comprising the VLCDR1 region 8B06 In one embodiment, an antigen binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16). In one embodiment, an antigen binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16).

[0114] In one embodiment, an antigen binding molecule is provided, such as an antibody, fragment or variant thereof, comprising a light chain variable region comprising the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16). In a specific embodiment, the antigen binding molecule is an antibody, fragment or variant thereof, and the VLCDR1 region of the antibody, fragment or variant thereof is RETELSRRLHYVR (SEQ ID NO: 16).

[0115] In some embodiments, antigen binding molecules derived from the 8B06 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least 30.

[0116] Amino acid substitutions may be made, for example, to reduce or eliminate the lesion in the 8B06 VLCDR1 region.

[0117] 8D06 In one embodiment, an antigen-binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12). In one embodiment, an antigen-binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12).

[0118] In one embodiment, an antigen-binding molecule is provided, such as an antibody, fragment or variant thereof, comprising a light chain variable region comprising the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12). In a specific embodiment, the antigen-binding molecule is an antibody, fragment or variant thereof, and the VLCDR1 region of the antibody, fragment or variant thereof is VLSPRTHAGHYYR (SEQ ID NO: 12).

[0119] In some embodiments, antigen binding molecules derived from the 8D06 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 5.

[0120] Amino acid substitutions may be made, for example, to reduce or eliminate the lesion in the 8D06 VLCDR1 region.

[0121] 8G08 In one embodiment, an antigen-binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10). In one embodiment, an antigen-binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10).

[0122] In one embodiment, an antigen binding molecule is provided, such as an antibody, fragment or variant thereof, comprising a light chain variable region comprising the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10). In a specific embodiment, the antigen binding molecule is an antibody, fragment or variant thereof, and the VLCDR1 region of the antibody, fragment or variant thereof is ISNDDPASGHYHR (SEQ ID NO: 10).

[0123] In some embodiments, antigen binding molecules derived from the 8G08 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 5.

[0124] Amino acid substitutions may be made, for example, to reduce or eliminate the lesion in the 8G08 VLCDR1 region.

[0125] 8A04 In one embodiment, an antigen binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence MRTGTNKGHYTR (SEQ ID NO: 14). In one embodiment, an antigen binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence MRTGTNKGHYTR (SEQ ID NO: 14).

[0126] In one embodiment, an antigen binding molecule is provided, such as an antibody, fragment or variant thereof, comprising a light chain variable region comprising the amino acid sequence MRTGTGNKGHYTR (SEQ ID NO: 14). In a specific embodiment, the antigen binding molecule is an antibody, fragment or variant thereof, and the VLCDR1 region of the antibody, fragment or variant thereof is MRTGTGNKGHYTR (SEQ ID NO: 14).

[0127] In some embodiments, antigen binding molecules derived from the 8A04 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 30.

[0128] Amino acid substitutions may be made, for example, to reduce or eliminate lesions in the 8A04 VLCDR1 region.

[0129] For example, in one embodiment, there is provided an antigen binding molecule, such as an antibody, fragment or variant thereof, comprising a VLCDR1 comprising the amino acid sequence XRTGTGNKGHYTR (SEQ ID NO: 44), where X can be any naturally occurring amino acid.

[0130] In one embodiment, there is provided an antigen binding molecule, such as an antibody, fragment or variant thereof, comprising a VLCDR1 comprising the amino acid sequence XRTGTGNKGHYTR (SEQ ID NO: 45), wherein X is selected from the group consisting of Met, Ala or Leu.

[0131] In one embodiment, there is provided an antigen binding molecule, such as an antibody, fragment or variant thereof, comprising a VLCDR1 comprising the amino acid sequence XRTGTGNKGHYTR (SEQ ID NO: 46), wherein X is selected from the group consisting of Ala or Leu.

[0132] 8D04 In one embodiment, an antigen-binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18). In one embodiment, an antigen-binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18).

[0133] In one embodiment, an antigen binding molecule is provided, such as an antibody, fragment or variant thereof, comprising a light chain variable region comprising the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18). In a specific embodiment, the antigen binding molecule is an antibody, fragment or variant thereof, and the VLCDR1 region of the antibody, fragment or variant thereof is RGTGSSFHHKYVR (SEQ ID NO: 18).

[0134] Amino acid substitutions may be made, for example, to reduce or eliminate the lesion in the 8D04 VLCDR1 region.

[0135] In some embodiments, antigen binding molecules derived from the 8D04 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 5.

[0136] 2A09_WT In one embodiment, an antigen-binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2). In one embodiment, an antigen-binding molecule, such as an antibody, a fragment or variant thereof, is provided that comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2).

[0137] In one embodiment, an antigen binding molecule is provided, such as an antibody, fragment or variant thereof, comprising a light chain variable region comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2). In a specific embodiment, the antigen binding molecule is an antibody, fragment or variant thereof, and the VLCDR1 region of the antibody, fragment or variant thereof is TRSSGSIASNYVQ (SEQ ID NO: 2).

[0138] Amino acid substitutions may be made, for example, to reduce or eliminate the lesion in the 2A09_WT VLCDR1 region.

[0139] Heavy and / or Light Chain CDRs 8B06 In one embodiment, VHCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SYGMY (SEQ ID NO: 6); 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or VLCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16); 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4), and a VLCDR3 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0140] In one embodiment, a heavy chain variable region comprising a VHCDR1 having at least 90% identity to the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 having at least 90% identity to the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 having at least 90% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or Provided is an antibody, fragment or variant thereof comprising a light chain variable region comprising a VLCDR1 having at least 90% identity to the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16), a VLCDR2 having at least 90% identity to the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 having at least 90% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0141] In some embodiments, antigen binding molecules derived from the 8B06 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 30.

[0142] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR1 comprising the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0143] Amino acid substitutions may be made, for example, to reduce or eliminate lesions in the 8B06 CDRs.

[0144] For example, in one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 29), where X1 and X2 can be any naturally occurring amino acid; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 32), where X can be any naturally occurring amino acid; VLCDR1 comprising the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:23), where X can be any naturally occurring amino acid.

[0145] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 30), wherein X1 and X2 are each independently selected from the group consisting of Asp, Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 33), where X is selected from the group consisting of Asp, Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:24), where X is selected from the group consisting of Asp, Gln, Glu and Ala.

[0146] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 31), wherein X1 and X2 are each independently selected from the group consisting of Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 34), where X is selected from the group consisting of Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:25), where X is selected from the group consisting of Gln, Glu and Ala.

[0147] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or a light chain variable region comprising a VLCDR1 comprising the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4); Optionally, an antibody, fragment or variant thereof is provided in which each Met residue is independently substituted with an amino acid selected from the group consisting of Ala and Leu, and each Asp residue is independently substituted with an amino acid selected from the group consisting of Ala, Gln and Glu.

[0148] 8D06 In one embodiment, VHCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SYGMY (SEQ ID NO: 6); 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or VLCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12); 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4), and a VLCDR3 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0149] In one embodiment, a heavy chain variable region comprising a VHCDR1 having at least 90% identity to the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 having at least 90% identity to the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 having at least 90% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or Provided is an antibody, fragment or variant thereof comprising a light chain variable region comprising a VLCDR1 having at least 90% identity to the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12), a VLCDR2 having at least 90% identity to the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 having at least 90% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0150] In some embodiments, antigen binding molecules derived from the 8D06 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 5.

[0151] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR1 comprising the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0152] Amino acid substitutions may be made, for example, to reduce or eliminate lesions in the 8D06 CDRs.

[0153] For example, in one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 29), where X1 and X2 can be any naturally occurring amino acid; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 32), where X can be any naturally occurring amino acid; VLCDR1 comprising the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:23), where X can be any naturally occurring amino acid.

[0154] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 30), wherein X1 and X2 are each independently selected from the group consisting of Asp, Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 33), where X is selected from the group consisting of Asp, Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:24), where X is selected from the group consisting of Asp, Gln, Glu and Ala.

[0155] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 31), wherein X1 and X2 are each independently selected from the group consisting of Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 34), where X is selected from the group consisting of Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:25), where X is selected from the group consisting of Gln, Glu and Ala.

[0156] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or a light chain variable region comprising a VLCDR1 comprising the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4); Optionally, an antibody, fragment or variant thereof is provided in which each Met residue is independently substituted with an amino acid selected from the group consisting of Ala and Leu, and each Asp residue is independently substituted with an amino acid selected from the group consisting of Ala, Gln and Glu.

[0157] 8G08 In one embodiment, VHCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SYGMY (SEQ ID NO: 6); 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or VLCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence ISNDDVASGHYHR (SEQ ID NO: 10); 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4), and a VLCDR3 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0158] In one embodiment, a heavy chain variable region comprising a VHCDR1 having at least 90% identity to the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 having at least 90% identity to the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 having at least 90% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or Provided is an antibody, fragment or variant thereof comprising a light chain variable region comprising a VLCDR1 having at least 90% identity to the amino acid sequence ISNDDVASGHYHR (SEQ ID NO: 10), a VLCDR2 having at least 90% identity to the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 having at least 90% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0159] In some embodiments, antigen binding molecules derived from the 8G08 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 5.

[0160] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR1 comprising the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0161] Amino acid substitutions may be made to reduce or eliminate the disorders in the 8G08 CDRs.

[0162] For example, in one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 29), where X1 and X2 can be any naturally occurring amino acid; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 32), where X can be any naturally occurring amino acid; VLCDR1 comprising the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:23), where X can be any naturally occurring amino acid.

[0163] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 30), wherein X1 and X2 are each independently selected from the group consisting of Asp, Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 33), where X is selected from the group consisting of Asp, Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:24), where X is selected from the group consisting of Asp, Gln, Glu and Ala.

[0164] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 31), wherein X1 and X2 are each independently selected from the group consisting of Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 34), where X is selected from the group consisting of Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:25), where X is selected from the group consisting of Gln, Glu and Ala.

[0165] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or a light chain variable region comprising a VLCDR1 comprising the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4); Optionally, an antibody, fragment or variant thereof is provided in which each Met residue is independently substituted with an amino acid selected from the group consisting of Ala and Leu, and each Asp residue is independently substituted with an amino acid selected from the group consisting of Ala, Gln and Glu.

[0166] 8A04 In one embodiment, VHCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SYGMY (SEQ ID NO: 6); 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or VLCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence MRTGTNKGHYTR (SEQ ID NO: 14); 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4), and a VLCDR3 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0167] In one embodiment, a heavy chain variable region comprising a VHCDR1 having at least 90% identity to the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 having at least 90% identity to the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 having at least 90% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or Provided is an antibody, fragment or variant thereof comprising a light chain variable region comprising a VLCDR1 having at least 90% identity to the amino acid sequence MRTGTGNKGHYTR (SEQ ID NO: 14), a VLCDR2 having at least 90% identity to the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 having at least 90% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0168] In some embodiments, antigen binding molecules derived from the 8A04 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 30.

[0169] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR1 comprising the amino acid sequence MRTGTGNKGHYTR (SEQ ID NO: 14), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0170] Amino acid substitutions may be made, for example, to reduce or eliminate lesions in the 8A04 CDRs.

[0171] For example, in one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 29), where X1 and X2 can be any naturally occurring amino acid; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 32), where X can be any naturally occurring amino acid; VLCDR1 comprising the amino acid sequence XRTGTGNKGHYTR (SEQ ID NO: 44), where X can be any naturally occurring amino acid; VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:23), where X can be any naturally occurring amino acid.

[0172] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 30), wherein X1 and X2 are each independently selected from the group consisting of Asp, Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 33), where X is selected from the group consisting of Asp, Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence XRTGTGNKGHYTR (SEQ ID NO: 45), wherein X is selected from the group consisting of Met, Ala, or Leu; VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:24), where X is selected from the group consisting of Asp, Gln, Glu and Ala.

[0173] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 31), wherein X1 and X2 are each independently selected from the group consisting of Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 34), where X is selected from the group consisting of Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence XRTGTGNKGHYTR (SEQ ID NO: 46), wherein X is selected from the group consisting of Ala or Leu; VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:25), where X is selected from the group consisting of Gln, Glu and Ala.

[0174] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or a light chain variable region comprising a VLCDR1 comprising the amino acid sequence MRTGTGNKGHYTR (SEQ ID NO: 14), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4); Optionally, an antibody, fragment or variant thereof is provided in which each Met residue is independently substituted with an amino acid selected from the group consisting of Ala and Leu, and each Asp residue is independently substituted with an amino acid selected from the group consisting of Ala, Gln and Glu.

[0175] 8D04 In one embodiment, VHCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SYGMY (SEQ ID NO: 6); 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or VLCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18); 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4), and a VLCDR3 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0176] In one embodiment, a heavy chain variable region comprising a VHCDR1 having at least 90% identity to the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 having at least 90% identity to the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 having at least 90% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or Provided is an antibody, fragment or variant thereof comprising a light chain variable region comprising a VLCDR1 having at least 90% identity to the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18), a VLCDR2 having at least 90% identity to the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 having at least 90% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0177] In some embodiments, antigen binding molecules derived from the 8D04 antibody, such as antibodies, fragments or variants thereof, are provided that may have a pH 6.0:7.4 EC50 ratio of at least about 5.

[0178] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR1 comprising the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0179] Amino acid substitutions may be made, for example, to reduce or eliminate lesions in the 8D04 CDRs.

[0180] For example, in one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 29), where X1 and X2 can be any naturally occurring amino acid; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 32), where X can be any naturally occurring amino acid; VLCDR1 comprising the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:24), where X can be any naturally occurring amino acid.

[0181] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 30), wherein X1 and X2 are each independently selected from the group consisting of Asp, Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 33), where X is selected from the group consisting of Asp, Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:25), where X is selected from the group consisting of Asp, Gln, Glu and Ala.

[0182] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 31), wherein X1 and X2 are each independently selected from the group consisting of Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 34), where X is selected from the group consisting of Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:26), where X is selected from the group consisting of Gln, Glu and Ala.

[0183] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or a light chain variable region comprising a VLCDR1 comprising the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4); Optionally, an antibody, fragment or variant thereof is provided in which each Met residue is independently substituted with an amino acid selected from the group consisting of Ala and Leu, and each Asp residue is independently substituted with an amino acid selected from the group consisting of Ala, Gln and Glu.

[0184] 2A09WT In one embodiment, VHCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SYGMY (SEQ ID NO: 6); 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or VLCDR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2); 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4), and a VLCDR3 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0185] In one embodiment, a heavy chain variable region comprising a VHCDR1 having at least 90% identity to the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 having at least 90% identity to the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 having at least 90% identity to the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); and / or Provided is an antibody, fragment or variant thereof comprising a light chain variable region comprising a VLCDR1 having at least 90% identity to the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2), a VLCDR2 having at least 90% identity to the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 having at least 90% identity to the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0186] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR1 comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4).

[0187] Amino acid substitutions may be made, for example, to reduce or eliminate impairments in the 2A09_WT CDRs.

[0188] For example, in one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 29), where X1 and X2 can be any naturally occurring amino acid; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 32), where X can be any naturally occurring amino acid; VLCDR1 comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:23), where X can be any naturally occurring amino acid.

[0189] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 30), wherein X1 and X2 are each independently selected from the group consisting of Asp, Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 33), where X is selected from the group consisting of Asp, Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:24), where X is selected from the group consisting of Asp, Gln, Glu and Ala.

[0190] In one embodiment, VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO:6), VHCDR2 comprising the amino acid sequence VISYX1GSNKYYAX2SVKG (SEQ ID NO: 31), wherein X1 and X2 are each independently selected from the group consisting of Gln, Glu, and Ala; and and / or a heavy chain variable region comprising a VHCDR3 comprising the amino acid sequence GALTHWGVVIGXGMDV (SEQ ID NO: 34), where X is selected from the group consisting of Gln, Glu and Ala; VLCDR1 comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2); VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO:3), and Antibodies, fragments or variants thereof are provided that comprise a light chain variable region comprising a VLCDR3 that comprises the amino acid sequence QSFXSTNPWV (SEQ ID NO:25), where X is selected from the group consisting of Gln, Glu and Ala.

[0191] In one embodiment, A heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8), and / or a light chain variable region comprising a VLCDR1 comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QSFDSTNPWV (SEQ ID NO: 4); Optionally, an antibody, fragment or variant thereof is provided in which each Met residue is independently substituted with an amino acid selected from the group consisting of Ala and Leu, and each Asp residue is independently substituted with an amino acid selected from the group consisting of Ala, Gln and Glu.

[0192] Heavy and / or light chain variable regions In one embodiment, the invention provides an antigen binding molecule, particularly an antibody, that binds to PD-L1, comprising a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:5, and / or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:1.

[0193] In such embodiments, antigen binding molecules, such as antibodies, fragments or variants thereof, having a pH 6.0:7.4 EC50 ratio of at least about 5 are provided.

[0194] In one embodiment, the antibody binds to PD-L1 and comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:5, and / or a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:1.

