JP6800191B2 - Anti-PD-1 antibody and its use for treatment and diagnosis - Google Patents

Description

The present invention relates to anti-PD-1 antibodies and their use for treatment and diagnosis.

Programmed death-1 (PD-1, also referred to as "CD279") is a 55KD receptor protein associated with the CD28 / CTLA4 co-stimulatory / inhibitory receptor family. See Non-Patent Document 1. The cDNA and gene encoding PD-1 were cloned and characterized in mice and humans. See Non-Patent Document 2 and Non-Patent Document 3. Full-length PD-1 contains 288 amino acid residues (NCBI Accession Number: NP_005009). Its extracellular domain is composed of amino acid residues 1-167, and the cytoplasmic C-terminal tail is composed of two hypothetical epidemiregulatory motifs, an immunoreceptor tyrosine system inhibitory motif (see Non-Patent Document 4), and It has an immunoreceptor tyrosine switch motif (see Non-Patent Document 5).

To date, two sequence-related ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), specifically interact with PD-1 and mediate CD3 and CD28. Induces intracellular signaling that inhibits sex T cell activation (see Non-Patent Document 6), thereby reducing cell proliferation, secreting IL-2 and IFN-γ, secreting other growth factors and cytokines, for example. It has been confirmed that the T cell activity is weakened.

Expression of PD-1 is often found in immune cells such as T cells, B cells, monocytes and natural killer (NK) cells. It is rarely expressed in other human tissues such as muscle, epithelium, nervous tissue and the like. Also, high levels of PD-1 expression are often associated with immune cell activation. For example, the human T cell line, Jurkat, is PD- when activated by phytohaemagglutinine (PHA) or phorbol ester (12-O-tetradecanoyl phorbol-13-acetate, or TPA). It was confirmed that the expression of 1 was upregulated in Western blot. See Non-Patent Document 7. Similar phenomena were observed in stimulated mouse T- and B-lymphocytes and primary human CD4 ⁺ T cells upon stimulation with anti-CD3 antibodies. See Non-Patent Document 8 and Non-Patent Document 9. Increased PD-1 expression after stimulation of T-effector cells directs activated T-effector cells towards exhaustion and decreased immune activity. Therefore, PD-1-mediated inhibitory signals play an important role in immune tolerance. See Non-Patent Document 10.

Increased PD-1 expression in tumor-infiltrating lymphocytes (TIL) and increased PD-1 ligand expression in tumor cells were found in lungs (see Non-Patent Document 11), liver (see Non-Patent Document 12 and Non-Patent Document 13), and stomach (see Non-Patent Document 12). Non-Patent Document 14), Kidney (See Non-Patent Document 15 and Non-Patent Document 16), Breast (See Non-Patent Document 17), Ovary (See Non-Patent Document 18), Pancreas (See Non-Patent Document 19), Melanosite (See Non-Patent Document 19) Non-Patent Document 20) and various cancers involving different types of organs and tissues such as the esophagus (see Non-Patent Document 21) have been reported. Increased expression of PD-1 and PD-L1 in these cancers is more often associated with a poor prognosis for patient survival outcomes. Transgenic mice with PD-1 gene knockout that inhibit the growth of xenograft cancer cells have demonstrated the importance of PD-1 signaling in the regulation of the immune system for cancer eradication or tolerance. See Non-Patent Document 22.

Upregulation of PD-1 signaling not only leads to immune tolerance, cancer growth, but also viral infection and its spread in humans. Epidemic liver infection viruses, HBV and HCV, induce overexpression of PD-1 ligands in hepatocytes and activate PD-1 signaling in T-effector cells, resulting in tolerance for T cell depletion and viral infections. Bring. See Non-Patent Document 23 and Non-Patent Document 24. Similarly, HIV infections frequently evade the human immune system by a similar mechanism. PD-1 signaling by antagonist molecules can bring immune cells back from tolerance and reactivate them to eradicate cancer and chronic viral infections. See Non-Patent Document 25 and Non-Patent Document 26.