[0195] In one embodiment, an antigen binding molecule, such as an antibody, variant or fragment thereof, is provided, wherein the antigen binding molecule comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: a) a VH comprising the amino acid sequence of SEQ ID NO: 5 and a VL comprising the amino acid sequence of SEQ ID NO: 15 (or comprising VH and VL sequences at least 90% identical to SEQ ID NO: 5 and SEQ ID NO: 15, respectively); b) a VH comprising the amino acid sequence of SEQ ID NO: 5 and a VL comprising the amino acid sequence of SEQ ID NO: 11 (or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO: 5 and SEQ ID NO: 11, respectively); c) a VH comprising the amino acid sequence of SEQ ID NO: 5 and a VL comprising the amino acid sequence of SEQ ID NO: 9 (or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO: 5 and SEQ ID NO: 9, respectively); d) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:13 (or comprising VH and VL sequences at least 90% identical to SEQ ID NO:5 and SEQ ID NO:13, respectively); e) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:17 (or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO:5 and SEQ ID NO:17, respectively); f) A VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:1 (or comprising VH and VL sequences at least 90% identical to SEQ ID NO:5 and SEQ ID NO:1, respectively).

[0196] In one embodiment, an antigen binding molecule, such as an antibody, variant or fragment thereof, is provided, wherein the antigen binding molecule comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: a) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a VH having up to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 5 (or a variant thereof such as an affinity matured variant thereof), and / or a VL comprising the amino acid sequence of SEQ ID NO: 15 or a VL sequence having up to 5 amino acid substitutions compared to the VL sequence of SEQ ID NO: 15 (or a variant thereof such as an affinity matured variant thereof), b) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a VH having up to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 5 (or a variant thereof, such as an affinity matured variant thereof), and / or a VL comprising the amino acid sequence of SEQ ID NO: 11 or a VL sequence having up to 5 amino acid substitutions compared to the VL sequence of SEQ ID NO: 11 (or a variant thereof, such as an affinity matured variant thereof), c) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a VH having up to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 5 (or a variant thereof, such as an affinity matured variant thereof), and / or a VL comprising the amino acid sequence of SEQ ID NO: 9 or a VL sequence having up to 5 amino acid substitutions compared to the VL sequence of SEQ ID NO: 9 (or a variant thereof, such as an affinity matured variant thereof), d) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a VH having up to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 5 (or a variant thereof, such as an affinity matured variant thereof), and / or a VL comprising the amino acid sequence of SEQ ID NO: 13 or a VL sequence having up to 5 amino acid substitutions compared to the VL sequence of SEQ ID NO: 13 (or a variant thereof, such as an affinity matured variant thereof), e) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a VH having up to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 5 (or a variant thereof, such as an affinity matured variant thereof), and / or a VL comprising the amino acid sequence of SEQ ID NO: 17 or a VL sequence having up to 5 amino acid substitutions compared to the VL sequence of SEQ ID NO: 17 (or a variant thereof, such as an affinity matured variant thereof), f) a VH comprising the amino acid sequence of SEQ ID NO:5 or a VH having up to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO:5 (or a variant thereof such as an affinity matured variant thereof), and / or a VL comprising the amino acid sequence of SEQ ID NO:1 or a VL sequence having up to 5 amino acid substitutions compared to the VL sequence of SEQ ID NO:1 (or a variant thereof such as an affinity matured variant thereof).

[0197] In some embodiments, all of the amino acid substitutions occur in the CDRs of the variable regions. In some embodiments, all of the amino acid substitutions occur in the framework regions of the variable regions.

[0198] In some embodiments, the amino acid substitutions may be conservative amino acid substitutions. In some embodiments, the amino acid substitutions may be to address one or more potential defects in the sequence. Some variant antibodies may have one or more amino acid substitutions to address one or more defects and may include one or more additional amino acid substitutions that are conservative amino acid substitutions.

[0199] In one embodiment, an antigen binding molecule, such as an antibody, fragment or variant thereof, is provided that has a pH 6.0:7.4 EC50 ratio of at least 5.

[0200] Thus, as discussed above, also provided are humanized and affinity matured variants thereof, as well as variants having lesser or greater percent identity or homology to the specified sequence(s), for example at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or homology. Variants having one or more amino acid substitutions are also provided.

[0201] In one embodiment of the invention, there is provided an anti-PD-L1 antigen binding molecule comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region that is at least 89% identical to SEQ ID NO: 1. Such antigen binding molecules may retain the functional activity (e.g. selective pH binding, EC50, IC50 and / or Kd) of the antigen binding molecule from which the variant antigen binding molecule is derived.

[0202] In some embodiments, the % sequence identity is calculated without including the sequence of all six CDRs of the designated heavy or light chain variable region. In such embodiments, sequence variations occur only in the framework regions. For example, an anti-PD-L1 antigen binding molecule may comprise a variable heavy chain region having at least 95% identity to the variable heavy chain region of SEQ ID NO: 5, and / or a variable light chain region having at least 95% identity to the variable light chain region of SEQ ID NO: 15, 11, 9, 13, 17, or 1, with any amino acid variations occurring only in the framework regions of the variable heavy and light chain sequences. In such embodiments, an anti-PD-L1 antigen binding molecule with a designated sequence identity comprises the complete heavy chain CDR1, CDR2, and CDR3 sequence of SEQ ID NO: 5, and the complete light chain CDR1, CDR2, and CDR3 sequence of SEQ ID NO: 15, 11, 9, 13, 17, or 1 (i.e., the complete heavy and light chain CDR sequences of the antibody from which the variants are derived).

[0203] As described above, amino acid substitutions can be made to reduce or eliminate the defects in the heavy chain variable region and / or the light chain variable region of the antigen-binding molecule of the present invention. Such substitutions to reduce or eliminate the defects can occur in the CDRs. Such substitutions to reduce or eliminate the defects can occur in the framework regions of the variable regions.

[0204] For example, in one embodiment, a heavy chain variable region that binds to PD-L1 and has the amino acid sequence: QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVISYX1GSNKYYAX2SVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCAKGALTHWGVVIGX3GMDVWGQGTTVTVSS (SEQ ID NO: 26), wherein X1, X2, and X3 can each independently be any naturally occurring amino acid; and / or a) NFX1LTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 20), where X1 and X2 can be any naturally occurring amino acid; b) NFX1LTQPHSVSESPGKTVTISCISNDVPASGHYHRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 35), where X1 and X2 can each independently be any naturally occurring amino acid; c) NFX1LTQPHSVSESPGKTVTISCVLSPRTHAGHYYRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 38), wherein X1 and X2 can each independently be any naturally occurring amino acid; d) NFX1LTQPHSVSESPGKTVTISCX2RTGTGNKGHYTRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX3STNPWVFGGGTKLTVL (SEQ ID NO: 41), where X1, X2 and X3 can each independently be any naturally occurring amino acid; e) NFX1LTQPHSVSESPGKTVTISCRETELSRRLHYVRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 47), where X1 and X2 can each independently be any naturally occurring amino acid; and f) An antigen-binding molecule (e.g., an antibody or antigen-binding fragment thereof) is provided, comprising a light chain variable region having an amino acid sequence selected from the group consisting of NFX1LTQPHSVSESPGKTVTISCRGTGSSFHHKYVRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 50), wherein X1 and X2 can each independently be any naturally occurring amino acid.

[0205] In one embodiment, a heavy chain variable region that binds to PD-L1 and has the amino acid sequence: QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVISYX1GSNKYYAX2SVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCAKGALTHWGVVIGX3GMDVWGQGTTVTVSS (SEQ ID NO: 27), and / or a) NFX1LTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 21), wherein X1 is selected from the group consisting of Met or Leu and X2 is selected from the group consisting of Asp, Gln, Glu and Ala; b) NFX1LTQPHSVSESPGKTVTISCISNDVPASGHYHRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 36), wherein X1 is selected from the group consisting of Met or Leu and X2 is selected from the group consisting of Asp, Gln, Glu and Ala; c) NFX1LTQPHSVSESPGKTVTISCVLSPRTHAGHYYRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 39), wherein X1 is selected from the group consisting of Met or Leu and X2 is selected from the group consisting of Asp, Gln, Glu and Ala; d) NFX1LTQPHSVSESPGKTVTISCX2RTGTGNKGHYTRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX3STNPWVFGGGTKLTVL (SEQ ID NO: 42), wherein X1 is selected from the group consisting of Met or Leu, X2 is selected from the group consisting of Met, Ala and Leu, and X3 is selected from the group consisting of Asp, Gln, Glu and Ala; e) NFX1LTQPHSVSESPGKTVTISCRETELSRRLHYVRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 48), in which X1 is selected from the group consisting of Met or Leu and X2 is selected from the group consisting of Asp, Gln, Glu and Ala; and f) An antigen-binding molecule (e.g., an antibody or an antigen-binding fragment thereof) is provided, comprising a light chain variable region having an amino acid sequence selected from the group consisting of NFX1LTQPHSVSESPGKTVTISCRGTGSSFHHKYVRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 51), wherein X1 is selected from the group consisting of Met or Leu, and X2 is selected from the group consisting of Asp, Gln, Glu, and Ala.

[0206] In one embodiment, a heavy chain variable region that binds to PD-L1 and has the amino acid sequence: QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVISYX1GSNKYYAX2SVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCAKGALTHWGVVIGX3GMDVWGQGTTVTVSS (SEQ ID NO: 28), and / or a) NFX1LTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 22), wherein X1 is Leu and X2 is selected from the group consisting of Gln, Glu, and Ala; b) NFX1LTQPHSVSESPGKTVTISCISNDVPASGHYHRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 37), in which X1 is Leu and X2 is selected from the group consisting of Gln, Glu, and Ala; c) NFX1LTQPHSVSESPGKTVTISCVLSPRTHAGHYYRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 40), wherein X1 is Leu and X2 is selected from the group consisting of Gln, Glu and Ala; d) NFX1LTQPHSVSESPGKTVTISCX2RTGTGNKGHYTRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX3STNPWVFGGGTKLTVL (SEQ ID NO: 43), wherein X1 is Leu, X2 is selected from the group consisting of Ala and Leu, and X3 is selected from the group consisting of Gln, Glu, and Ala; e) NFX1LTQPHSVSESPGKTVTISCRETELSRRLHYVRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 49), in which X1 is Leu and X2 is selected from the group consisting of Gln, Glu, and Ala; and f) An antigen-binding molecule (e.g., an antibody or an antigen-binding fragment thereof) is provided, comprising a light chain variable region having an amino acid sequence selected from the group consisting of: NFX1LTQPHSVSESPGKTVTISCRGTGSSFHHKYVRWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX2STNPWVFGGGTKLTVL (SEQ ID NO: 52), wherein X1 is Leu and X2 is selected from the group consisting of Gln, Glu, and Ala.

[0207] In one embodiment, an antigen binding molecule, such as an antibody, variant or fragment thereof, is provided, the antigen binding molecule comprising: a) a VH comprising the amino acid sequence of SEQ ID NO: 5 and a VL comprising the amino acid sequence of SEQ ID NO: 15; b) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:11; c) VH comprising the amino acid sequence of SEQ ID NO: 5 and VL comprising the amino acid sequence of SEQ ID NO: 9; d) VH comprising the amino acid sequence of SEQ ID NO:5 and VL comprising the amino acid sequence of SEQ ID NO:13; e) VH comprising the amino acid sequence of SEQ ID NO: 5 and VL comprising the amino acid sequence of SEQ ID NO: 17; f) VH comprising the amino acid sequence of SEQ ID NO:5 and VL comprising the amino acid sequence of SEQ ID NO:1; and optionally, each Met residue is independently substituted with an amino acid selected from the group consisting of Ala and Leu, and each Asp residue is independently substituted with an amino acid selected from the group consisting of Ala, Gln, and Glu.

[0208] A variant antigen-binding molecule having one or more amino acid substitutions may retain the functional activity (e.g., selective pH binding, EC50, IC50 and / or Kd) of the antigen-binding molecule from which the variant antigen-binding molecule is derived. The variant antigen-binding molecules of the present invention can be used and formulated in the same manner as described for the antigen-binding molecule from which they are derived.

[0209] Other antigen-binding molecules of the present invention The invention also provides an anti-PD-L1 antigen binding molecule having the following CDRH and CDRL sequences:

[0210] TIFF0007653408000005.tif28170

[0211] Here, X1~X 16 can each be any amino acid, preferably any naturally occurring amino acid.

[0212] In a preferred embodiment, X1 to X 16 are each independently selected from the group consisting of T, I, V, M, R, S, L, E, G, N, D, P, A, H, F, K, Y, and Q.

[0213] In one embodiment, X1 is selected from the group consisting of A, L, T, I, V, M, R or conservative amino acid substitutions thereof; X2 is selected from the group consisting of R, S, L, E, G, or a conservative amino acid substitution thereof; X3 is selected from the group consisting of S, N, T or a conservative amino acid substitution thereof; X4 is selected from the group consisting of S, D, P, G, E or a conservative amino acid substitution thereof; X5 is selected from the group consisting of G, V, R, T, L and S, or conservative amino acid substitutions thereof; X6 is selected from the group consisting of S, P, T, G or a conservative amino acid substitution thereof; X7 is selected from the group consisting of I, A, H, N, R, F or a conservative amino acid substitution thereof; X8 is selected from the group consisting of A, S, K, R, H or a conservative amino acid substitution thereof; X9 is selected from the group consisting of S, G, L, H or a conservative amino acid substitution thereof; X 10 is selected from the group consisting of N, H, K or conservative amino acid substitutions thereof; X 11 is selected from the group consisting of V, H, Y, T or conservative amino acid substitutions thereof; X 12 is selected from the group consisting of Q, R or a conservative amino acid substitution thereof; X 13 is selected from the group consisting of D, Q, E, A or conservative amino acid substitutions thereof; X 14 is selected from the group consisting of D, Q, E, A or conservative amino acid substitutions thereof; X 15 is selected from the group consisting of D, Q, E, A or conservative amino acid substitutions thereof; X 16 is selected from the group consisting of D, Q, E, A, or conservative amino acid substitutions thereof.

[0214] In one embodiment, X1 is selected from the group consisting of A, L, T, I, V, M and R; X2 is selected from the group consisting of R, S, L, E and G; X3 is selected from the group consisting of S, N and T; X4 is selected from the group consisting of S, D, P, G and E; X5 is selected from the group consisting of G, V, R, T, L and S; X6 is selected from the group consisting of S, P, T and G; X7 is selected from the group consisting of I, A, H, N, R and F; X8 is selected from the group consisting of A, S, K, R and H; X9 is selected from the group consisting of S, G, L and H; X 10 is selected from the group consisting of N, H and K; X 11 is selected from the group consisting of V, H, Y and T; X 12 is selected from the group consisting of Q and R; X 13 is selected from the group consisting of D, Q, E and A; X 14 is selected from the group consisting of D, Q, E and A; X 15 is selected from the group consisting of D, Q, E and A; X 16 is selected from the group consisting of D, Q, E and A.

[0215] In one embodiment (SEQ ID NO:57): X1 is selected from the group consisting of A, L, T, I, V and R; X2 is selected from the group consisting of R, S, L, E and G; X3 is selected from the group consisting of S, N and T; X4 is selected from the group consisting of S, D, P, G and E; X5 is selected from the group consisting of G, V, R, T, L and S; X6 is selected from the group consisting of S, P, T and G; X7 is selected from the group consisting of I, A, H, N, R and F; X8 is selected from the group consisting of A, S, K, R and H; X9 is selected from the group consisting of S, G, L and H; X10 is selected from the group consisting of N, H and K; X 11 is selected from the group consisting of V, H, Y and T; X 12 is selected from the group consisting of Q and R; X 13 is selected from the group consisting of Q, E and A; X 14 is selected from the group consisting of Q, E and A; X 15 is selected from the group consisting of Q, E and A; X 16 is selected from the group consisting of Q, E and A.

[0216] Such antibodies may preferably have the functional profile of one or more of 8B06, 8D06, 8G08, 8A04, 8D04 or 2A09.

[0217] The invention also provides an anti-PD-L1 antigen binding molecule having the following VH and VL sequences: VH:QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVISYX1GSNKYYAX2SVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCAKGALTHWGVVIGX3GMDVWGQGTTVTVSS VL:NFX4LTQPHSVSESPGKTVTISCX5X6X7X8X9X 10 X 11 X 12 X 13 X 14 YX 15 X 16 WYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSFX 17 STNPWVFGGGTKLTVL Here, X1~X 17 can each be any amino acid, preferably any naturally occurring amino acid (ie, the VH sequence comprises SEQ ID NO:53 and the VL sequence comprises SEQ ID NO:58).

[0218] In a preferred embodiment, X1 to X 17 are each independently selected from the group consisting of T, I, V, M, R, S, L, E, G, N, D, P, A, H, F, K, Y, Q (i.e., the VH sequence comprises SEQ ID NO:54 and the VL sequence comprises SEQ ID NO:59).