Blank et al. , 2005 Cancer Immunol Immunother 54: 307-314 Ishida et al. , 1992 EMBO J 11: 3887-3395 Shinohara et al. , 1994 Genomics 23: 704-706 ITIM; Vivier et al. , 1997 Immunol Today 18: 286-291 ITSM; Chemnitz et al. , 2004 J Immunol 173: 945-954 Riley, 2009 Immunol Rev 229: 114-125 Vibharka et al. , 1997 Exp Cell Res 232: 25-28 Agata et al. , 1996 Int Immunol 8: 765-772 Bennett et al. , 2003 J Immunol 170: 711-118 Bour-Jordan et al. , 2011 Immunol Rev 241: 180-205 Konishi et al. , 2004 Clin Cancer Res 10: 5094-5100 Shi et al. , 2008 Int J Cancer 128: 887-896 Gao et al. , 2009 Clin Cancer Res 15: 971-979) 5100 Wu et al. , 2006 Acta Histochem 108: 19-24 Thompson et al. , 2004 Proc Natl Acad Sci 101: 17174-17179 Thompson et al. , 2007 Clin Cancer Res 13: 1757-1761 Ghebeh et al. , 2006 Neoplasmia 8: 190-198 Hamanishi et al. 2007 Proc Natl Acad Sci 104: 3360-3365 Nomi et al. , 2007 Clin Cancer Res 13: 2151-2157 Hino et al. , 2010 Cancer 116: 1757-1766 Ohigashi et al. , 2005 Clin Cancer Res 11: 2947-2953 Zhang et al. , 2009 Blood 114: 1545-1552 Boni et al. , 2007 J Visual 81: 4215-4225 Golden-Mason et al. , 2008 J Immunol 180: 3637-3641 Blank et al. , 2005 Cancer Immunol Immunother 54: 307-314 Okazaki et al. , 2007 Int Immunol 19: 815-824

The present invention provides methods and compositions for immunosuppression (inhibition) of PD-1. In one aspect, the invention comprises a complementarity determining region (CDR) having a sequence selected from SEQ ID NOs: 11-22, 31-42 and 59-63 that binds human PD-1. I will provide a.
CDRs are delivered to the heavy and light chain variable regions (Vh) and light chain variable regions (Vk) containing the (CDR-H1, CDR-H2 and CDR-H3) and (CDR-L1, CDR-L2 and CDR-L3) sequences, respectively. It is suitable for recombination and retains PD-1-specific binding and / or functionality.

In certain embodiments, the domain comprises a heavy chain variable region (Vh) or a light chain variable region (Vk) that includes:
a) CDR-H1 (SEQ ID NO: 11, 17, 31, or 37),
b) CDR-H2 (SEQ ID NO: 12, 18, 32, or 38),
c) CDR-H3 (SEQ ID NO: 13, 19, 33, or 39),
d) CDR-L1 (SEQ ID NO: 14, 20, 34, or 40),
e) CDR-L2 (SEQ ID NO: 15, 21, 35, or 41), or f) CDR-L3 (SEQ ID NO: 16, 22, 36, or 42).

In certain embodiments, the domain comprises a heavy chain variable region (Vh) and / or a light chain variable region (Vk) including:
a) mu317: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NO: 11-13);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NO: 14-16);
b) mu326: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NO: 17-19);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NO: 20-22);
c) 317-4B6: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NO: 31-33);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NO: 34-36);
d) 326-4A3: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NO: 37-39);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NO: 40-42);
e) 317-1H: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 11, 59, 13);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NO: 14-16);
f) 317-4B2: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 11, 60, 13);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NOs: 61, 15, 16);
g) 317-4B5: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 11, 60, 13);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NOs: 61, 15, 16);
h) 317-4B6: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 11, 32, 13);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NOs: 61, 15, 16);
i) 326-1: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 17, 62, 19);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NO: 20-22);
j) 326-3B1: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 17, 62, 19);
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NO: 20-22);
k) 326-3G1: CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 17, 62, 19); or
CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NO: 20-22).

In certain embodiments, the domain comprises a heavy chain variable region (Vh) or a light chain variable region (Vk) that includes:
(A) CDR-H1 (SEQ ID NO: 31), CDR-H2 (SEQ ID NO: 12, 32, 59 or 60), and CDR-H3 (SEQ ID NO: 33),
CDR-L1 (SEQ ID NO: 14, 34, or 61), CDR-L2 (SEQ ID NO: 35), and CDR-L3 (SEQ ID NO: 36); or (b) CDR-H1 (SEQ ID NO: 37), CDR-H2 (SEQ ID NO: 37). SEQ ID NO: 18, 38, or 62), and CDR-H3 (SEQ ID NO: 39),
CDR-L1 (SEQ ID NO: 40), CDR-L2 (SEQ ID NO: 41), and CDR-L3 (SEQ ID NO: 42).