[0219] In one embodiment, X1 is selected from the group consisting of D, Q, E, A or conservative amino acid substitutions thereof; X2 is selected from the group consisting of D, Q, E, A, or a conservative amino acid substitution thereof; X3 is selected from the group consisting of D, Q, E, A or a conservative amino acid substitution thereof; X4 is selected from the group consisting of M, L, or a conservative amino acid substitution thereof; X5 is selected from the group consisting of A, L, T, I, V, M, R or conservative amino acid substitutions thereof; X6 is selected from the group consisting of R, S, L, E, G or a conservative amino acid substitution thereof; X7 is selected from the group consisting of S, N, T or a conservative amino acid substitution thereof; X8 is selected from the group consisting of S, D, P, G, E or a conservative amino acid substitution thereof; X9 is selected from the group consisting of G, V, R, T, L and S or conservative amino acid substitutions thereof; X 10 is selected from the group consisting of S, P, T, G or conservative amino acid substitutions thereof; X 11 is selected from the group consisting of I, A, H, N, R, F or conservative amino acid substitutions thereof; X 12 is selected from the group consisting of A, S, K, R, H or conservative amino acid substitutions thereof; X 13 is selected from the group consisting of S, G, L, H or conservative amino acid substitutions thereof; X 14 is selected from the group consisting of N, H, K or conservative amino acid substitutions thereof; X 15 is selected from the group consisting of V, H, Y, T or conservative amino acid substitutions thereof; X 16 is selected from the group consisting of Q, R or a conservative amino acid substitution thereof; X 17 is selected from the group consisting of D, Q, E, A, or conservative amino acid substitutions thereof (i.e., the VH sequence comprises SEQ ID NO:55 and the VL sequence comprises SEQ ID NO:60).

[0220] In one embodiment, X1 is selected from the group consisting of D, Q, E and A; X2 is selected from the group consisting of D, Q, E and A; X3 is selected from the group consisting of D, Q, E and A; X4 is selected from the group consisting of M and L; X5 is selected from the group consisting of A, L, T, I, V, M and R; X6 is selected from the group consisting of R, S, L, E and G; X7 is selected from the group consisting of S, N and T; X8 is selected from the group consisting of S, D, P, G and E; X9 is selected from the group consisting of G, V, R, T, L and S; X 10 is selected from the group consisting of S, P, T and G; X 11 is selected from the group consisting of I, A, H, N, R and F; X 12 is selected from the group consisting of A, S, K, R and H; X 13 is selected from the group consisting of S, G, L and H; X 14 is selected from the group consisting of N, H and K; X 15 is selected from the group consisting of V, H, Y and T; X 16 is selected from the group consisting of Q and R; X 17 is selected from the group consisting of D, Q, E and A (i.e., the VH sequence comprises SEQ ID NO:56 and the VL sequence comprises SEQ ID NO:61).

[0221] In one embodiment, X1 is selected from the group consisting of Q, E and A; X2 is selected from the group consisting of Q, E and A; X3 is selected from the group consisting of Q, E and A; X4 is L, X5 is selected from the group consisting of L, T, I, V, M and R; X6 is selected from the group consisting of S, L, E and G; X7 is selected from the group consisting of N and T; X8 is selected from the group consisting of D, P, G and E; X9 is selected from the group consisting of V, R, T, L and S; X 10 is selected from the group consisting of P, T and G; X 11 is selected from the group consisting of A, H, N, R and F; X 12 is selected from the group consisting of S, K, R and H; X 13 is selected from the group consisting of G, L and H; X 14 is selected from the group consisting of H and K; X 15 is selected from the group consisting of H, Y and T; X 16 is R, X 17 is selected from the group consisting of E and A (i.e., the VH sequence comprises SEQ ID NO:57 and the VL sequence comprises SEQ ID NO:62).

[0222] Nucleic acid sequences encoding antigen-binding molecules In one aspect of the present invention, a nucleic acid sequence encoding an antigen-binding molecule of the present invention (including fragments and variants thereof) is provided.

[0223] In one embodiment, there is provided a nucleotide sequence encoding an antigen binding molecule that binds to PD-L1, comprising a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:5, and / or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:1.

[0224] In one embodiment, there is provided a nucleotide sequence encoding an antigen binding molecule that binds to PD-L1, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:5, and / or a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:1.

[0225] The present invention also provides nucleic acid molecules encoding any of the variant antibody sequences disclosed herein that contain one or more amino acid substitutions.

[0226] Nucleic acid molecules encoding an amino acid sequence according to any one of SEQ ID NOs: 1-62 are also provided.

[0227] Plasmids and vectors containing a nucleic acid sequence encoding the antigen-binding molecule of the present invention are also provided. The nucleic acid may be incorporated into a plasmid or vector for expression, particularly in a eukaryotic expression system, more particularly in a mammalian cell line. Thus, host cells transfected with the plasmid or vector of the present invention, such as HEK cells, NS0 mouse myeloma cells or CHO cells, are also provided.

[0228] Also provided is a method for producing an anti-PD-L1 antigen binding molecule, comprising culturing a host of the invention in a cell culture medium under conditions allowing intracellular expression of the encoding nucleic acid sequence of the plasmid or vector. The method may further comprise obtaining the anti-PD-L1 antigen binding molecule from the cell culture supernatant. Further provided is a method for producing a cell expressing an anti-PD-L1 antigen binding molecule, comprising transfecting a cell with a plasmid or vector of the invention. The cell may then be cultured for production of the antigen binding molecule.

[0229] antigen The antigen-binding molecules of the invention specifically bind to PD-L1, in particular human PD-L1 or hPD-L1. The antigen-binding molecules of the invention also specifically bind to cynomolgus PD-L1. Most preferably, the antigen-binding molecules of the invention specifically bind to human PD-L1.

[0230] The antigen binding molecule of the present invention generally does not bind to mouse PD-L1. PD-L1 (programmed death ligand 1) is one of two ligands described for the inhibitory receptor PD-1 (programmed death 1 polypeptide). PD-L1 is a type 1 transmembrane protein involved in suppressing the adaptive immune system. Upregulation of PD-L1 may allow cancer to evade the immune system.

[0231] PD-L1 is expressed at low levels on immune cells such as B cells, dendritic cells, macrophages, and T cells, and is upregulated after activation. PD-L1 is also expressed in non-lymphoid organs such as endothelial cells, heart, lung, pancreas, muscle, keratinocytes, and placenta. Expression in non-lymphoid tissues suggests that PD-L1 may regulate the function of autoreactive T and B cells and myeloid cells in peripheral tissues, or inflammatory responses in target organs. PD-L1 expression is primarily regulated by type 1 and type 2 interferons, which are the master regulators of PD-L1 in endothelial and epithelial cells. PD-L1 is abundantly found in a variety of human cancers, and the interaction between PD-1 and PD-L1 results in a reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and immune evasion by cancerous cells. PD-L1 is expressed in tumor samples and is associated with poor prognosis. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1. Inhibition of PD-L1 signaling has been shown to enhance T cell immunity for the treatment of cancer.

[0232] The amino acid sequence of human PD-L1 that binds to the antigen-binding molecules of the invention is provided below.

[0233] Human PD-L1 (NCBI reference sequence: NP_054862.1) Amino acid sequence (SEQ ID NO: 19) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVD PVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

[0234] The anti-PD-L1 antigen-binding molecules of the invention specifically bind to PD-L1 in a pH-dependent manner.

[0235] In one embodiment, the anti-PD-L1 antigen binding molecules of the invention specifically bind to PD-L1 in a pH-dependent manner, for example in a monovalent format. Monovalent formats include formats such as Fab fragments. It is hypothesized that this may be due to the lower affinity of Fabs than monoclonal antibodies (e.g., approximately 100-fold lower) and / or the presence of histidine residues in the CDRs.

[0236] The antigen-binding molecules of the present invention exhibited significantly higher affinity for PD-L1 at acidic pH (about pH 6.0) compared to physiological pH (about pH 7.4), which is in contrast to other PD-L1-binding molecules known in the art, which do not exhibit pH-specific binding, and many actually exhibit lower affinity at pH 6.0 compared to pH 7.4.

[0237] The antigen-binding molecules of the present invention also showed clear pH-dependent inhibition. The antigen-binding molecules of the present invention have significantly higher inhibitory activity against PD-L1 at acidic pH (about pH 6.0) compared to physiological pH (about pH 7.4). At pH 7.4, the antigen-binding molecules of the present invention have almost no inhibitory activity against PD-L1. This is in contrast to other PD-L1-binding molecules known in the art that do not show pH-dependent inhibition.

[0238] This pH-specific binding is important because tumours and their microenvironments are acidic (e.g., about pH 6.0 to about pH 6.5). Thus, anti-PD-L1 binding molecules that have maximal activity at pH 6 to 6.5 (the acidic pH of tumours) and reduced activity at a physiological pH of 7.4 may reduce the toxicity and other side effects of peripheral checkpoint inhibitor therapy. Thus, the antigen-binding molecules of the invention are particularly useful as potential anti-cancer agents.

[0239] Thus, antigen-binding molecules of the invention that bind to PD-L1 also bind to cells that express PD-L1.

[0240] EC50 In one embodiment, the antigen-binding molecule of the present invention has an EC50 value of less than about 15 nM against PD-L1 or PD-L1-positive cells at pH 6.0. In one embodiment, the antigen-binding molecule of the present invention has an EC50 value of about 1.45 nM to about 15 nM against PD-L1 at pH 6.0.

[0241] In one embodiment, an antigen-binding molecule of the invention has an EC50 value for PD-L1 of at least about 10 nM at pH 7.4. In one embodiment, an antigen-binding molecule of the invention has an EC50 value for PD-L1 of about 13.2 nM to about 100 nM at pH 7.4.

[0242] The antigen-binding molecules of the invention have a significantly higher potency (EC50) in binding to PD-L1 at acidic pH (pH 6.0) than at physiological pH (pH 7.4). The antigen-binding molecules of the invention have a pH 6.0:7.4 binding ratio of at least about 5, i.e., the EC50 at pH 6.0 is at least about 5-fold lower than at pH 7.4, and the antigen-binding molecules are at least about 5-fold more potent at pH 6.0 than at pH 7.4.

[0243] In a preferred embodiment, an antigen binding molecule of the invention has an EC50 value for PD-L1 at pH 6.0 of less than about 15 nM and an EC50 value for PD-L1 at pH 7.4 of at least about 10 nM, and the antigen binding molecule has a pH 6.0:7.4 binding ratio of at least about 5.

[0244] The term EC50 is known to those skilled in the art and refers to the 50% effective concentration of a drug or substance, i.e., the concentration of that substance that induces a response halfway between the maximum response and the baseline after a specified exposure time. EC50 is a measure of potency. The lower the EC50, the more potent the drug or substance. EC50 can be measured by any suitable means known to those skilled in the art.

[0245] In a cell binding assay, EC50 may refer to the concentration of Fab that induces a response approximately halfway between the baseline and maximum (MFI) after about 60 minutes of incubation with 0.60E05 MDA-MB-231 cells at about 4° C. In an ELISA assay, EC50 may refer to the concentration of Fab that induces a response approximately halfway between the baseline and maximum (OD) after about 2 hours of incubation with about 1 μg / ml human PD-L1-Fc protein coated wells at room temperature (e.g., about 15° C. to about 25° C.).

[0246] IC50 In one embodiment, an antigen-binding molecule of the invention has an IC50 value of less than about 50 nM at pH 6.0 for inhibition of PD-L1:PD-1 binding. In one embodiment, an antigen-binding molecule of the invention has an IC50 value of about 12.1 nM to about 42.3 nM against PD-L1 at pH 6.0.

[0247] In one embodiment, an antigen-binding molecule of the invention has an IC50 value for inhibition of PD-L1:PD-1 binding of at least about 50 nM at pH 7.4. In one embodiment, an antigen-binding molecule of the invention has an IC50 value for PD-L1 of about 81.2 nM to about 100 nM at pH 7.4.

[0248] The antigen-binding molecules of the present invention have significantly higher inhibitory activity (IC50) against PD-L1:PD-1 binding at acidic pH (pH 6.0) than at physiological pH (pH 7.4). The antigen-binding molecules of the present invention have a pH 6.0:7.4 inhibition ratio of at least 2, i.e., the IC50 at pH 6.0 is at least 2-fold lower than at pH 7.4, and the antigen-binding molecules are at least 5-fold more potent at inhibition at pH 6.0 than at pH 7.4.

[0249] In a preferred embodiment, an antigen binding molecule of the invention has an IC50 value against PD-L1 at pH 6.0 of less than about 50 nM and an IC50 value against PD-L1 at pH 7.4 of at least about 50 nM, and the antigen binding molecule has a pH 6.0:7.4 inhibition ratio of at least about 2.

[0250] The term IC50 is known to those skilled in the art and refers to the 50% inhibitory concentration of a drug or substance, i.e., the concentration of that substance that induces 50% inhibition. IC50 is a measure of the potency of a substance in inhibiting a particular biological or biochemical function. The lower the IC50, the more potent the antagonist or substance is as an inhibitor. IC50 can be measured by any suitable means known to those skilled in the art. For example, in a PD-L1-PD-1 inhibition ELISA, IC50 can be the 50% inhibitory concentration of Fab that inhibits PD-L1 biotin binding at about 1 μg / ml to wells coated with PD-1-Fc protein at about 1 μg / ml.

[0251] Of course, for any of the functional characteristics of the antibodies, the comparison of the functional characteristics is performed under substantially the same experimental conditions, except for pH, since the effect of changes in pH is measured. Functional characteristics (IC50, EC50 or K D Other features of the assay (e.g., the amount of protein present ... etc.) are kept the same as much as possible.

[0252] In some embodiments of the present invention, the inventors have discovered that antigen binding molecules compete with PD-L1 binding to the inhibitory receptor programmed death 1 polypeptide (PD-1) and the costimulatory molecule CD80. This mechanism is important because PD-L1 is abundantly found in various human cancers, and the interaction between PD-1 and PD-L1 results in a reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and immune evasion by cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 and PD-L1. Inhibition of PD-L1 signaling has been shown to enhance T cell immunity for the treatment of cancer. The antigen binding molecules of the present invention inhibit the binding of PD-L1 and its receptor by at least 40%, at least 50%, or at least 80%.

[0253] The present inventors also found that the antigen-binding molecules of the present invention inhibit the binding of PD-L1-expressing cells to PD-1 and / or CD80.

[0254] The pH-sensitive binding of the antigen-binding molecules of the present invention may result in the antigen-binding molecules having at least 5-fold higher affinity at acidic pH (e.g., pH 6) than at physiological pH (e.g., about pH 7.4). The affinity of the antigen-binding molecules of the present invention to their epitopes can be measured using surface plasmon resonance (SPR). For example, a BIAcore SPR system (BIAcore 3000, GE Healthcare) can be used. The BIAcore system is used to monitor molecular interactions in real time, and the detection principle is based on surface plasmon resonance (SPR), which is sensitive to changes in refractive index within about 150 nm from the sensor surface. For example, PD-L1-Fc can be attached to a surface, and the test anti-PD-L1 antigen-binding molecules can be passed over the surface in a continuous flow of sample solution. The SPR response is directly proportional to the change in mass concentration near the surface, and kinetic parameters are evaluated from the association and dissociation phases of the sensogram.

[0255] In one aspect, an anti-PD-L1 antigen binding molecule, such as an antibody, fragment or variant thereof, is provided, which antigen binding molecule competes with an antigen binding molecule of the invention for binding to PD-L1 as defined above.

[0256] For example, in one embodiment, the invention provides an antigen binding molecule (preferably an antibody) that specifically binds to PD-L1, particularly human PD-L1, and competes with an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, and 2A09 for binding to PD-L1. Also provided are antigen binding molecules (e.g., antigen binding molecules comprising the six CDR regions or the VH and VL sequences of the above antibodies, as well as other variants) that compete with fragments and variants thereof for binding to PD-L1.

[0257] To determine whether a test antibody is able to compete for binding to the same epitope as that bound by an antibody of the invention, a cross-blocking assay, such as a competitive ELISA assay, can be performed. In an exemplary competitive ELISA assay, PD-L1-coated wells of a microtiter plate or PD-L1-coated sepharose beads are pre-incubated with or without a candidate competing antibody, followed by the addition of a biotin-labeled anti-PD-L1 antibody of the invention. The amount of labeled anti-PD-L1 antibody bound to the PD-L1 antigen in the wells or on the beads can be measured using an avidin-peroxidase conjugate and an appropriate substrate.

[0258] Alternatively, the anti-PD-L1 antibody can be labeled, for example with a radioactive or fluorescent label, or some other detectable and measurable label. The amount of labeled anti-PD-L1 antibody that binds to the antigen is inversely correlated with the ability of the candidate competing antibody (test antibody) to compete for binding to the same epitope on the antigen, i.e., the higher the affinity of the test antibody for the same epitope, the less labeled anti-PD-L1 antibody that binds to the antigen-coated well. A candidate competing antibody is considered to be an antibody that substantially binds to the same epitope as, or competes for binding to, the same epitope as, an anti-PD-L1 antibody of the invention if the candidate competing antibody is able to block binding of the anti-PD-L1 antibody by at least 20%, preferably at least 20%-50%, and even more preferably at least 50%, compared to a control run in parallel in the absence of the candidate competing antibody (but which may be in the presence of a known non-competing antibody). It is understood that this assay can be performed in a variety of ways to arrive at the same quantitative value.

[0259] Also provided is an antigen binding molecule that specifically binds to PD-L1 and inhibits binding of PD-L1 to an antigen binding molecule of the invention.

[0260] For example, in one embodiment, the antigen binding molecule (preferably an antibody) specifically binds to PD-L1 and inhibits binding of PD-L1 to an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, and 2A09. Also provided are antigen binding molecules that specifically bind to PD-L1 and inhibit binding of PD-L1 to fragments and variants thereof (e.g., antigen binding molecules comprising the six CDR regions or the VH and VL sequences of the above antibodies, as well as other variants).

[0261] Also provided are antigen-binding molecules that specifically bind to the epitope of PD-L1 to which an antigen-binding molecule of the invention binds.

[0262] For example, in one embodiment, the invention provides an antigen binding molecule (preferably an antibody) that specifically binds to an epitope of PD-L1 bound by an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, and 2A09. Also provided are antigen binding molecules that specifically bind to an epitope of PD-L1 bound by fragments and variants thereof (e.g., antigen binding molecules comprising the six CDR regions or the VH and VL sequences of the above antibodies, as well as other variants).

[0263] composition In one aspect of the present invention, a pharmaceutical composition comprising an antigen-binding molecule of the present invention is provided.

[0264] The composition according to this aspect of the invention can be formulated for use by any convenient route. The pharmaceutical composition of the invention typically comprises a pharma- ceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, buffer or stabilizer in addition to the antigen-binding molecule of the invention. Such carriers include, but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, polyethylene glycol, ethanol, and combinations thereof.

[0265] The pharmaceutical composition may be in any suitable form depending on the desired method for administering it to a patient.

[0266] The pharmaceutical compositions of the present invention can be provided in unit dosage forms containing a predetermined amount of each active ingredient per administration. Such units can be adapted to provide any of the compounds at 0.5 mg / kg to 50 mg / kg, preferably 1 mg / kg to 10 mg / kg, 1 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg or 10 mg / kg to 50 mg / kg. Such doses can be given in a single administration or as multiple individual administrations. The final dose will of course vary depending on the condition being treated, the route of administration, and the age, weight and condition of the patient, and is at the discretion of the physician.

[0267] The pharmaceutical compositions of the present invention may be adapted for administration by any suitable route, such as oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes. IV administration may be preferred. Such formulations may be prepared by any method known in the art of pharmacy, for example by combining the active ingredient with the carrier(s) or excipient(s).

[0268] Pharmaceutical formulations suitable for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water or water-in-oil liquid emulsions.

[0269] Pharmaceutical formulations suitable for transdermal administration can be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient can be delivered from the patch by iontophoresis, as generally described in Pharmaceutical Research, 3(6), 318 (1986).

[0270] Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0271] For application to the eye or other external tissues, such as the mouth and skin, the formulation is preferably applied as a topical ointment or cream.When formulated in an ointment, the active ingredient can be used with either a paraffinic or water-miscible ointment base.Alternatively, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

[0272] Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

[0273] Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles and mouthwashes.

[0274] Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas.

[0275] Pharmaceutical formulations suitable for nasal administration where the carrier is a solid include coarse powder having a particle size in the range, for example, 20 to 500 microns, administered in the manner of snuff, i.e. rapid inhalation through the nasal passage from a container of the powder held close to the nose. Formulations suitable for administration as a nasal spray or nasal drops, where the carrier is a liquid, include aqueous or oily solutions of the active ingredient.

[0276] Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0277] Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0278] Pharmaceutical formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in single-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may also be prepared from sterile powders, granules, and tablets.

[0279] Pharmaceutical compositions of the present invention may contain, in addition to a molecule of the present invention, one or more other therapeutically active agents.

[0280] In some embodiments, the active drug concentrate formulation may include a pharma- ceutically acceptable tonicity agent, a buffering agent, and a pharma- ceutically acceptable surfactant.

[0281] Alternatively, the formulation may contain, in addition to the active ingredient, sodium phosphate monobasic, sodium phosphate dibasic, sodium chloride, polysorbate 80 or polysorbate 20 (surfactant to minimize the risk of aggregation on agitation) and water (USP / Ph.Eur), optionally with a pH adjusted to about 6.0-7.0, e.g. around 6.5.

[0282] Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, of an active ingredient, or an appropriate fraction thereof.

[0283] It will be appreciated that the formulations may contain, in addition to the ingredients particularly mentioned above, other agents conventional in the art having regard to the type of formulation in question; for example, formulations suitable for oral administration may include flavoring agents.

[0284] Treatment method The antigen-binding molecules of the invention are useful for the prevention and / or treatment of PD-L1 mediated disorders or diseases, in particular cancer. Accordingly, this aspect of the invention also includes a method of treating a PD-L1 mediated disorder or disease (such as cancer) in a subject, comprising administering to the subject an antigen-binding molecule of the invention. Thus, the invention also extends to the use of the antigen-binding molecules of the invention in the manufacture of a medicament for use in the treatment and / or prevention of PD-L1 mediated disorders or diseases (such as cancer), as well as the use of the antigen-binding molecules of the invention in the prevention and / or treatment of such pathologies.

[0285] The treatment method may be of a human or animal subject, and the invention extends equally to use in both human and / or veterinary medicine. The antigen-binding molecules of the invention are preferably administered to an individual in a "therapeutically effective amount" sufficient to show benefit to the individual. As used herein, "treatment" includes any regime that may benefit a human or non-human animal, preferably a mammal. Treatment may be for an existing condition or may be preventative (prophylactic treatment). The method may be an in vitro method. The method may be an in vivo method.

[0286] In one embodiment, the antigen binding molecules of the present invention enhance T cell immunity when administered in vitro or in vivo.

[0287] In one embodiment, an antigen binding molecule of the invention reverses immunosuppression when administered in vitro or in vivo.

[0288] In one embodiment, an antigen binding molecule of the invention is an immune checkpoint inhibitor.

[0289] In one embodiment, the antigen binding molecules of the present invention are used to treat or prevent cancer. As used herein, "cancer" refers to diseases caused by the uncontrolled division of abnormal cells. These include sarcomas (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, metastatic rhabdomyoma, fibroma, lipoma and teratoma of the heart; bronchogenic carcinoma of the lung (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondrotic hamartoma, mesothelioma; gastrointestinal tract: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (tubular adenocarcinoma, insulinoma), and pulmonary sarcoma (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymph ... linoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colorectal cancer; genitourinary tract: kidney (adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma) ), Testis (Seminoma, Teratoma, Embryonal Carcinoma, Teratocarcinoma, Choriocarcinoma, Sarcoma, Stromal Cell Carcinoma, Fibroma, Fibroadenoma, Adenomatoid Tumor, Lipoma); Liver: Hepatocellular Carcinoma, Cholangiocarcinoma, Hepatoblastoma, Angiosarcoma, Hepatocellular Adenoma, Hemangioma; Bone: Osteogenic Sarcoma, Fibrosarcoma, Malignant Fibrous Histiocytoma, Chondrosarcoma, Ewing's Sarcoma, Malignant Lymphoma (Reticulum Cell Sarcoma), Multiple Myeloma, Malignant Giant Cell Tumor, Chordoma, Osteochondroma (Osteochondral Exostosis), Benign Chondroma, Chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumor; Nervous system: skull (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), meninges (meningioma, meningeal sarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal neurofibroma, meningioma, glioma, sarcoma);Female reproductive system: uterus (endometrial carcinoma), uterus (cervical carcinoma of the cervix, preneoplastic cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, sarcoma botryoides) (embryonal rhabdomyosarcoma), Fallopian tube (cancer), breast cancer;Hematologic system: Blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma);Skin: Malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi (moles dysplastic nevi), lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland: neuroblastoma. In another embodiment, the cancer is carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (tract) cancer, renal cancer. , ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colon cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer. Another specific example of cancer is renal cell carcinoma. Yet another specific example of cancer is non-Hodgkin's lymphoma or cutaneous T-cell lymphoma.;

[0290] Of particular interest is the treatment or prevention of melanoma, metastatic cancer, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, hepatocellular carcinoma and bladder cancer.

[0291] In one embodiment, the cancer may be selected from the group consisting of melanoma, metastatic cancer, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, hepatocellular carcinoma, and bladder cancer.

[0292] Depending on the pathology to be treated, the antigen-binding molecules of the present invention can be used in combination with other pharma- ceutically active ingredients for simultaneous, separate or sequential use. For example, when treating or preventing cancer, the antigen-binding molecules of the present invention can be used in combination with another therapy or additional therapeutically active agent. Suitable therapies or additional therapeutically active agents include radiation therapy, chemotherapy treatment, targeted therapy, immunotherapy, monoclonal antibody therapy, hormonal therapy, angiogenesis inhibition, cancer vaccines, oncolytic viruses, toll-like receptor agonists, epigenetic modifications, modified T cells, T cell costimulatory agonists, tyrosine kinase inhibitors, other anti-cancer chemicals, palliative care for cancer therapy, immune checkpoint inhibitors, immunosuppressants, anti-inflammatory drugs, immunomodulators, immunostimulators, and / or inhibitors, such as IDO inhibitors, CSF-1R inhibitors, TGFB inhibitors, T cell costimulatory antagonists, Treg inhibitors, macrophage regulators, natural killer cell regulators, and chemokine receptor inhibitors.

[0293] Suitable chemotherapy treatments include gemcitabine, cyclophosphamide, doxorubicin, paclitaxel, and cisplatin. Suitable T cell costimulatory agonists include 4-1BB, OX40, CD40, GITR, and ICOS. Immune checkpoint inhibitors may act on members of the group consisting of PD-1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, CD70, and TIGIT.

[0294] The other therapy or additional therapeutically active agent may be another antigen binding molecule, which may be selected from the group consisting of an anti-PD-L1 antigen binding molecule, an anti-PD-1 antigen binding molecule, an anti-CTLA-4 antigen binding molecule, an anti-OX40 antigen binding molecule, an anti-ICOS antigen binding molecule, and an anti-GITR antigen binding molecule.

[0295] The pharmaceutical composition of the present invention can be formulated to include one or more additional pharma- ceutical active ingredients, such as those listed above. The antigen-binding molecule of the present invention may be provided as a part of a kit. Such a kit may include instructions for use and / or additional pharma- ceutical active ingredients. The antigen-binding molecule and the additional pharma-ceutical active ingredient may be arranged separately in the kit, or in some embodiments, the antigen-binding molecule and the additional pharma-ceutical active ingredient may be formulated together.

[0296] In one embodiment of the invention, there is provided an antibody, particularly a monovalent antibody such as a Fab fragment, that specifically binds to PD-L1. The antibody is selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, and 2A09. The antibody is used for the treatment or prevention of cancer.

[0297] The present invention will now be further described with reference to a number of specific examples which are presented for purposes of illustration and are not to be construed as limiting the scope of the invention. EXAMPLES

[0298] Construction of human naive libraries: RNA was extracted from PBMCs (0.5L blood / donor) of six healthy donors. 240 μg RNA was used for cDNA synthesis using random primers. cDNA was used for primary PCR amplification using non-tagged primers annealing to the VH, Vκ and Vλ and hinge CH1 regions of FR1, followed by secondary PCR amplification to introduce restriction endonuclease sites for cloning the VH, Vλ and Vκ genes into the pCB3 phagemid vector. Libraries were electroporated into TG1 E. coli cells and bacterial glycerol stocks of human libraries were stored at -80°C (human naive Fab library pool H002κ-H007κ and human naive Fab library pool H002λ-H007λ).

[0299] Selection: pH-selective anti-PD-L1 Fab Phage generation from human naive Fab library pools H002κ-H007κ and human naive Fab library pools H002λ-H007λ were used for two consecutive rounds of panning phage display selection with human PD-L1, followed by a third round of panning selection with cynomolgus and mouse PD-L1 proteins, and a fourth round of panning with mouse PD-L1 to increase the possibility of cross-reactive clones (human and mouse PD-L1 Fc were obtained from R&D Systems, and cynomolgus PD-L1 Fc was obtained from Sino biological). All selection rounds were performed with 10μg / ml recombinant PD-L1 protein at pH 6.0 (CPA buffer; 10mM sodium citrate, 10mM sodium phosphate, 10mM sodium acetate, 115mM sodium chloride), followed by washing away non-specific phages, followed by elution of specific phages by trypsin (complete elution). Serial dilutions of the eluted phages were performed and used to infect exponentially growing TG1. Infected TG1 were plated on LBCarb100Glu2% plates and enrichment values ​​over background (no antigen used for selection) were calculated.

[0300] ELISA Screening: Individual clones from the output of the third and fourth round panning selection conditions were picked into 96-well master plates and tested by binding ELISA for binding to human, cynomolgus monkey and mouse PD-L1 at pH 6.0 versus pH 7.4 as phage and periplasmic extracts (PE).

[0301] For phage binding ELISA, MaxiSorp™ high protein binding capacity 96-well ELISA plates were coated with 1μg / ml PD-L1-Fc protein diluted in PBS overnight at 4℃. The next day, plates were washed 3 times with CPA Tween 0.05% (pH 6.0) and blocked with 250μl / well 4% Marvel / CPA (pH 6.0) at room temperature for 1 hour. After blocking, plates were washed 3 times with CPA Tween 0.05% (pH 6.0) and incubated with 10μl phage per well in 90μl 1% Marvel / CPA (pH 6.0) for 1 hour at room temperature with shaking. Plates were then washed 3 times with CPA Tween 0.05% (pH 6.0) and incubated with 100 μl of anti-M13-HRP (GE catalog number 27-9421-01) per well in 1% Marvel / CPA (pH 6.0) for 1 h at RT with shaking. Plates were washed 3 times with CPA Tween 0.05% (pH 6.0), substrate solution (TMB, ThermoFisher) was added, the reaction was stopped with H2SO4 (ThermoFisher), and absorbance was read at 450 nm.

[0302] For the periplasmic extract (PE) binding ELISA, MaxiSorp™ high protein binding capacity 96-well ELISA plates were coated with 1 μg / ml PD-L1-Fc protein diluted in PBS overnight at 4° C. The next day, plates were washed three times with CPA Tween 0.05% (pH 6.0) or PBS Tween 0.05% (pH 7.4) and blocked with 250 μl / well of 4% Marvel / CPA (pH 6.0) or 4% Marvel / PBS (pH 7.4) for 1 hour at room temperature.

[0303] After blocking, the plates were washed 3 times with CPA Tween 0.05% (pH 6.0) or PBS Tween 0.05% (pH 7.4) and incubated with 20 μl P.E + 80 μl 1% Marvel / CPA (pH 6.0) or 1% Marvel / PBS (pH 7.4) per well for 1 hour at RT with shaking. The plates were then washed 3 times with CPA Tween 0.05% (pH 6.0) or PBS Tween 0.05% (pH 7.4) and incubated with 100 μl anti-c-Myc-HRP (Bethyl Cat. No. A190-105P) per well in 1% Marvel / CPA (pH 6.0) or 1% Marvel / PBS (pH 7.4) for 1 hour at RT with shaking. The plates were then washed three times with CPA Tween 0.05% (pH 6.0) or PBS Tween 0.05% (pH 7.4), and substrate solution (TMB solution) was added to the plates. The reaction was stopped with H2SO4, and absorbance was read at 450 nm. For most of the clones, binding was observed at both pHs. Positive binders to human PD-L1 were sequenced, and clones were grouped into families according to different HCDR3 sequences.

[0304] Clones from families with histidine in CDR3 showed higher OD values ​​at pH 6.0 than at pH 7.4 and bound to human, cynomolgus monkey and / or mouse PD-L1 in ELISA. A panel of family representative clones was selected to be screened for binding to human, cynomolgus monkey and mouse PD-L1 transfected HEK293FF cells.

[0305] Screening of selected clones in cells: Transfection of HEK293FF cells with human, cynomolgus and mouse PD-L1. HEK293FF cells were cultured in Freestyle™ 293 Expression Medium and seeded at 0.8E+06 cells / ml on the day of transfection. A mix of (Lipofectamine 2000 + 70μg DNA of human, cynomolgus or mouse plasmid) was prepared and added to the cells (mammalian expression plasmids were obtained from Sino Biological). Cells were incubated at 37°C, 5% CO2 for 18 hours and QC for human, cynomolgus and mouse PD-L1 expression by flow cytometry. Briefly, 2.0E+05 HEK293FF WT, human PD-L1, cynomolgus PD-L1 and mouse PD-L1 cells were incubated with 2.5μg / ml anti-human / mouse / cynomolgus (R&D Systems) PD-L1 (goat IgG) in a final volume of 50μl / well for 30min at 4℃ with gentle shaking. Cells were washed 3 times with 150μl FACS buffer (PBS / 0.5%FBS) at 300g for 3min and incubated with secondary anti-goat Alexa Fluor 647 (ThermoFisher) in a final volume of 50μl / well for 30min at 4℃ with gentle shaking in the dark. The cells were then washed three times with 150 μl / well FACS buffer, resuspended in 75 μl / well FACS buffer, and measured in the FL-4 channel (APC channel) of a FACS instrument (Accuri C6), acquiring a total of 10,000 cells per sample.

[0306] Binding of periplasmic extract (PE) to PD-L1 transfected cells: Periplasmic extracts (PE) from selected clones were incubated with anti-c-myc antibody specific for the c-myc tag present in the soluble Fab for 30 minutes at room temperature (RT) with agitation. The mix (PE + anti-c-myc antibody) was added to WT or human, cynomolgus monkey and mouse PD-L1 HEK293FF transfected cells and incubated for 60 minutes at 4°C with gentle shaking. The cells were then washed three times with 150μl / well FACS buffer, and then incubated with 50μl / well of secondary antibody goat anti-mouse APC (ThermoFisher) for 30 minutes at 4°C with shaking, protected from light. The cells were washed again and centrifuged three times with 150μl / well FACS buffer, then resuspended in 75μl / well FACS buffer and measured in the FL-4 channel (APC channel) of a FACS instrument (Accuri C6), acquiring a total of 10,000 cells per sample. Several clones bound to human and cynomolgus monkey PD-L1 cells. No mouse PD-L1 binders were found.