In certain embodiments, the heavy chain variable region (Vh) or light chain variable region (Vk) includes:
a) mu317 (SEQ ID NO: 4 or 6);
b) mu326 (SEQ ID NO: 8 or 10);
c) 317-4B6 (SEQ ID NO: 24 or 26);
d) 326-4A3 (SEQ ID NO: 28 or 30);
e) 317-4B2 (SEQ ID NO: 43 or 44);
f) 317-4B5 (SEQ ID NO: 45 or 46);
g) 317-1 (SEQ ID NO: 48 or 50);
h) 326-3B1 (SEQ ID NO: 51 or 52);
i) 326-3G1 (SEQ ID NO: 53 or 54);
j) 326-1 (SEQ ID NO: 56 or 58);
k) 317-3A1 (SEQ ID NO: 64);
l) 317-3C1 (SEQ ID NO: 65);
m) 317-3E1 (SEQ ID NO: 66);
n) 317-3F1 (SEQ ID NO: 67);
o) 317-3G1 (SEQ ID NO: 68);
p) 317-3H1 (SEQ ID NO: 69);
q) 317-3I1 (SEQ ID NO: 70);
r) 317-4B1 (SEQ ID NO: 71);
s) 317-4B3 (SEQ ID NO: 72);
t) 317-4B4 (SEQ ID NO: 73);
u) 317-4A2 (SEQ ID NO: 74);
v) 326-3A1 (SEQ ID NO: 75);
w) 326-3C1 (SEQ ID NO: 76);
x) 326-3D1 (SEQ ID NO: 77);
y) 326-3E1 (SEQ ID NO: 78);
z) 326-3F1 (SEQ ID NO: 79);
aa) 326-3B N55D (SEQ ID NO: 80);
ab) 326-4A1 (SEQ ID NO: 81); or ac) 326-4A2 (SEQ ID NO: 82).

In certain embodiments, the domain comprises a heavy chain variable region (Vh) and a light chain variable region (Vk) including:
a) mu317 (SEQ ID NOs: 4 and 6);
b) mu326 (SEQ ID NOs: 8 and 10);
c) 317-4B6 (SEQ ID NOs: 24 and 26);
d) 326-4A3 (SEQ ID NOs: 28 and 30);
e) 317-4B2 (SEQ ID NOs: 43 and 44);
f) 317-4B5 (SEQ ID NOs: 45 and 46);
g) 317-1 (SEQ ID NOs: 48 and 50);
h) 326-3B1 (SEQ ID NO: 51 or 52);
i) 326-3G1 (SEQ ID NOs: 53 and 54);
j) 326-1 (SEQ ID NOs: 56 and 58);
k) 317-3A1 (SEQ ID NOs: 64 and 26);
l) 317-3C1 (SEQ ID NOs: 65 and 26);
m) 317-3E1 (SEQ ID NOs: 66 and 26);
n) 317-3F1 (SEQ ID NOs: 67 and 26);
o) 317-3G1 (SEQ ID NOs: 68 and 26);
p) 317-3H1 (SEQ ID NOs: 69 and 26);
q) 317-3I1 (SEQ ID NOs: 70 and 26);
r) 317-4B1 (SEQ ID NOs: 71 and 26);
s) 317-4B3 (SEQ ID NOs: 72 and 26);
t) 317-4B4 (SEQ ID NOs: 73 and 26);
u) 317-4A2 (SEQ ID NOs: 74 and 26);
v) 326-3A1 (SEQ ID NOs: 75 and 30);
w) 326-3C1 (SEQ ID NOs: 76 and 30);
x) 326-3D1 (SEQ ID NOs: 77 and 30);
y) 326-3E1 (SEQ ID NOs: 78 and 30);
z) 326-3F1 (SEQ ID NOs: 79 and 30);
aa) 326-3B N55D (SEQ ID NOs: 80 and 30);
ab) 326-4A1 (SEQ ID NOs: 28 and 81); or ac) 326-4A2 (SEQ ID NOs: 28 and 82).

In certain embodiments, the domain specifically binds to the following PD1 residues:
(A) K45 and I93 (amino acid numbering based on Ref. [2008 PNAS, 105: 10483]; corresponding to K58 and I106 in SEQ ID NO: 2); or (b) I93, L95 and P97 (Ref. [2008 PNAS, 105] Amino acid numbering based on: 10483]; corresponding to I106, L108 and P110 in SEQ ID NO: 2).

In certain embodiments, the domain induces IL-2 release in HuT78 / PD-1 cells co-cultured with HEK293 / OS8 / PD-L1 cells or HEK293 / OS8 / PD-L2 cells and / or HEK293. Inhibits IL-2 secretion in HuT78 / P3Z cells co-cultured with / PD-L1 cells or HEK293 / PD-L2 cells.

The present invention also provides antibody IgG4 heavy chain effectors or constant regions comprising either SEQ ID NO: 83-88, particularly SEQ ID NO: 87 or 88.

The present invention also provides an antibody, F (ab) or F (ab) ₂ , comprising a subject PD-1 binding domain.

The present invention also provides an antibody comprising an IgG4 heavy chain effector or constant region comprising SEQ ID NO: 83-88, in particular SEQ ID NO: 87 or 88, and a PD-1 binding domain of interest.

The present invention also provides a polynucleotide encoding a subject PD-1 binding domain, particularly a cDNA sequence.

The present invention also provides a method of using a target domain, which comprises administering the target domain to an individual who has been determined to have cancer or a viral infection, or an individual who requires PD-1 antagonism.

The present invention also relates to (a) a single chain variable fragment (scFv) (SEQ ID NO: 89) of an anti-human CD3 monoclonal antibody OKT3 fused to the C-terminal domain (113-220) of mouse CD8α; or (b) human CD3ζ. Provided is a fusion protein containing an extracellular and transmembrane domain (SEQ ID NO: 90) of human PD-1 fused to the cytoplasmic domain of the chain.

The present invention also provides a method using a subject fusion protein, comprising assaying, screening, or selecting an anti-PD-1 antibody in a cell line expressing the fusion protein.

It is the schematic of PD-1 / Fc (top) and PD-1 / His (bottom). ECD: extracellular domain; L: linker; H: His tag; Fc: γ4Fc fragment from human IgG4; N: N-terminus; C: C-terminus. FIG. 6 is a dose-dependent response curve of a mouse monoclonal antibody that binds to human PD-1 in ELISA. Mouse monoclonal antibodies are displayed in the upper left corner of each figure. Monoclonal antibodies 317 and 517 share high homology in the variable regions of the heavy and light chains. The binding signal intensity is represented by a direct OD ₄₅₀ reading (value). The antigen, PD-1 / His, was coated in a volume of 50 μl at a concentration increasing up to 70 μg per well. This method will be described in Example 1. A dose-dependent response curve of mouse mAbs that bind to human PD-1 expressed on living cells, based on FACS analysis. Mouse antibody coding and EC ₅₀ was displayed on each panel. MFI represents the average fluorescence intensity. HuT78 / PD-1 cells were suspended in 96-well plates at 5 × 10 ⁴ cells per well for FACS. As described in Example 1, binding of the PD-1 monoclonal antibody to the cell surface target and FACS detection were performed. FIG. 6 is a schematic representation of a cell co-culture system used to assay the functional activity of an anti-PD-1 monoclonal antibody. T cells (either CD4 ⁺ or CD8 ⁺ ) represent HuT78 / PD-1 or primary T cells in PBMC. TCR: T cell receptor; N: nucleus; C: cytoplasm. FIG. 8 is a dose-dependent response curve of mouse monoclonal antibody-induced IL-2 secretion in HuT78 / PD-1 cells co-cultured with HEK293 / OS8 / PD-L1 cells. Baseline: Average IL-2 release induced by mIgG at all test concentrations. Topline: Best IL-2 emission based on regression calculation by prism software. FIG. 6A is a histogram showing IFN-γ secretion induced by an anti-PD-1 monoclonal antibody in PBMC (donor 19) co-cultured with the cell line HEK293 / OS8 / PD-L1. FIG. 6B is a histogram showing IFN-γ secretion induced by an anti-PD-1 monoclonal antibody in PBMC (donor 20) co-cultured with cell line HEK293 / OS8 / PD-L1. 7 (A) and 7 (B) show the activity of the anti-PD-1 monoclonal antibody by co-culture of effector cells (NK92MI / PD-1) and target cells (HuT78 / PD-1). The average was calculated from two data points of a typical experiment. Monoclonal antibody was added to a concentration of 10 μg / ml. Experiments were performed as described in Example 9. Mapping and binding epitopes of anti-PD-1 monoclonal antibody by ELISA (upper panel) and Western blot (lower panel). Binding activity was evaluated by ELISA and Western blotting using conditioned medium containing WT or Mt PD-1. ^** indicates an AA residue whose monoclonal antibody binding activity was reduced to 25-50% of WT PD-1. ^*** indicates AA residues with monoclonal antibody binding activity reduced to less than 25% of WT PD-1. FIG. FIG. IFN-γ release induced by humanized anti-PD-1 monoclonal antibody in primary human PBMC from another healthy donor co-cultured with HEK293 / OS8 / PD-L1 cells. It shows cytotoxicity of NK92MI / PD-1 cells enhanced by humanized anti-PD-1 monoclonal antibodies, hu317 (A) and hu326 (B). The target lung cancer cells, SK-MES-1 / PD-L1, were co-cultured with effector cells at a ratio of (T to E) 1: 2 and assayed as described in Example 12. Individual tumor growth (growth) curves in three treatment groups, vehicle (PBS), human IgG (huIgG) and anti-PD-1 monoclonal antibody (hu317-1 / IgG4mt2). Each curve represents the tumor growth pathway of tumor-bearing mice indicated by numbers on the right side of each panel. Hep3B / OS8 / PD-L1 cells (established from the hepatocellular carcinoma strain Hep3B) were seeded on day 1, PBMC was transplanted on day 15, and three doses of hu317-1 / IgG4mt2 were administered at 18, 28 and 38. Each was injected on the day. The method is described in Example 12.