[0307] Screening selected clones for PD-1:PD-L1 blockade: PD-L1 cell-binding clones were screened by ELISA for PD-1:PD-L1 inhibition at pH 6.0. MaxiSorp™ high protein binding capacity 96-well ELISA plates were coated with 1 μg / ml human PD-1 Fc protein (R&D Systems) diluted in PBS overnight at 4° C. The next day, plates were washed three times with CPA Tween 0.05% (pH 6.0) and blocked with 250 μl / well of 1% casein / CPA (pH 6.0) for 1 h at room temperature. After blocking, the plates were washed 3 times with CPA Tween 0.05% (pH 6.0) and incubated with 50μl biotinylated PD-L1 (2μg / ml, R&D Systems) in CPA / 0.1% casein + 50μl PE (1:2.5) in CPA / 0.1% casein for competition in a final volume of 100μl for 1 hour at RT with shaking. The plates were washed 3 times with CPA Tween 0.05% (pH 6.0) and 50μl Extravidin-HRP (Sigma-Aldrich) diluted 2000-fold in 0.1% casein / CPA (pH 6.0) was added to the wells to detect binding of biotinylated human PD-L1 to coated PD-1 Fc in the presence of PE. The plates were incubated at room temperature for 1 hour. The plates were then washed three times with CPA Tween 0.05% (pH 6.0), substrate solution (TMB) was added to the plates, the reaction was stopped with H2SO4, and the plates were read at 450 nm. The ability of the clones to block PD-1:PD-L1 interaction was assessed by comparing the OD values ​​of wells with biotinylated PD-L1 in the presence of relevant and irrelevant PE. As a control and to compare the inhibitory effect of PE, a neutralizing anti-PD-L1 antibody was also used in the assay. Four clones, 1E08, 1A06, 2A09, and 2C11, were able to inhibit PD-1:PD-L1 interaction by 40%-80%. These clones were used for BIAcore analysis at pH 6.0 and pH 7.4 after IgG formatting.

[0308] IgG formatting, production and purification: Synthetic genes encoding VH, Vκ and Vλ antibody variable domains were purchased from Invitrogen. Each DNA was reconstituted according to the manufacturer's instructions. 200 ng of DNA was transformed into E. coli TOP10 chemically competent cells. DNA purification was performed using QIAGEN's QIAprep Spin Miniprep kit. Each DNA construct was restriction enzyme digested, the inserts gel purified, and each variable domain insert was ligated with a mammalian expression vector carrying an antibody heavy chain constant domain for the VH insert or a light chain constant domain for the Vκ or Vλ insert. ExpiCHO-S cells were transfected with the clones according to the manufacturer's instructions (ThermoFisher). 40 μg of all DNA plasmid constructs carrying the VH and Vk chains were used for protein production (32° C., 5% CO2) for 4 to 10 days in a total volume of 50 mL cells. After 4 and 10 days of protein production, quality control was performed by SDS-Page by analyzing the presence of a 150 kDa band in non-reducing conditions and 50 and 25 kDa bands in reducing conditions, corresponding to the molecular weight of IgG. Antibodies were purified using Hitrap MabSelect Sure columns on an AKTA Pure 25 system. IgG was subsequently eluted at pH 2.7 with 0.1 M glycine and 1.0 ml fractions were collected in tubes containing 0.1 ml Tris-HCl (pH 9.0) for neutralization. Antibody-containing fractions were pooled, desalted using HiTrap desalting columns on an AKTA Pure and buffer exchanged into 1x PBS (phosphate-buffered saline, pH 7.4) solution. Preparative size-exclusion chromatography was used to remove aggregates from the Protein A fraction. Protein concentration was determined by measuring absorbance at 280 nm.

[0309] Binding of purified antibodies to WT, human, cynomolgus and mouse PD-L1 transfected HEK293FF Transfection of HEK293FF cells with human, cynomolgus and mouse PD-L1 was performed as described above. Briefly, 2 × 10 5HEK293FF WT, human PD-L1, cynomolgus monkey PD-L1 and mouse PD-L1 cells were incubated with purified anti-PD-L1 IgG1 clones (500 nM-0.69 nM) in FACS buffer (PBS / 0.5% FBS) and incubated at 2 × 10 5 The plates were incubated with 100 μl / well of WT or human, cynomolgus monkey and mouse HEK293FF transfected cells for 1 h at 4° C. with gentle shaking in a final volume of 100 μl / well. An irrelevant antibody was used as a specificity control. After incubation, the cells were washed three times with 150 μl FACS buffer and 50 μl / well of anti-human IgG-Fc FITC-labeled antibody (ThermoFisher) was added to the cells for 1 h at 4° C. with gentle shaking. After washing, the cells were resuspended in 75 μl / well FACS buffer and measured in the FL-1 channel (FITC channel) of a FACS instrument (Accuri C6), acquiring a total of 10,000 cells per sample.

[0310] ELISA binding to PD-L1 at different pH: Purified anti-PD-L1 IgG1 clones were tested by binding ELISA for binding to human PD-L1 at pH 6.0 versus pH 7.4. MaxiSorp™ high protein binding capacity 96-well ELISA plates were coated with 1μg / ml human PD-L1-Fc protein diluted in PBS overnight at 4℃. The next day, plates were washed 3 times with CPA Tween 0.05% (pH 6.0) or PBS Tween 0.05% (pH 7.4) and blocked with 250μl / well of 4% Marvel / CPA (pH 6.0) or PBS (pH 7.4) for 1 hour at room temperature. After blocking, the plates were washed three times with CPA Tween 0.05% (pH 6.0) or PBS Tween 0.05% (pH 7.4) and incubated with 100 μl of serial dilutions of antibodies (3-fold dilutions; 100 nM to 0.137 nM) prepared in 1% Marvel CPA (pH 6.0) or PBS (pH 7.4) and incubated for 2 hours at room temperature (RT) with shaking. The plates were then washed three times with CPA Tween 0.05% (pH 6.0) or PBS Tween 0.05% (pH 7.4) and 100 μl / well of secondary antibody anti-human CH1 specific HRP (BD Pharmingen catalog number 555788) prepared in 1% Marvel CPA (pH 6.0) or PBS (pH 7.4) was added and incubated for 1 hour at RT with shaking. Plates were washed three times with CPA Tween 0.05% (pH 6.0) or PBS Tween 0.05% (pH 7.4), substrate solution (TMB) was added, the reaction was stopped with H2SO4, and plates were read at 450 nm. Krebs Ringer Bicarbonate Buffer (KRB, Amsbio Cat. No. KRB-1000) containing 1 mM lactate (Sigma Cat. No. 252476-500G) for pH 7.4, KRB + 15 mM lactate for pH 6.5, and KRB + 20 mM lactate for pH 6.0 were also used in the final screening assay.

[0311] Screening for antibodies for PD-1:PD-L1 blockade: Antibodies were screened by ELISA for PD-1:PD-L1 inhibition at pH 6.0.

[0312] MaxiSorp™ high protein binding capacity 96-well ELISA plates were coated with 1μg / ml human PD-1-Fc protein diluted in PBS overnight at 4℃. The next day, plates were washed 3 times with CPA Tween 0.05% (pH 6.0) and blocked with 250μl / well of 1% casein / CPA (pH 6.0) at room temperature for 1 hour. After blocking, plates were washed 3 times with CPA Tween 0.05% (pH 6.0) and incubated with 3-fold dilutions of anti-PD-L1 IgG1 clones starting from 100nM to 0.05nM for 1 hour (RT) with 1μg / ml human PD-L1 biotin in 0.1% casein / CPA (pH 6.0) in a total volume of 100μl / well. Plates were washed three times with CPA Tween 0.05% (pH 6.0) and Extravidin-HRP (1:2000 dilution) in 0.1% casein / CPA (pH 6.0) was added to the wells for detection of biotinylated human PD-L1 that may bind to coated PD-1-Fc in the presence of antibody. Plates were incubated at room temperature for 1 hour, then washed three times with CPA Tween 0.05% (pH 6.0), substrate solution (TMB) was added, the reaction was stopped with H2SO4, and plates were read at 450 nm. The ability of antibodies to block PD-1:PD-L1 interaction was assessed by comparing the OD values ​​of wells with biotinylated PD-L1 in the presence of anti-PD-L1 antibody versus irrelevant antibody. This assay was also performed at pH 6.5 and pH 7.4, substituting PBS for CPA in the above buffer. In the final screening assay, Krebs-Ringer bicarbonate buffer (KRB, Amsbio catalog number KRB-1000) containing 1 mM lactate (Sigma catalog number 252476-500G) for pH 7.4, KRB+15 mM lactate for pH 6.5, and KRB+20 mM lactate for pH 6.0 were also used.

[0313] Screening antibodies for CD80:PD-L1 blockade The ELISA assay was the same as above "Screening of antibodies for PD-1:PD-L1 blockade" except the coating antigen was human B7-1-Fc(CD80) at 1 μg / ml (R&D Systems catalog number 140-B1).

[0314] BIAcore analysis: To assess the affinity of selected IgG format purified clones to human PD-L1 at pH 6.0 and pH 7.4, human PD-L1-Fc protein was coated by amine coupling. Surface plasmon resonance (SPR) (BIAcore 3000, GE Healthcare) was used to determine the binding kinetics of selected IgG format purified clones to human PD-L1 at pH 6.0 and pH 7.4. Approximately 3000RU of human PD-L1 Fc (R&D Systems) at 50μg / ml in acetate buffer at pH 5.0 was immobilized onto a CM5 chip using standard amine coupling procedures. QC of PD-L1 immobilization was performed using a commercial anti-PD-L1 antibody (R&D systems catalogue no. AF156) at 2.0μg / ml. 1x HBS-EP (pH 7.4) or HBS-EP (pH 7.0) or HBS-EP 10 nM citrate buffer (pH 6.5) or HBS-EP 10 nM citrate buffer (pH 6.0) were used as running buffers in the binding kinetics measurements. Antibody gradients consisted of 3-fold dilutions starting from 50 nM to 0.13 nM in the appropriate pH buffer and were injected for 120 seconds at a flow rate of 30 μl / min. After dissociation, regeneration of the PD-L1-Fc surface was achieved by a single injection of 10 mM NaOH in 1 M NaCl (30 seconds injection at 20 μl / min) followed by 10 mM glycine (pH 1.5) at 20 μl / min for 30 seconds. A 1:1 binding fit with mass transfer was applied to the curves using BIAevaluation software to obtain association rate (ka), dissociation rate (kd) and affinity (K D The rate constants of the antibody-antigen interaction, including

[0315] Generation of affinity maturation libraries: The construction of the phage display library was generated by gene assembly. V genes were synthetically generated by PCR using overlapping oligonucleotides and / or trimers with specific mutations at certain positions defined in the library design. Oligonucleotides were designed to assemble the complete VH and Vlambda sequences, and primers specific for the HCDR1, HCDR2, HCDR3 or LCDR1, LCDR2, LCDR3 regions contained either parental nucleotides or degenerate codons to allow the construction of different variants. Cloning restriction sites were introduced into the VH and Vlambda genes and further inserted into the phagemid vector. All gene assembly PCRs were performed using three different annealing temperatures, and PCR products were pooled if no differences were observed between the PCR products of different annealing temperatures. Gel-purified Vlambda genes were cloned into pCB13_2A09VH_WT phagemid vector with constant human heavy and light genes (CH and CL) and parental VH chain domains to construct AFF2A09_LCDR1, AFF2A09_LCDR2 and AFF2A09_LCDR3 affinity maturation libraries. Gel-purified VH randomized genes were cloned into pCB13_2A09Vlambda_WT phagemid vector with CH and CL human constant regions and parental Vlambda domains to construct AFF2A09_HCDR1, AFF2A09_HCDR2 and AFF2A09_HCDR3 affinity maturation libraries. All Fab phage display libraries had more than 80% transformants containing complete Fab inserts, and 48 transformants per library were sent for sequencing and analyzed for amino acid frequency introduction in the target heavy and light CDRs compared to the WT sequence of the parent clone 2A09.

[0316] Affinity maturation phage display selection: Round I: Panning selection. The first round of panning selection with the 2A09 affinity maturation sub-library was performed to select Fabs against human PD-L1 at pH 6.0. Plates were coated with 10 μg / ml human PD-L1 in 1×PBS at 100 μl / well O / N at 4°C, followed by blocking with 250 μl / well of 4% skim milk (Marvel, Cat. No. 928964) in PBS for 2 hours at RT. Phages from the 2A09 affinity maturation sub-library were blocked in 4% skim milk / CPA (pH 6.0) per selection condition for 30 minutes at RT with rotation. 50 μl of phage from the affinity maturation sub-library in 4% skim milk / 1×PBS per selection condition were incubated on the antigen-coated plates at 100 μl / well for 2 hours at RT. After incubation, wells were washed 15 times with 1x CPA-Tween 0.05% (including 5 min shaking incubation after every 5 washes) and 5 times with 250 μl / well of 1x CPA (pH 6.0). Complete elution of phages bound to PD-L1 was performed using 1 mg / ml trypsin (Sigma, Cat. No. T1426-5G). 150 μl / well trypsin was added and incubated for 20 min at RT. Trypsin protease activity was inhibited with 10 μl of AEBSF (Sigma, Cat. No. A8456). Serial dilutions of eluted phages were performed and used to infect exponentially growing TG1. Infected TG1 were plated on LBCarb100Glu2% plates and enrichment was calculated as the ratio of the number of phages eluted with antigen selection conditions to the number of phages eluted without antigen selection conditions. Good enrichment was observed for all sub-libraries and the output was subjected to a second round in solution.

[0317] Round II: Affinity selection in solution. For selection of affinity matured Fabs against PD-L1 recognizing antigen in solution, a second round was performed with biotinylated PD-L1 and streptavidin coated magnetic beads. Antigen blocking: 10nM and 1nM biotinylated PD-L1 were blocked with 2% Marvel CPA buffer (pH6.0) for 30min at RT with rotation. Phage blocking: 10μl input phage from output from the first round was blocked with 4% Marvel CPA buffer (per condition) for 30min at RT with rotation. Phage+antigen mix: 100μl blocking antigen (bio-PD-L1) + 100μl blocking phage (per condition) were incubated for 2h at RT with rotation. Bead washing / blocking: 30μl Dynabeads™ MyOne™ Streptavidin T1 magnetic beads (Invitrogen, Cat. No. 65601) per selection condition were washed 3 times with CPA buffer-Tween 0.05% and blocked in 2% Marvel CPA buffer for 30 minutes at RT with rotation. Mixed phage+antigen bead capture: 200μl of mix (phage+antigen bio-PD-L1) was added to the magnetic beads for 15 minutes at RT with rotation. After incubation, the beads were washed 5 times with CPA buffer-Tween 0.05% and then twice with 1ml CPA buffer. Elution: Complete elution of phages bound to PD-L1 was performed with 200μl / condition of 1mg / ml trypsin for 20 minutes at RT with rotation. Inhibition of trypsin protease activity was performed with 10 μl of AEBSF. Enrichment: Serial dilutions of eluted phages were made and used to infect exponentially growing TG1. Infected TG1 were plated on LBCarb100Glu2% plates and enrichment during recombinant protein selection was calculated as the ratio of the number of phages eluted with antigen selection conditions to the number of phages eluted without antigen selection conditions.

[0318] Round 3: Affinity selection in solution with 2h or 20h off-rate wash. To select affinity matured Fabs against PD-L1 recognizing antigen in solution, a third round of selection in solution was performed using streptavidin coated magnetic beads with 100x excess of non-biotinylated PD-L1 with 2h or 20h off-rate wash to select high affinity clones. Antigen blocking: Biotinylated PD-L1 at 1nM, 0.1nM, 0.01nM and 0nM was blocked with 2% Marvel CPA buffer (pH 6.0) for 30min at RT with rotation. Phage blocking: 10μl of input phage from output from round 2 was blocked with 4% Marvel CPA buffer (per condition) for 30min at RT with rotation. Phage+antigen mix: 100μl blocking antigen (bio-PD-L1) + 100μl blocking phage (per condition) were incubated for 2 hours at RT with rotation. Beads washing / blocking: 30μl Dynabeads™ MyOne™ Streptavidin T1 magnetic beads (Invitrogen, Cat. No. 65601) per selection condition were washed 3 times with CPA buffer-Tween 0.05% and blocked in 2% Marvel CPA buffer for 30 minutes at RT with rotation. Mixed phage+antigen bead capture: 200μl mix (phage+antigen bio-PD-L1) was added to the magnetic beads for 15 minutes at RT with rotation. After incubation, the beads were washed 5 times with CPA buffer-Tween 0.05% and twice with CPA buffer. Off-rate wash: 100-fold excess of non-biotinylated PD-L1 (10 nM, 1 nM and 0.1 nM) or CPA buffer was added to selected wells and incubated with rotation at RT for 2 or 20 hours. Elution: Complete elution of phages bound to PD-L1 was performed with 1 mg / ml trypsin at 200 μl / condition for 20 minutes at RT with rotation. Inhibition of trypsin protease activity was performed with 10 μl AEBSF. Enrichment: Serial dilutions of eluted phages were performed and used to infect exponentially growing TG1.Infected TG1 were plated on LBCarb100Glu2% plates and enrichment during recombinant protein selection was calculated as the ratio of the number of phages eluted with antigen selection conditions to the number of phages eluted without antigen selection conditions.Outputs selected with 1 nM biotinylated PD-L1 and 100 nM PD-L1 for 2 h or 20 h off-rate washes were subjected to round IV selection in cells.