When PD-1 binds to its ligand, PD-L1 or PD-L2, it initiates inhibitory signaling in immune cells. In the case of cancer growth or viral infection, activation of PD-1 signaling promotes immune tolerance, allowing cancer or virally infected cells to escape immune surveillance and metastatic cancer. Alternatively, it also results in an increase in viral load. Inhibition of PD-1 mediated intracellular signal transduction by therapeutic agents enhances immune cell function that inhibits cancer cell proliferation or viral infection by activating immune cells including T cells, B cells and NK cells. Treat these human diseases by restoring immune surveillance and immune memory function.

The present invention provides an antibody that has the function of antagonizing PD-1-mediated intracellular signal transduction induced by a ligand in immune cells. Mouse anti-PD-1 antibodies have been humanized to be highly similar to human antibodies within the framework region. Complete antibodies made to the mutated human IgG4 variant format have unique features in terms of effector function and physicochemical properties. The disclosed anti-PD-1 antibodies are suitable for therapeutic use in the treatment of cancer, control of viral infections, and other human diseases that are mechanically involved in exacerbated immune tolerance.

Definition

Unless otherwise indicated, the term "antibody" is used in the broadest sense and specifically includes antibodies (including full-length monoclonal antibodies) and antibody fragments capable of recognizing PD-1. The antibody molecule is usually monospecific, but can also be described as unique (idiospicific), heterospecific (heterospecific), or polyspecific (polyspecific). Antibody molecules bind by specific binding sites to specific antigenic determinants or epitopes on the antigen. An "antibody fragment" comprises a portion of a full-length antibody and is generally its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F (ab') ₂ , and Fv fragments; bispecific antibodies (diabody); linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antigen fragments. There is.

Monoclonal antibodies (mAbs) can be obtained by methods well known to those of skill in the art. For example, reference [Kohler et al (1975)]; US Pat. No. 4,376,110; reference [Ausube et al (1987-1999)]; reference [Hallow et al (1988)]; and reference [Colligan et al (1993)]. )]reference. The monoclonal antibody of the present invention may be any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. Hybridomas that produce monoclonal antibodies can be cultured in vitro or in vivo. Monoclonal high-titer antibodies are obtained by intraperitoneally injecting cells from individual hybridomas into mice, such as pristine-primed Balb / c mice, to ascites containing high concentrations of the desired monoclonal antibody. It can be obtained by producing in vivo production. Monoclonal antibodies of isotype IgM or IgG can be purified from ascites or from culture supernatants using column chromatography methods well known to those of skill in the art.

An "isolated (isolated) polynucleotide" is a polynucleotide segment or fragment isolated from a sequence flanking it in its native state, eg, usually flanking a sequence flanking the fragment (eg flanking a fragment in the native genome) DNA fragment removed from the sequence). Thus, the term refers to, for example, a vector, self-replicating plasmid or virus, or a segregated molecule that has been integrated into prokaryotic or eukaryotic genomic DNA or is independent of other sequences (eg, cDNA fragments or genomic fragments, or Contains recombinant DNA, which is present as a cDNA fragment produced by PCR or restriction enzymatic digestion. It also contains recombinant DNA that is part of a hybrid gene that encodes an additional polypeptide sequence.

A "configuration" is derived from any source, including polynucleotide molecules, in which one or more polynucleotide molecules are operatively linked, i.e. operably linked. Any set of linear or circular single- or double-stranded DNA or RNA polynucleotide molecules, plasmids, cosmids, viruses, self-replicating polynucleotide molecules, or phages that can integrate or self-replicate the genome. It means a replacement polynucleotide molecule. The recombinant structure will typically contain a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell. Heterologous and non-heterologous (ie, endogenous) promoters can be used to direct expression of the nucleic acids of the invention.

By "vector" is meant any recombinant polynucleotide structure that can be used for light conversion, i.e., introduction of heterologous DNA into a host cell. One type of vector is a "plasmid" that refers to a circular double-stranded DNA loop to which additional DNA segments can be linked. Another type of vector is a viral vector in which additional DNA segments can be linked to the viral genome. Certain vectors are self-renewable within the host cell into which they are introduced (eg, a bacterial vector having a bacterial origin of replication and an episomal mammalian vector). Other vectors (eg, non-episome mammalian vectors) are integrated into the host cell upon introduction into the host cell, thereby replicating with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such a vector is referred to as an "expression vector" herein.

As used herein, an "expression vector" is one that replicates and expresses a gene of interest when it is transformed, transfected, or transduced into a host cell. A nucleic acid molecule that can be used. The expression vector comprises an origin of replication and one or more phenotypic selectable markers to reliably maintain the vector and, if necessary, amplify it in the host. In addition, the expression vector contains a promoter that drives the expression of the polypeptide in the cell. Suitable expression vectors may be, for example, various commercially available pUC plasmids or plasmids derived from pBR322. Other expression vectors may be derived from bacteriophage, phagemid, or cosmid expression vectors.

Further Embodiments of the present invention

In certain embodiments, the present invention provides mouse monoclonal antibodies identified from the screening of mouse hybridoma clones disclosed herein.

In other embodiments, the invention provides the composition of the following polynucleotide and protein sequences:

a) cDNA sequence encoding the heavy chain variable region of mouse monoclonal antibody 317, SEQ ID NO: 3;

b) Protein sequence of the heavy chain variable region of mouse monoclonal antibody 317 or mu317_Vh (SEQ ID NO: 4);

c) cDNA sequence encoding the light chain variable region of mouse monoclonal antibody 317, SEQ ID NO: 5;

d) Protein sequence of the light chain variable region of mouse monoclonal antibody 317 or mu317_Vk (SEQ ID NO: 6);

e) cDNA sequence encoding the heavy chain variable region of mouse monoclonal antibody 326, SEQ ID NO: 7;

f) Protein sequence of the heavy chain variable region of mouse monoclonal antibody 326 or mu326_Vh (SEQ ID NO: 8);

g) cDNA sequence encoding the light chain variable region of mouse monoclonal antibody 326, SEQ ID NO: 9;

h) Protein sequence of the light chain variable region of mouse monoclonal antibody 326 or mu326_Vk (SEQ ID NO: 10);

In one aspect, the invention provides a composition comprising a complementarity determining region (CDR) sequence that mediates binding to the target antigen PD-1, including the CDR sequences of mu317 and m326.

a) The mu317 heavy chain CDR1 (mu317H-CDR1) contains the amino acid GFSLTSYGVH (SEQ ID NO: 11).

b) mu317H-CDR2 comprises the amino acid sequence of VIWAGGSTNYNSALMS (SEQ ID NO: 12).

c) mu317H-CDR3 comprises the amino acid sequence of ARAYGNYWYIDV (SEQ ID NO: 13).

d) CDR1 (mu317L-CDR1) of the mu317 light chain comprises the amino acid sequence of KASQSVSNDVA (SEQ ID NO: 14).

e) mu317L-CDR2 comprises the amino acid sequence of YAFHRFT (SEQ ID NO: 15).

f) mu317L-CDR3 comprises the amino acid sequence of HQAYSSPYT (SEQ ID NO: 16).

g) mu326H-CDR1 comprises the amino acid sequence of GYTFTNYGMN (SEQ ID NO: 17).

h) mu326H-CDR2 comprises the amino acid sequence of WINNNNGEPTYAEEFKG (SEQ ID NO: 18).

i) mu326H-CDR3 comprises the amino acid sequence of ARDVMDY (SEQ ID NO: 19).

j) mu326L-CDR1 comprises the amino acid sequence of RASESVDNYGYSFMH (SEQ ID NO: 20).

k) mu326L-CDR2 comprises the amino acid sequence of RASNLES (SEQ ID NO: 21).

l) mu326L-CDR3 comprises the amino acid sequence of QQSKEYPT (SEQ ID NO: 22).

In another embodiment, the invention provides a composition comprising a sequence of humanized monoclonal antibodies produced from mouse monoclonal antibodies mu317 and mu326, including:

a) The humanized monoclonal antibody hu317-4B6 comprises the protein sequence of the heavy chain variable region (Vh) as SEQ ID NO: 24 encoded by:

b) cDNA of hu317-4B6_Vh (SEQ ID NO: 23);

c) The humanized monoclonal antibody hu317-4B6 comprises the protein sequence of the light chain variable region (Vk) as SEQ ID NO: 26 encoded by:

d) cDNA of hu317-4B6 (SEQ ID NO: 25);

e) Humanized mAbhu326-4A3 comprises the protein sequence of Vh as SEQ ID NO: 28 encoded by:

f) cDNA of hu326-4A3-Vh (SEQ ID NO: 27);

g) The humanized monoclonal antibody hu326-4A3 comprises the protein sequence of Vk as SEQ ID NO: 30 encoded by:

h) cDNA of hu326-4A3_Vk (SEQ ID NO: 29);

i) Protein sequence of hu317-4B2_Vh (SEQ ID NO: 43) and protein of hu317-4B2_Vk (SEQ ID NO: 44);

j) Protein sequence of hu317-4B5_Vh (SEQ ID NO: 45) and protein sequence of hu317-4B5_Vk (SEQ ID NO: 46);

k) The protein sequence of hu317-1_Vh (SEQ ID NO: 48) and the cDNA encoding hu317-1_Vh (SEQ ID NO: 47);

l) The protein sequence of hu317-1_Vk (SEQ ID NO: 50) and the cDNA encoding hu317-1_Vk (SEQ ID NO: 49);

m) Protein sequence of hu326-3B1_Vh (SEQ ID NO: 51) and protein sequence of hu326-3B1_Vk (SEQ ID NO: 52);