[0319] Round 4: Cell selection. To select affinity matured Fabs against native PD-L1 present on the cell surface, a selection round was performed at pH 6.0 using MDA-MB-231 cells (endogenous cells expressing PD-L1). Cells: 5.0E+06 MDA-MB-231 cells in 10% FBS / PBS (pH 6.0) (acidified with 1M HCL) were used per sub-library condition. Cells were also QCed for PD-L1 expression using secondary antibody (Alexa Fluor™ 488 AffiniPure Mouse Anti-Human IgG; Jackson ImmunoResearch Laboratories Catalog No. 209-545-098) for detection by incubating with mAb 2A09 or mAb 12A4 (according to US Pat. No. 9,856,320) at 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0 nM in FACS buffer pH 7.4 or pH 6.0 for 1 hour with shaking at 4°C. Phage blocking: Output phages from round 3 selection (100 nM PD-L1 for 2 hours or 20 hours off-rate wash) per sub-library were pooled and blocked with 10% FBS / PBS (pH 6.0) for 20 minutes at 4°C with rotation. Phage+ cells: 5E+06 MDA-MB-231 cells per condition were resuspended with blocking phage and incubated for 2 hours at 4°C with rotation. After incubation, cells were washed 4 times with 10% FBS / PBS (pH 6.0) and once more with 1 ml PBS (pH 6.0) per wash. Elution: Complete elution of PD-L1-bound phages was performed with 200 μl of 1 mg / ml trypsin per condition for 20 minutes at RT with rotation, and trypsin protease activity was inhibited with 10 μl AEBSF. Enrichment: Serial dilutions of eluted phages were performed and used to infect exponentially growing TG1. Infected TG1 were plated on LBCarb100Glu2% plates and enrichment values ​​relative to background (no antigen used for selection) were calculated.

[0320] The HCDR1 and HCDR2 libraries and the LCDR3 library did not yield any significant hits compared to 2A09 WT. The HCDR3 library yielded one primary hit and three other unique sequences were isolated with improved binding compared to 2A09, the LCDR1 library yielded 36 unique hits, and the LCDR2 library yielded 6 unique hits with improved binding.

[0321] These hits were further analyzed by cell binding assays at pH 6 and pH 7.4, BIAcore assays and PD-L1 neutralization assays as described above.

[0322] The lead Fab was generated by Pierce™ Fab Preparation Kit (cat no. 44985) from purified IgG as described above.

[0323] Binding of anti-PD-L1 mAb or Fab to MDA-MB-231 cells in tumor-like buffer To mimic the acidic tumor environment (The metabolism of tumors in the body, Warburg et al. J Gen Physiol. 1927;8:519-30) using tumor cells expressing PD-L1, the following buffers were used: Krebs-Ringer bicarbonate buffer (KRB, Amsbio catalog number KRB-1000) with 1 mM lactate (Sigma catalog number 252476-500G) for pH 7.4, KRB + 15 mM lactate for pH 6.5, and KRB + 20 mM lactate for pH 6.0. Three-fold dilutions of Fab or IgG (100 nM to 0.0005 nM; 0) were prepared in the respective pH buffers and incubated with 0.60E05 MDA-MB-231 cells for 60 min at 4 °C. Cells were then washed three times with the respective pH buffers at 300 g for 3 min. The cells were then incubated with 50 μl of anti-human IgG (Fab specific)-FITC (Sigma, Cat. No. F5512-1ML) diluted 100-fold in the respective pH buffer. Binding of IgG or Fab to PD-L1 expressing cells was measured by FACS (Attune Nxt), acquiring a total of 10,000 cells per sample.

[0324] statistical analysis GraphPad Prism 7 software (GraphPad 50 Software, Inc., La Jolla, Calif., USA) was used for IC50, EC50 analysis and statistical analysis.

[0325] result The initial screening cascade of the Fab library was phage ELISA at pH6 against human and cynomolgus PD-L1, followed by PD-L1-PD-1 inhibition ELISA and binding to PD-L1 positive cells (MDA-MB-231). Twenty clones from different families that were positive by these assays were rescreened as PEs. Fab generation at pH6 in ELISA against human and cynomolgus PD-L1, followed by PD-L1-PD-1 inhibition ELISA and cell binding assay in MDA-MB-231 cells. From these screens, 16 clones were found to bind to human and cynomolgus cells as both phage and PEs, seven had weak neutralizing activity, and four Fabs with high neutralizing activity were selected. As two of these clones had identical CDRs, three Fabs were converted to IgG for further analysis. All three mAbs inhibited PD-L1 binding to PD-1 by ELISA (Figure 1A), with IC50 values ​​calculated from Figure 1A of 0.4 nM for 2A09, 0.9 nM for 1A06, and 1.1 nM for 1E08. All three mAbs also inhibited PD-L1 binding to CD80 by ELISA as shown in Figure 1B, with IC50 values ​​calculated from Figure 1B of 1.2 nM for 2A09, 6.3 nM for 1A06, and 2.8 nM for 1E08. mAb 2A09 also showed comparable binding to human and cynomolgus PD-L1.

[0326] The Fab sequence of 2A09 is shown in Table 4.

[0327] TIFF0007653408000006.tif28170

[0328] Initial analysis of off-rates by BIAcore (Figure 2) revealed that two of the mAbs (1A06 and 1E08) have selective binding to PD-L1 at pH 7.4, as indicated by lower Kd values ​​at pH 7.4 compared to pH 6. In stark contrast, mAb 2A09 has better binding to PD-L1 at pH 6.0, with an off-rate that was approximately 5-fold better. The BIAcore analysis was repeated over a wider range of pH and compared with anti-PD-L1 mAb 12A4 (from US Patent No. 9,856,320) and the results are shown in Table 5.

[0329] mAb 2A09 showed approximately 20-fold higher affinity at pH 6 compared to pH 7.4, which is in stark contrast to mAb 12A4, which is not selected for pH-specific binding and actually shows lower affinity at pH 6 compared to pH 7.4.

[0330] TIFF0007653408000007.tif49170

[0331] The data presented in Table 5 reflects a pH dependence of binding, however there is an avidity component to the binding of mAbs to PD-L1 on the BIAcore surface. To circumvent this issue and analyze the binding of 2A09 to cells in buffers that resemble the tumor microenvironment, PD-L1-expressing MDA-MB-231 cells were incubated with 2A09 mAb and Fab in Krebs buffer containing lactate at pH 6, 6.5, and 7.4 (Figure 3). The EC50 for 2A09 binding to PD-L1 was calculated from Figure 3 to be 16.3 nM at pH 7.4, 6.1 nM at pH 6.6, and 2.4 nM at pH 6.0. Under these conditions, the Fab of 2A09 had little binding activity (Figure 3).

[0332] As a result of the low affinity of the 2A09 Fab, an affinity maturation campaign was performed on this clone. A screening campaign was performed as outlined above for 2A09, and five Fab affinity matured leads were selected that bound to PD-L1, showing pH dependence of binding and increased affinity compared to the parental 2A09 clone (Table 6). All five leads were selected, including mutations in CDR1 of the light chain.

[0333] TIFF0007653408000008.tif43170

[0334] All five selected Fabs were purified and initially examined by ELISA (Figure 4). All Fabs demonstrated pH-specific binding, with the positive control anti-PD-L1 Fab or mAb 2.7A04OPT (from WO2011066389) showing no pH-dependent binding. The negative control Fab (2A11, anti-HEL) showed no specific binding. Initial assays by this ELISA demonstrated that the Fabs have much greater affinity at pH 6 (Table 7) across the concentrations examined, and weak non-saturable binding at pH 7.4. Although EC50s could not be accurately determined by this methodology at pH 7.4, the ratios of binding are shown in Table 7, and comparison of OD readings on both pH curves demonstrated that the Fabs bound 9- to 53-fold better at pH 6. In this assay, all Fabs reached saturation of binding at 4 nM at pH 6, whereas none of the five Fabs reached saturation at pH 7.4 (Figure 4).

[0335] TIFF0007653408000009.tif34170

[0336] The anti-PD-L1 affinity matured Fabs were tested for their inhibitory activity at pH 6.0, 6.5 and 7.4 in a PD-L1 inhibition ELISA (Figure 5). Anti-PD-L1 Fab 2.7A04OPT was used as a positive control and Fab 2A11 was used as a negative control in this assay. The IC50s were calculated from the curves and are shown in Table 8. The IC50s for inhibition of PD-L1 binding to PD-1 by anti-PD-L1 Fabs 8D06, 8A04, 8G08 and 8B06 at pH 6 are only approximately 3-fold lower than that of the control Fab 2.7A04OPT. At pH 6, 6.5 and 7.4, anti-PD-L1 Fab 2.7A04OPT shows no pH preference for inhibition. This is in stark contrast to the Fabs of the present invention, which show a clear pH-dependence of inhibition (Figure 5 and Table 8). Fabs 8D06, 8A04, 8G08 and 8B06 have approximately 3-fold lower inhibitory activity at pH 6.5 compared to pH 6, and have little activity at pH 7.4. Anti-PD-L1 Fab 8D04 is approximately 3-fold less potent than Fabs 8D06, 8A04, 8G08 and 8B06 in this assay. It is important to note that at 100 nM, none of the Fabs completely inhibit PD-L1 binding to PD-1 at pH 7.4, with Fab 8A04 being the best with approximately 60% inhibition (Figure 5).

[0337] TIFF0007653408000010.tif38170

[0338] The anti-PD-L1 affinity matured Fabs were tested for their ability to bind native PD-L1 in a cell line (MDA-MB-231 cells) in a buffer (Krebs-lactate) that resembles the tumor microenvironment. Again, all five affinity matured Fabs showed pH-dependent binding (Figure 6 and Table 9A). The positive control anti-PD-L1 Fab 2.7A04OPT showed no pH binding preference. The pH binding preference ranged from 8-fold (Fab 8G08) and 11-fold (Fab 8D06) to over 100-fold (Fabs 8A04, 8D04 and 8B06). For all Fabs, the total binding (MFI) at pH 7.4 and 100 nM Fab was much lower than at pH 6 and did not reach saturation. The Fabs were also formatted into IgG1 and binding to MDA-MB-231 cells at pH 7.4, 6.5 and 6.0 was investigated (Table 9B). Surprisingly, the mAb did not show a pH-dependence of binding (Table 9B). The pH-dependence of binding is related to the format (Fab, monomeric binder), affinity since the EC50 binding of the Fab is approximately 100-fold lower than the mAb, and the introduction of histidine residues in the CDRs (Table 10).

[0339] TIFF0007653408000011.tif50170

[0340] TIFF0007653408000012.tif42170

[0341] Proton-related binding events play an important role in biological regulation, including, for example, the Bohr effect in hemoglobin (Perutz MF et al. J Mol Biol. 1980;138:649-68), pH-dependent binding of serine protease inhibitors (Ascenzi P. et al. J Mol Recognit. 1991, 4:113-9), and human prolactin, which has been reported to show a large decrease in binding affinity to its receptor with a slight decrease in pH from 8 to 6.7 (Kulkarni MV et al. J Biol Chem. 2010;285:38524-33). Highly pH-dependent binding events require multiple ionizable residues (e.g., histidines), which can be successfully introduced by the screening methodology used in the methodology described herein. Imidazole forms the side chain of histidine, and in addition to its pK (≈6.0) being within the physiological pH range, the protonated and unprotonated forms of imidazole are chemically very different. The unprotonated form is hydrophobic and aromatic, whereas the protonated form is hydrophilic and positively charged. Thus, chemical interactions differ significantly at pHs around the pK. At pH 7.0, the unprotonated form predominates, favoring interactions with other hydrophobic groups, whereas at pH 5.0 the imidazole group is protonated, favoring a hydrophilic environment.

[0342] The HCDR1, HCDR2, and HCDR3, and LCDR2 and LCDR3 sequences of the five affinity matured anti-PD-L1 Fabs are identical to the wild-type 2A09 Fab. There is only one His residue in HCDR3, which may explain the low pH sensitivity of this clone. The five affinity matured anti-PD-L1 Fabs have one or two additional His residues in the CDR1 sequence (Table 10), and the VL and VH sequences are shown in Table 11.

[0343] The additional pH specificity and affinity may be due, in part, to the selection of clones with an additional His residue in LCDR1 that can interact with the PD-L1 epitope and create a "pH switch".

[0344] TIFF0007653408000013.tif39170

[0345] TIFF0007653408000014.tif100170

[0346] Evaluation of Ab-52 and Ab-55 according to WO 2014055897 WO2014055897 (DANA FARBER CANCER INST INC) discloses human monoclonal antibodies that bind to PD-L1. The VL sequences of Ab-52 and Ab-55 disclosed in WO2014055897 comprise the sequence of SEQ ID NO: 2. These antibodies were evaluated to determine whether there was evidence of pH-dependent binding.

[0347] method Ab-52 and Ab-55 full-length anti-PD-L1 antibodies were generated from sequences in WO2014055897 (see WO2014055897, page 16). The positive control anti-PD-L1 mAb, 2.7A04OPT, is from WO2011066389 (see WO2011066389, page 71), and the negative control mAb (2A11) is anti-HEL (hen egg lysozyme).

[0348] Fab was generated from intact mAb using a commercially available kit.

[0349] PD-L1 neutralization ELISA mAbs or Fabs (100nM-0.14nM) were added to 96-well plates coated with 1μg / ml PD-1, and the inhibition of 1μg / ml PD-L1-biotin was measured to determine the neutralizing capacity of each mAb or Fab. PD-L1 interaction with PD-L1-biotin was detected using Extravidin-HRP. The buffer used was 0.1% casein / Krebs / lactate (pH 6.0 or pH 7.4).

[0350] The complete methodology for the assays performed is presented in the Examples section of this application under the headings "ELISA binding to PD-L1 at different pH" and "Screening of antibodies for PD-1:PD-L1 blockade."

[0351] result In the first experiment (Figure 8), the Fabs of Ab-52 and Ab-55 were compared against each of the five Fabs of the invention (8B06, 8D06, 8G08, 8A04, 8D04) for their ability to neutralize PD-L1 at pH 6 or pH 7.4. Fab 2.7A04OPT from WO2011066389 was used as a positive control.

[0352] The positive control comparator mAb, 2.7A04OPT, equally inhibited PD-L1 binding to PD-1 at pH 6 compared to pH 7.4.

[0353] Five Fabs of the invention (8D06, 8A04, 8G08, 8D04, 8B06) inhibited PD-L1 binding to PD-1 at pH 6, with little inhibition observed at pH 7.4 as expected. In contrast, no inhibition of binding was observed at either pH for Fabs Ab-52 and Ab-55. This may be due to the lower neutralization affinity of Fabs Ab-52 and Ab-55 compared to the other Fabs used in Figure 8.

[0354] For this reason, Ab-52 and Ab-55 mAbs were used in a second experiment to determine whether they possess pH-dependent neutralizing activity (FIG. 9).

[0355] Two of the Fabs of the invention (8G08 and 8B06) were used in this assay and again demonstrated pH-dependent neutralization at pH 6 and pH 6.5, with little activity at pH 7.4. In stark contrast, mAbs Ab-52 and Ab-55 demonstrated no pH-dependent neutralization of PD-L1 binding to PD-1. This data is comparable to the positive control Fab 2.7A04OPT.