n) The protein sequence of hu326-3G1_Vh (SEQ ID NO: 53) and the protein sequence of hu326-3G1_Vk (SEQ ID NO: 54);

o) The protein sequence of hu326-1_Vh (SEQ ID NO: 56) and the cDNA encoding hu326-1_Vh (SEQ ID NO: 55);

p) The protein sequence of hu326-1_Vk (SEQ ID NO: 58) and the cDNA encoding hu326-1_Vk (SEQ ID NO: 57);

q) Protein sequence of another humanized monoclonal antibody produced from mu317 (SEQ ID NO: 63-74);

r) Protein sequence of another humanized monoclonal antibody produced from mu326 (SEQ ID NO: 75-82).

In one aspect, the invention provides a composition comprising a CDR sequence of a humanized monoclonal antibody. CDRs can be shared between humanized monoclonal antibodies of the same series, such as hu317 or hu326 (see Tables 15-18). The non-redundant CDRs are:

a) H-CDR1 sequence shared through humanized monoclonal antibodies, hu317 and mu317 in the heavy chain, GFSLTSYGVH (SEQ ID NO: 31);

b) H-CDR3 sequence shared through humanized monoclonal antibodies in heavy chains, hu317 and mu317, ARAYGNYWYIDV (SEQ ID NO: 33);

c) L-CDR1 sequence shared through the humanized monoclonal antibodies in the light chain, hu317-4B2, hu317-4B5, and hu317-4B6, KSSESVSNDVA (SEQ ID NO: 34);

d) L-CDR2 sequence shared through humanized monoclonal antibodies, hu317 and mu317 in the light chain, YAHFRT (SEQ ID NO: 35);

e) L-CDR3 sequence shared through humanized monoclonal antibodies, hu317 and mu317 in the light chain, HQAYSSPYT (SEQ ID NO: 36);

f) H-CDR2 sequence in hu317-4B6_Vh, VIYADGSTNYNPSLKS (SEQ ID NO: 32);

g) H-CDR2 sequence in hu317-4B2_Vh and hu317-4B5_Vh, VIYAGGSTNYNPSLKS (SEQ ID NO: 60);

h) H-CDR2 sequence in hu317-1_Vh, VIWAGGSTNYNPSLKS (SEQ ID NO: 59);

L-CDR1 sequence in hu317-1_Vk, KASQSVSNDVA (SEQ ID NO: 11);

j) H-CDR1 sequence shared through humanized monoclonal antibodies, hu326 and mu326 in the heavy chain, GYTFTNYGMN (SEQ ID NO: 37);

k) H-CDR3 sequence shared through humanized monoclonal antibodies, hu326 and mu326 in the heavy chain, ARDVMDY (SEQ ID NO: 39);

l) L-CDR1 sequence RASESVDNYGYSFMH (SEQ ID NO: 40) shared through humanized monoclonal antibodies, hu326 and mu326 in the light chain;

m) L-CDR2 sequence shared through humanized monoclonal antibodies, hu326 and mu326 in the light chain, RASNLES (SEQ ID NO: 41);

n) L-CDR3 sequence shared through humanized monoclonal antibodies, hu326 and mu326, in the light chain, QQSKEYPT (SEQ ID NO: 42).

o) H-CDR2 sequence in hu326_4A3_Vh, WINNNNAEPTYAQDFRG (SEQ ID NO: 38);

p) H-CDR2 sequence in Vh of hu326_1 and other hu317 monoclonal antibodies, WINNNNGEPTYAQGFRG (SEQ ID NO: 62).

In another aspect, the invention provides a particular binding epitope of a humanized anti-PD-1 monoclonal antibody on an antigen, and its functional use. Six important amino acid (AA) residues in PD-1 required for ligand binding were individually mutated and mutant and wild-type PD-1 proteins were used to evaluate binding epitopes. Residues whose antibody binding is significantly impaired by mutation are recognized as important binding epitopes. Important binding epitopes of the monoclonal antibodies, hu317-4B5 and hu317-4B6, are K45 and I93 (amino acid numbering based on 2008 PNAS, 105: 10483; corresponding to K58 and I106 in SEQ ID NO: 2 (equivalents)); Significant binding epitopes for the monoclonal antibodies hu326-3B1 and hu317-4A3 are I93, L95 and P97 (amino acid numbering based on 2008 PNAS, 105: 10483; equivalents of I106, L108 and P110 in SEQ ID NO: 2).