[0356] Thus, despite the slight sequence overlap, Ab-52 and Ab-55 do not share the novel properties of the anti-PD-L1 antigen binding molecules of the invention. In particular, Ab-52 and Ab-55 do not share the higher affinity for PD-L1 at pH 6.0 compared to pH 7.4 exhibited by the anti-PD-L1 antigen binding molecules of the invention. In certain embodiments, for example, the following items are provided: (Item 1) An anti-PD-L1 antigen binding molecule, comprising a VLCDR1 having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 16, 12, 10, 14, 18, or 2. (Item 2) The anti-PD-L1 antigen binding molecule of item 1, comprising a VLCDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 16, 12, 10, 14, 18 or 2. (Item 3) VHCDR1 having at least 80% identity to the amino acid sequence of SEQ ID NO:6; VHCDR2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 7; And, VHCDR3 having at least 80% identity to the amino acid sequence of SEQ ID NO:8; and / or VLCDR1 having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 16, 12, 10, 14, 18 or 2; VLCDR2 having at least 80% identity to the amino acid sequence of SEQ ID NO:3; And, comprising a VLCDR3 having at least 80% identity to the amino acid sequence of SEQ ID NO:4; 3. The anti-PD-L1 antigen-binding molecule of item 1 or 2. (Item 4) VHCDR1 comprising the amino acid sequence of SEQ ID NO:6, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and VHCDR3 comprising the amino acid sequence of SEQ ID NO:8, and / or VLCDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 16, 12, 10, 14, 18 or 2; VLCDR2 comprising the amino acid sequence of SEQ ID NO:3, and VLCDR3 comprising the amino acid sequence of SEQ ID NO:4, The anti-PD-L1 antigen-binding molecule of any one of items 1 to 3. (Item 5) A heavy chain variable region having at least 80% identity to the amino acid sequence of SEQ ID NO:5; and / or comprising a light chain variable region having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, and SEQ ID NO:1; The anti-PD-L1 antigen-binding molecule of any one of items 1 to 4. (Item 6) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5, and / or a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, and SEQ ID NO:1; The anti-PD-L1 antigen-binding molecule of any one of items 1 to 5. (Item 7) (a) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (b) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (c) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (d) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence MRTGTGNKGHYTR (SEQ ID NO: 14), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (e) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (f) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; 7. The anti-PD-L1 antigen-binding molecule of any one of items 1 to 6, selected from the group consisting of: (Item 8) (a) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:15, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:15, respectively; (b) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:11, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:11, respectively; (c) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:9, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:9, respectively; (d) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:13, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:13, respectively; (e) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:17, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:17, respectively; (f) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:1, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:1, respectively; 8. The anti-PD-L1 antigen-binding molecule of any one of items 1 to 7, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: (Item 9) The anti-PD-L1 antigen-binding molecule of any one of items 1 to 8, which is an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, 2A09. (Item 10) 10. The anti-PD-L1 antigen-binding molecule of item 9, wherein the antibody contains 1 to 10, 1 to 5, or 1 to 2 conservative amino acid substitutions across all six CDR regions. (Item 11) An anti-PD-L1 antigen-binding molecule that specifically binds to PD-L1 and inhibits binding of PD-L1 to the antigen-binding molecule of any one of items 1 to 10. (Item 12) An anti-PD-L1 antigen binding molecule that specifically binds to an epitope on human PD-L1 and competes for binding to PD-L1 with an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, and 2A09. (Item 13) 13. The anti-PD-L1 antigen-binding molecule of any one of items 1 to 12, wherein the antigen-binding molecule is an antibody or an antigen-binding fragment or derivative thereof, and optionally the antibody fragment or derivative is a Fab, F(ab')2, Fv, scFv, dAb, Fd or diabody. (Item 14) 14. The anti-PD-L1 antigen-binding molecule of item 13, wherein the antibody or antigen-binding fragment or derivative thereof is monovalent. (Item 15) 15. The anti-PD-L1 antibody of item 14, wherein the antibody, or antigen-binding fragment or derivative thereof, is fully human. (Item 16) 16. The anti-PD-L1 antibody or antigen-binding fragment or derivative of any one of items 13 to 15, wherein the antibody or antigen-binding fragment or derivative is an IgA, IgD, IgE, IgG, IgM or IgY antibody, or an antigen-binding fragment or derivative thereof, optionally wherein the antibody or antigen-binding fragment or derivative is an IgG antibody, and further optionally wherein the IgG antibody or antigen-binding fragment or derivative is an IgG1 antibody or antigen-binding fragment or derivative thereof. (Item 17) 17. The anti-PD-L1 antibody or antibody fragment of paragraph 16, wherein the antibody is a bispecific antibody or a bispecific antigen-binding fragment or derivative thereof. (Item 18) 18. The antigen-binding molecule of any one of items 1 to 17, which specifically binds to PD-L1 in a pH-dependent manner. (Item 19) 19. The antigen-binding molecule of any one of items 1 to 18, which has a higher affinity for PD-L1 at pH 6.0 than at pH 7.4. (Item 20) 20. The antigen-binding molecule of any one of items 1 to 19, which has at least about 5-fold higher affinity for PD-L1 at pH 6.0 than at a pH of 7.4. (Item 21) 21. The antigen-binding molecule of any one of items 1 to 20, having a pH 6.0:7.4 binding ratio of at least 5. (Item 22) 22. The antigen-binding molecule of any one of items 1 to 21, wherein the IC50 inhibition value of PD-L1 binding to PD-1 is higher at pH 7.4 than at pH 6.0. (Item 23) When administered in vivo or in vitro, a. reverse immune suppression; and / or Enhances bT cell immunity, The antigen-binding molecule according to any one of items 1 to 22. (Item 24) 24. A pharmaceutical composition comprising the antigen-binding molecule according to any one of items 1 to 23 and a pharma- ceutical acceptable excipient. (Item 25) 25. The pharmaceutical composition according to item 24, further comprising an additional therapeutically active agent or used in combination with another therapy or an additional therapeutically active agent. (Item 26) 26. The antigen-binding molecule according to any one of items 1 to 23 or the pharmaceutical composition according to item 24 or 25, for use in medicine. (Item 27) The antigen-binding molecule according to any one of items 1 to 23 or the pharmaceutical composition according to item 24 or 25, for use in the treatment or prevention of cancer. (Item 28) 28. The antigen-binding molecule or pharmaceutical composition for use according to Item 27, wherein the cancer is selected from the group consisting of melanoma, metastatic cancer, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, hepatocellular carcinoma and bladder cancer.

[0357] EMBODIMENTS OF THE PRESENT DISCLOSURE The present invention provides at least the following embodiments, which are listed as numbered items: 1. An anti-PD-L1 antigen binding molecule, comprising a VLCDR1 having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 16, 12, 10, 14, 18 or 2. 2. The anti-PD-L1 antigen binding molecule of item 1, comprising a VLCDR1 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences of SEQ ID NOs: 16, 12, 10, 14, 18, and 2. 3. The anti-PD-L1 antigen binding molecule of item 1, comprising a VLCDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 16, 12, 10, 14, 18, or 2. 4. A VHCDR1 having at least 80% identity to the amino acid sequence of SEQ ID NO:6; A VHCDR2 having at least 80% identity to the amino acid sequence of SEQ ID NO:7, and VHCDR3 having at least 80% identity to the amino acid sequence of SEQ ID NO: 8, and / or VLCDR1 having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 16, 12, 10, 14, 18 and 2; VLCDR2 having at least 80% identity to the amino acid sequence of SEQ ID NO:3; 4. The anti-PD-L1 antigen-binding molecule of any one of items 1 to 3, comprising a VLCDR3 having at least 80% identity to the amino acid sequence of SEQ ID NO:4. 5. A VH CDR1 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:6; A VH CDR2 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 7, and A VH CDR3 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 8, and / or VLCDR1 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences of SEQ ID NOs: 16, 12, 10, 14, 18 and 2; A VLCDR2 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:3, and 5. The anti-PD-L1 antigen-binding molecule of any one of items 1 to 4, comprising a VLCDR3 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:4. 6. VHCDR1 comprising the amino acid sequence of SEQ ID NO:6; VHCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 8, and / or VLCDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 16, 12, 10, 14, 18 or 2; VLCDR2 comprising the amino acid sequence of SEQ ID NO:3, and 6. The anti-PD-L1 antigen-binding molecule of any one of items 1 to 5, comprising a VLCDR3 comprising the amino acid sequence of SEQ ID NO:4. 7. A heavy chain variable region having at least 80% identity to the amino acid sequence of SEQ ID NO:5, and / or 7. The anti-PD-L1 antigen-binding molecule of any one of items 1 to 6, comprising a light chain variable region having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 1. 8. A heavy chain variable region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:5, and / or 8. The anti-PD-L1 antigen-binding molecule of any one of items 1 to 7, comprising a light chain variable region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:1. 9. A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5, and / or 9. The anti-PD-L1 antigen-binding molecule of any one of Items 1 to 8, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 1. 10. (a) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence RETELSRRLHYVR (SEQ ID NO: 16), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (b) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence VLSPRTHAGHYYR (SEQ ID NO: 12), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (c) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence ISNDDPASGHYHR (SEQ ID NO: 10), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (d) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence MRTGTGNKGHYTR (SEQ ID NO: 14), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (e) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence RGTGSSFHHKYVR (SEQ ID NO: 18), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule comprising VH CDR and VL CDR sequences at least 90%, at least 95%, at least 98% or at least 99% identical thereto; (f) a heavy chain variable region comprising a VHCDR1 comprising the amino acid sequence SYGMY (SEQ ID NO: 6), a VHCDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 7), and a VHCDR3 comprising the amino acid sequence GALTHWGVVIGDGMDV (SEQ ID NO: 8); a light chain variable region comprising a VLCDR1 comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 2), a VLCDR2 comprising the amino acid sequence EDDQRPS (SEQ ID NO: 3), and a VLCDR3 comprising the amino acid sequence QNVLTTPWT (SEQ ID NO: 4); or an anti-PD-L1 antigen binding molecule that comprises VH CDR and VL CDR sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. 11. (a) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:15, or a VH and VL sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:15, respectively; (b) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:11, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:11, respectively; (c) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:9, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:9, respectively; (d) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:13, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:13, respectively; (e) a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:17, or a VH and VL sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:17, respectively; (f) the anti-PD-L1 antigen-binding molecule of any of the preceding items, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of a VH comprising the amino acid sequence of SEQ ID NO:5 and a VL comprising the amino acid sequence of SEQ ID NO:1, or a VH and VL sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:5 and SEQ ID NO:1, respectively. 12. The anti-PD-L1 antigen-binding molecule of any of the previous items, which is an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, and 2A09. 13. The anti-PD-L1 antigen binding molecule of item 12, wherein the antibody contains 1-10, 1-5, or 1 or 2 conservative amino acid substitutions across all six CDR regions. 14. The anti-PD-L1 antigen-binding molecule of item 12, wherein the antibody comprises 1-10, 1-5, or 1 or 2 conservative amino acid substitutions in one or both of the variable heavy and variable light regions. 15. The anti-PD-L1 antigen binding molecule of item 12, wherein the antibody contains 1-10, 1-5, or 1 or 2 conservative amino acid substitutions in the framework regions. 16. An antigen-binding molecule that specifically binds to PD-L1 and inhibits binding of PD-L1 to the antigen-binding molecule of any one of items 1 to 15. 17. The antigen-binding molecule of item 16, which specifically binds to PD-L1 and inhibits binding of PD-L1 to an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, and 2A09. 18. An antigen-binding molecule that specifically binds to the epitope of PD-L1 to which the antigen-binding molecule of any one of items 1 to 15 binds. 19. The antigen-binding molecule of item 18, which specifically binds to an epitope of PD-L1 bound by an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, and 2A09. 20. An antigen binding molecule that specifically binds to an epitope of human PD-L1, wherein the epitope is composed of sequence number 19. 21. An antigen-binding molecule that specifically binds to an epitope of cynomolgus monkey PD-L1. 22. The antigen-binding molecule of item 20, selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, 2A09, and fragments and variants thereof. 23. The antigen-binding molecule of item 20, which is 8B06. 24. The antigen-binding molecule of item 20, which is 8A04. 25. An antigen-binding molecule that specifically binds to PD-L1 and competes with the antigen-binding molecule of any one of items 1 to 15 for binding to PD-L1. 26. The antigen-binding molecule of item 25, which specifically binds to PD-L1 and competes for binding to PD-L1 with an antibody selected from the group consisting of 8B06, 8D06, 8G08, 8A04, 8D04, 2A09. 27. The anti-PD-L1 antigen-binding molecule according to any one of paragraphs 1 to 15, comprising 1 to 10 amino acid substitutions in the specified sequence(s). 28. The anti-PD-L1 antigen-binding molecule according to item 27, comprising 1 to 5 amino acid substitutions in the specified sequence(s). 29. The anti-PD-L1 antigen-binding molecule according to item 27, comprising one or two amino acid substitutions in the specified sequence(s). 30. The anti-PD-L1 antigen-binding molecule according to any one of paragraphs 27 to 29, wherein there are 1 to 10, 1 to 5, or 1 or 2 amino acid substitutions in the CDR region(s) of the antigen-binding molecule. 31. The anti-PD-L1 antigen-binding molecule according to any one of paragraphs 27 to 29, wherein there are 1 to 10, 1 to 5, or 1 or 2 amino acid substitutions in one or both of the variable regions of the antigen-binding molecule. 32. The anti-PD-L1 antigen-binding molecule according to any one of paragraphs 27 to 29, wherein the 1-10, 1-5, or 1 or 2 amino acid substitutions are in a framework region of the antigen-binding molecule. 33. The anti-PD-L1 antigen binding molecule according to any one of paragraphs 27 to 29, which is a variant derived from an antibody selected from the group consisting of: 8B06, 8D06, 8G08, 8A04, 8D04, 2A09. 34. The anti-PD-L1 antigen-binding molecule according to any one of paragraphs 13 to 15 or 27 to 29, wherein the amino acid substitutions are conservative amino acid substitutions. 35. The anti-PD-L1 antigen-binding molecule of any of the above items which is an antibody, or an antigen-binding fragment, or derivative thereof. 36. The anti-PD-L1 antigen-binding molecule of item 35, wherein the antigen-binding antibody fragment or derivative is a Fab, F(ab')2, Fv, scFv, dAb, Fd, or diabody. 37. The anti-PD-L1 antigen-binding molecule of item 35, wherein the antigen-binding antibody fragment is a Fab. 38. The anti-PD-L1 antigen-binding molecule of item 35, wherein the antibody or antigen-binding fragment or derivative thereof is monovalent. 39. The anti-PD-L1 antibody of paragraph 35, wherein the antibody, or antigen-binding fragment or derivative thereof, is fully human. 40. The anti-PD-L1 antibody or antigen-binding antibody fragment or derivative thereof of any one of paragraphs 35 to 39, which is an IgA, IgD, IgE, IgG, IgM, or IgY antibody, or an antigen-binding antibody fragment or derivative thereof. 41. The anti-PD-L1 antibody or antibody fragment of any one of paragraphs 35 to 39, wherein the antibody or antigen-binding fragment or derivative thereof is an IgG antibody or antigen-binding fragment or derivative thereof. 42. The anti-PD-L1 antibody or antigen-binding antibody fragment or derivative of item 41, wherein the IgG antibody or antigen-binding antibody fragment or derivative is an IgG1 antibody or antigen-binding antibody fragment or derivative thereof. 43. An anti-PD-L1 antigen binding molecule, which is an affinity matured mutant of the antibody 2A09. 44. The anti-PD-L1 antigen binding molecule of item 43, wherein the affinity matured mutant is selected from the group consisting of 8B06, 8D06, 8G08, 8A04, and 8D04. 45. An antigen-binding molecule according to any of the above items, which specifically binds to human or cynomolgus PD-L1. 46. ​​An antigen-binding molecule according to any of the above items, which specifically binds to PD-L1 in a pH-dependent manner. 47. An antigen-binding molecule according to any of the above items, which specifically binds to PD-L1 at acidic pH. 48. The antigen-binding molecule of any of the preceding items, which specifically binds to PD-L1 at a pH of about pH 6 to about pH 6.5. 49. An antigen-binding molecule according to any of the preceding items, which has a higher affinity for PD-L1 at acidic pH than physiological pH. 50. The antigen-binding molecule of any of the preceding items, which has a higher affinity for PD-L1 at about pH 6.0 than at about pH 7.4. 51. The antigen-binding molecule of item 49, wherein the acidic pH is about pH 6.5 or less. 52. The antigen-binding molecule of item 49, wherein the physiological pH is about pH 7.4. 53. The antigen-binding molecule of any of the preceding items, which has at least about 5-fold higher affinity for PD-L1 at about pH 6.0 than at a pH of about 7.4. 54. The antigen-binding molecule of any of the preceding items, which has an EC50 value against PD-L1 of less than about 15 nM at pH 6.0. 55. The antigen-binding molecule of any of the preceding items, which has an EC50 value against PD-L1 of about 1.45 nM to about 15 nM at pH 6.0. 56. The antigen-binding molecule of any of the preceding items, which has an EC50 value against PD-L1 of at least about 10 nM at pH 7.4. 57. The antigen-binding molecule of any of the preceding items, which has an EC50 value against PD-L1 of about 13.2 nM to about 100 nM at pH 7.4. 58. The antigen-binding molecule of any of the previous items, having a pH 6.0:7.4 binding ratio of at least about 5. 59. The antigen-binding molecule of any of the preceding items, which has an EC50 value for PD-L1 of less than about 15 nM at pH 6.0 and an EC50 value for PD-L1 of at least about 10 nM at pH 7.4, and has a pH 6.0:7.4 binding ratio of at least 5. 60. An antigen-binding molecule according to any of the preceding items, which inhibits binding of PD-L1 to PD-1 and / or CD80, or binding of a PD-L1-expressing cell to PD-1 and / or CD80. 61. The antigen-binding molecule of item 60, which inhibits binding by at least about 40%, at least about 50%, or at least about 80%. 62. An antigen-binding molecule according to any of the preceding items, which has a higher IC50 inhibitory value of PD-L1 binding to PD-1 at about pH 7.4 than at about pH 6.0. 63. The antigen-binding molecule of any of the preceding items, which has an IC50 inhibition value of PD-L1 binding to PD-1 at about pH 6.0 of about 12.1 nM to about 42.3 nM. 64. The antigen-binding molecule of any of the preceding items, which has an IC50 inhibition value of PD-L1 binding to PD-1 at about pH 6.0 of less than about 50 nM. 65. The antigen-binding molecule of any of the preceding items, which has an IC50 inhibition value of PD-L1 binding to PD-1 at about pH 7.4 of about 81.2 nM to about 100 nM. 66. An antigen-binding molecule according to any of the preceding items, having an IC50 inhibition value of PD-L1 binding to PD-1 at pH 7.4 of at least 50 nM. 67. An antigen-binding molecule according to any of the preceding items, having an IC50 inhibition value of PD-L1 binding to PD-1 at pH 7.4 of at least 80 nM. 68. The antigen-binding molecule of any of the preceding items, having a pH 6.0:7.4 inhibition ratio of at least about 2. 69. The antigen-binding molecule of any of the preceding items, wherein the IC50 inhibitory value of PD-L1 binding to PD-1 at pH 6.0 is less than about 50 nM and the IC50 inhibitory value of PD-L1 binding to PD-1 at pH 7.4 is at least about 80 nM, and the antigen-binding molecule has a pH 6.0:7.4 inhibitory ratio of at least about 2. 70. The antigen-binding molecule of any of the preceding items, wherein the PD-L1 is human PD-L1 or cynomolgus PD-L1. 71. An antigen-binding molecule according to any of the above items, which reverses immunosuppression when administered in vivo or in vitro. 72. An antigen-binding molecule according to any of the above items, which enhances T cell immunity when administered in vivo or in vitro. 73. An antigen-binding molecule according to any one of the above items, which is an immune checkpoint inhibitor. 74. A pharmaceutical composition comprising an antigen-binding molecule of any of the above items, optionally further comprising one or more pharma- ceutically acceptable excipients. 75. The pharmaceutical composition of item 74, further comprising an additional therapeutically active agent or used in combination with another therapy or an additional therapeutically active agent. 76. The pharmaceutical composition according to item 75, wherein the other therapy or additional therapeutically active agent is selected from the group consisting of radiation therapy, chemotherapy treatment, targeted therapy, immunotherapy, monoclonal antibody therapy, hormonal therapy, angiogenesis inhibition, cancer vaccines, oncolytic viruses, toll-like receptor agonists, epigenetic modifications, modified T cells, T cell costimulatory agonists, tyrosine kinase inhibitors, other anti-cancer chemicals, palliative care for cancer therapy, immune checkpoint inhibitors, immunosuppressants, anti-inflammatory drugs, immunomodulators, immunostimulators and / or inhibitors, such as IDO inhibitors, CSF-1R inhibitors, TGFB inhibitors, T cell costimulatory antagonists, Treg inhibitors, macrophage modulators, natural killer cell modulators or chemokine receptor inhibitors. 77. The pharmaceutical composition of item 76, wherein the chemotherapy treatment is selected from the group consisting of gemcitabine, cyclophosphamide, doxorubicin, paclitaxel, and cisplatin. 78. The pharmaceutical composition of item 76, wherein the T cell costimulatory agonist is selected from the group consisting of 4-1BB, OX40, CD40, GITR, BTLA, CD70 and ICOS. 79. The pharmaceutical composition of item 76, wherein the immune checkpoint inhibitor acts on a member of the group consisting of PD-1, CTLA-4, TIM-3, LAG-3, VISTA and TIGIT. 80. The pharmaceutical composition of item 75, wherein the other therapy or additional therapeutically active agent is another antigen-binding molecule. 81. The pharmaceutical composition of item 80, wherein the additional antigen-binding molecule is selected from the group consisting of an anti-PD-L1 antigen-binding molecule, an anti-PD-1 antigen-binding molecule, an anti-CTLA-4 antigen-binding molecule, an anti-OX40 antigen-binding molecule, an anti-ICOS antigen-binding molecule, and an anti-GITR antigen-binding molecule, and optionally, the additional antigen-binding molecule is an antibody. 82. A kit comprising the antigen-binding molecule of any one of items 1 to 73 or the pharmaceutical composition according to any one of items 74 to 81, and further comprising an additional therapeutically active agent. 83. The kit of item 82, further comprising instructions for use. 84. The kit of items 82 or 83, wherein the pharmaceutical ingredients are arranged separately within the kit. 85. The kit of any of items 82 to 84, wherein the additional therapeutically active agent is selected from the list consisting of immune checkpoint inhibitors, immunosuppressants, anti-inflammatory drugs, immunomodulators, immunostimulators, and / or inhibitors, such as IDO inhibitors, CSF-1R inhibitors, TGFB inhibitors, T cell costimulatory antagonists, Treg inhibitors, macrophage modulators, natural killer cell modulators, or chemokine receptor inhibitors. 86. The kit of any of items 82 to 84, wherein the additional therapeutically active agent is an immune checkpoint inhibitor. 87. The kit of any of items 82 to 86, wherein the antigen-binding molecule or pharmaceutical composition and the additional therapeutically active agent are for separate, sequential or simultaneous administration. 88. The antigen-binding molecule of any one of items 1 to 73 or the pharmaceutical composition of any one of items 74 to 81 for use in medicine. 89. The antigen-binding molecule of any one of items 1 to 73 or the pharmaceutical composition of any one of items 78 to 81, for use in the treatment or prevention of cancer. 90. Cancers of the heart: sarcoma, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, metastatic rhabdomyoma, fibroma, lipoma and teratoma; pulmonary bronchogenic carcinoma, squamous cell carcinoma, undifferentiated small cell carcinoma, non-small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma, alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondroitin hamartoma, mesothelioma; gastrointestinal tract: esophageal squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma, stomach carcinoma, lymphoma, leiomyosarcoma, head and neck, gastric, pancreatic tubular adenocarcinoma, insulinoma, glucagonoma, gastrinoma , carcinoid tumor, vipoma, adenocarcinoma of the small intestine, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma, adenocarcinoma of the large intestine, tubular adenoma, villous adenoma, hamartoma, leiomyoma, colorectal carcinoma; genitourinary tract: adenocarcinoma of the kidney, Wilms' tumor (nephroblastoma), lymphoma, leukemia, squamous cell carcinoma of the bladder and urethra, transitional cell carcinoma, adenocarcinoma, urothelial carcinoma, adenocarcinoma of the prostate, sarcoma, seminoma of the testis, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma; hepatoma of the liver, hepatoma Cell carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondroid exostosis), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor; nervous system: osteoma of the skull, hemangioma, granuloma, xanthomas, osteitis deformans, meningioma of the meninges, meningeal sarcoma, gliomatosis, astrocytoma of the brain, medulloblastoma, glioma, ependymoma, germinoma (pineal tumor), pleomorphic Glioblastoma, oligodendroglioma, Schwannoma, retinoblastoma, congenital tumors, spinal neurofibroma, meningioma, glioma, sarcoma; female reproductive organs: endometrial carcinoma of the uterus, cervical carcinoma of the cervix, preneoplastic cervical dysplasia, ovarian carcinoma of the ovaries, serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma, squamous cell carcinoma of the vulva, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma, clear cell carcinoma of the vagina, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube carcinoma, breast cancer;The antigen-binding molecule or pharmaceutical composition for use as described in item 89, selected from the group consisting of blood system: myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma); skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland: neuroblastoma. 91. An antigen-binding molecule, affinity maturation mutant or pharmaceutical composition for use as in item 89 or 90, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, leukemia, blastoma and sarcoma. 92. The antigen-binding molecule or pharmaceutical composition for use according to any one of items 89 to 91, wherein the cancer is selected from the group consisting of melanoma, metastatic cancer, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, hepatocellular carcinoma and bladder cancer. 93. Use of an antigen-binding molecule according to any one of items 1 to 73 in the manufacture of a medicament for use in the treatment of cancer. 94. Cancers include: cardiac sarcoma, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, metastatic rhabdomyoma, fibroma, lipoma and teratoma; pulmonary bronchogenic carcinoma, squamous cell carcinoma, undifferentiated small cell carcinoma, non-small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma, alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondroitin hamartoma, mesothelioma; gastrointestinal tract: esophageal squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma, gastric cancer, lymphoma, leiomyosarcoma, head and neck, stomach, tubular adenocarcinoma of the pancreas, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma, adenocarcinoma of the small intestine, lymphoma, carcinoid tumor, Kaposi's tumor tumors, leiomyomas, hemangiomas, lipomas, neurofibromas, fibromas, adenocarcinomas of the colon, tubular adenomas, villous adenomas, hamartomas, leiomyomas, colorectal carcinoma; genitourinary tract: adenocarcinomas of the kidney, Wilms' tumor (nephroblastoma), lymphomas, leukemias, squamous cell carcinomas of the bladder and urethra, transitional cell carcinomas, adenocarcinomas, urothelial carcinomas, adenocarcinomas of the prostate, sarcomas, seminoma of the testis, teratomas, embryonal carcinomas, teratocarcinomas, choriocarcinomas, sarcomas, interstitial cell carcinomas, fibromas, fibroadenomas, adenomatous tumors, lipomas; hepatomas of the liver, hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, angiosarcomas, hepatocellular adenomas, hemangiomas; bones: osteogenic sarcomas (osteosarcoma), fibrosarcomas, malignant fibrous histiocytoma, chondrosarcomas, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondroid exostosis), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor; nervous system: osteoma of the skull, hemangioma, granuloma, xanthomas, osteitis deformans, meningioma of the meninges, meningeal sarcoma, gliomatosis, astrocytoma of the brain, medulloblastoma, glioma, ependymoma, germinoma (pineal tumor), glioblastoma multiforme, oligodendroglioma, Schwannoma, retinoblastoma, congenital tumors, spinal neurofibroma, meningioma, glioma, sarcoma; female reproductive system: endometrial carcinoma of the uterus, cervical carcinoma of the cervix, preneoplastic Cervical dysplasia, ovarian carcinoma of the ovary, serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma, squamous cell carcinoma of the vulva, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma, clear cell carcinoma of the vagina, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube carcinoma, breast cancer; blood system: myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma);Use of an antigen-binding molecule according to item 93, selected from the group consisting of skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland: neuroblastoma. 95. Use of an antigen-binding molecule according to item 93, wherein the cancer is selected from the group consisting of melanoma, metastatic cancer, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, hepatocellular carcinoma and bladder cancer. 96. A method for treating or preventing a PD-L1-mediated disease or disorder, comprising administering to a subject an antigen-binding molecule according to any one of items 1 to 73 or a pharmaceutical composition according to any one of items 74 to 81. 97. The method of item 96, wherein the PD-L1 mediated disease or disorder is cancer. 98. Cancers include: cardiac sarcoma, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, metastatic rhabdomyoma, fibroma, lipoma and teratoma; pulmonary bronchogenic carcinoma, squamous cell carcinoma, undifferentiated small cell carcinoma, non-small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma, alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondroitin hamartoma, mesothelioma; gastrointestinal tract: esophageal squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma, gastric cancer, lymphoma, leiomyosarcoma, head and neck, stomach, tubular adenocarcinoma of the pancreas, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma, adenocarcinoma of the small intestine, lymphoma, carcinoid tumor, Kaposi's tumor tumors, leiomyomas, hemangiomas, lipomas, neurofibromas, fibromas, adenocarcinomas of the colon, tubular adenomas, villous adenomas, hamartomas, leiomyomas, colorectal carcinoma; genitourinary tract: adenocarcinomas of the kidney, Wilms' tumor (nephroblastoma), lymphomas, leukemias, squamous cell carcinomas of the bladder and urethra, transitional cell carcinomas, adenocarcinomas, urothelial carcinomas, adenocarcinomas of the prostate, sarcomas, seminoma of the testis, teratomas, embryonal carcinomas, teratocarcinomas, choriocarcinomas, sarcomas, interstitial cell carcinomas, fibromas, fibroadenomas, adenomatous tumors, lipomas; hepatomas of the liver, hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, angiosarcomas, hepatocellular adenomas, hemangiomas; bones: osteogenic sarcomas (osteosarcoma), fibrosarcomas, malignant fibrous histiocytoma, chondrosarcomas, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondroid exostosis), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor; nervous system: osteoma of the skull, hemangioma, granuloma, xanthomas, osteitis deformans, meningioma of the meninges, meningeal sarcoma, gliomatosis, astrocytoma of the brain, medulloblastoma, glioma, ependymoma, germinoma (pineal tumor), glioblastoma multiforme, oligodendroglioma, Schwannoma, retinoblastoma, congenital tumors, spinal neurofibroma, meningioma, glioma, sarcoma; female reproductive system: endometrial carcinoma of the uterus, cervical carcinoma of the cervix, preneoplastic Cervical dysplasia, ovarian carcinoma of the ovary, serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma, squamous cell carcinoma of the vulva, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma, clear cell carcinoma of the vagina, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube carcinoma, breast cancer; blood system: myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma);The method of treating skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland: neuroblastoma. 99. The method of treatment of item 97, wherein the cancer is selected from the group consisting of melanoma, metastatic cancer, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, hepatocellular carcinoma and bladder cancer. 100. A method for inhibiting binding of human PD-L1 to PD-1 and / or CD80, or binding of a PD-L1-expressing cell to PD-1 and / or CD80, comprising contacting human PD-L1 or a PD-L1-expressing cell with an antigen-binding molecule according to any one of items 1 to 73. 101. The method of item 100, which is an in vitro method. 102. The method of item 100, which is an in vivo method. 103. A nucleic acid encoding the antigen-binding molecule of any one of items 1 to 73. 104. A plasmid containing the nucleic acid of item 103. 105. A vector comprising the nucleic acid of item 103. 106. A host cell containing a plasmid or vector according to item 104 or item 105. 107. A method for producing a cell that expresses an anti-PD-L1 antigen-binding molecule, comprising transfecting a cell with a plasmid or vector according to item 104 or 105. 108. A method for producing an anti-PD-L1 antigen-binding molecule, comprising culturing a host cell according to item 106 in a cell culture medium under conditions that allow intracellular expression of the encoding nucleic acid sequence of the plasmid or vector. 109. The method of item 108, further comprising obtaining the anti-PD-L1 antigen binding molecule from the cell culture supernatant. 110. The method of item 109, further comprising formulating the resulting anti-PD-L1 antigen binding molecule into a pharmaceutical composition together with one or more pharma- ceutically acceptable excipients.

Claims

1. An anti-PD-L1 antigen binding molecule, comprising: (a) a heavy chain variable (VH) region comprising: (i) a heavy chain complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO:6; (ii) a heavy chain complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO:7; And, (iii) a heavy chain complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO:8; A VH region comprising: and (b) a light chain variable (VL) region comprising: (i) a light chain complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 16; (ii) a light chain complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO:3; And, (iii) a light chain complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO:4; A VL region comprising: and having a higher affinity for PD-L1 at pH 6.0 than at pH 7.

4.

2. the VH region comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:5; The VL region comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:

15. The anti-PD-L1 antigen-binding molecule of claim 1.

3. The VH region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5, and the VL region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

15. The anti-PD-L1 antigen-binding molecule of claim 1 or 2.

4. The anti-PD-L1 antigen-binding molecule of any one of claims 1 to 3, wherein the VH region comprises the amino acid sequence of SEQ ID NO:5 and the VL region comprises the amino acid sequence of SEQ ID NO:

15.

5. The anti-PD-L1 antigen-binding molecule of any one of claims 1 to 4, wherein the antigen-binding molecule is an antibody or an antigen-binding fragment or derivative thereof, and the antibody fragment or derivative is a Fab, F(ab')2, Fv, scFv, dAb, Fd, or a diabody.

6. The anti-PD-L1 antigen-binding molecule of claim 5, wherein the antibody, or antigen-binding fragment or derivative thereof, is monovalent.

7. The anti-PD-L1 antigen-binding molecule of claim 5 or 6, wherein the antibody or antigen-binding fragment or derivative thereof is fully human.

8. The anti-PD-L1 antigen-binding molecule of any one of claims 5 to 7, wherein the antibody or antigen-binding fragment or derivative thereof is an IgA, IgD, IgE, IgG, IgM, or IgY antibody, or an antigen-binding fragment or derivative thereof.

9. The anti-PD-L1 antigen-binding molecule of claim 8, wherein the antibody or antigen-binding fragment or derivative thereof is an IgG antibody or antigen-binding fragment or derivative thereof.

10. 10. The anti-PD-L1 antigen-binding molecule of claim 9, wherein the IgG antibody, or antigen-binding fragment or derivative thereof, is an IgG1 antibody, or antigen-binding fragment thereof.

11. The anti-PD-L1 antigen-binding molecule of any one of claims 8 to 10, wherein the antibody or antigen-binding fragment or derivative thereof is a bispecific antibody or a bispecific antigen-binding fragment or derivative thereof.

12. The anti-PD-L1 antigen-binding molecule of any one of claims 1 to 11, having at least 5-fold higher affinity for PD-L1 at pH 6.0 than at a pH of 7.

4.

13. 13. The anti-PD-L1 antigen binding molecule of any one of claims 1 to 12, having a pH 6.0:7.4 binding ratio of at least 5.

14. The anti-PD-L1 antigen-binding molecule of any one of claims 1 to 13, wherein the IC50 inhibition value of PD-L1 binding to PD-1 is higher at pH 7.4 than at pH 6.

0.

15. A pharmaceutical composition comprising the anti-PD-L1 antigen-binding molecule of any one of claims 1 to 14 and a pharma- ceutical acceptable excipient.

16. 16. The pharmaceutical composition of claim 15, further comprising an additional therapeutically active agent or used in combination with another therapy or an additional therapeutically active agent.

17. 17. The pharmaceutical composition according to claim 15 or 16, for the treatment or prevention of cancer, wherein the cancer is selected from the group consisting of melanoma, metastatic cancer, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, hepatocellular carcinoma and bladder cancer.

Images