In yet another aspect, the invention is of a recombinant human IgG4 variant that can be linked to the variable region of an antibody of interest, including a humanized anti-PD-1 monoclonal antibody that has demonstrated favorable effector function and physicochemical properties. A composition comprising a constant region sequence is provided. The sequence is as follows:

Constant region sequence of IgG4mt10 (SEQ ID NO: 88);
a) Reference sequence of IgG4mt1 (SEQ ID NO: 83);
b) Reference sequence for IgG4mt2 (SEQ ID NO: 84);
c) Reference sequence of IgG4mt6 (SEQ ID NO: 85);
d) Reference sequence for IgG4mt8 (SEQ ID NO: 86);
e) Reference sequence for IgG4mt9 (SEQ ID NO: 87).

In another embodiment, the invention produces a stable cell line, HEK293 / OS8 / PD-L2 or HEK293 / OS8 / PD-L1, that co-expresses OS8 (T cell activating molecule) and PD-1 ligand. Provided is a method for analyzing anti-PD-1 antibody function using a plasmid expressing the recombinant fusion protein OS8. Using this cell line, T-cells and PBMCs were bound by co-culture to evaluate the function of the anti-PD-1 monoclonal antibody (see Examples 3 and 4). Alternatively, a stable cell line, HuT78 / P3Z, was prepared using another plasmid expressing the recombinant fusion protein, P3Z. Here, P3Z functions as a molecular sensor and a signal transduction mediator. When the PD-1 ligand binds to P3Z, it sends an intracellular signal to activate the release of IL-2 in HuT78 cells. This system may be used to evaluate the inhibitory effect of anti-PD-1 monoclonal antibody (see Example 3).

In one aspect, the invention provides a composition comprising the amino acid sequence of a recombinant fusion protein as follows.
a) OS8 protein sequence (SEQ ID NO: 89);
b) Protein sequence of P3Z (SEQ ID NO: 90)

In another aspect, the invention is for quantitatively assaying methods for producing stable cell lines expressing the recombinant fusion proteins described herein, and the functional activity of anti-PD-1 monoclonal antibodies. Provides a way to use the system.

In another embodiment, the invention provides a polynucleotide encoding a protein of interest. The polynucleotide may be operably linked to a heterologous transcriptional regulatory sequence for expression, or may be integrated into a vector, cell, or the like.

In another embodiment, the invention activates immune cells that act to suppress PD-1-mediated signaling and trigger a cascade of immune responses, including cytokine secretion and cytotoxicity to target cells such as cancer cells. Mouse anti-PD-1 antibody and humanized version anti-PD-1 antibody, including functional hu317-4B6, hu317-4B5, hu317-4B2, etc., and hu326-4A3, hu326-3B1, hu326-3G1, etc. , Provide such functional use of the antibody.

In one aspect, the invention comprises human T cells, NK cells and PBMCs that have the function of amplifying immune response signals, mobilizing the immune system and acting as immune effector cells for clearance of cancer cells and viral infections. Provided are humanized anti-PD-1 antibodies that activate several types of immune cells expressing PD-1, including, and such functional use of such antibodies.

In another aspect, humanized anti-PD-1 monoclonal antibodies treat human diseases involved in the suppression of immune cells by PD-1-mediated intracellular signaling that results in disease progression, in particular cancer and viral infections. It is used as a remedy for the disease.

In the compositions of the present invention, cancer, neurodegenerative diseases, and infectious diseases, particularly viral infectious diseases, and inappropriate or harmful expression of human PD-1 are elements of the etiology or pathophysiology of the condition. It is useful for the treatment of diseases. Therefore, the present invention provides a method of treating cancer in a subject (subject) who needs it with an anti-PD-1 protein or inhibiting tumor progression. The present invention further provides the use of a subject polynucleotide in the manufacture of a medicament for treating cancer or inhibiting tumor progression in a subject.

The present invention includes all combinations of the particular embodiments listed. Further embodiments and the full scope of application of the present invention will become apparent from the detailed description below. Although various modifications and modifications within the spirit and scope of the invention are apparent to those skilled in the art, preferred embodiments of the invention are shown, but detailed description and specific examples of the invention are provided by way of example. You should understand that. All references, patents, and patent applications referred to herein, including cited references, are hereby incorporated by reference in their entirety for all purposes.

Example 1 Preparation of anti-PD-1 monoclonal antibody

The anti-PD-1 monoclonal antibody (mAb) was made based on the conventional hybridoma fusion technique (with minor changes). References [Kohler and Milstein 1976 Eur J Immunol 6: 511-519], References [de St Groth and Sheidegger 1980, J Immunol Methods 35: 1-21], and References [Mechener] .. Monoclonal antibodies with high binding activity in the enzyme-linked immunosorbent assay (ELISA) and fluorescence activated cell sorting (FACS) assays were selected for further characterization.

PD-1 recombinant protein for immunization and binding assays

An expression plasmid containing full-length human PD-1 cDNA was obtained from Origin (Cat. No. SC117011, NCBI Accession No .: NM_005018.1, Beijing, China). The extracellular domain (SEQ ID NO: 1, SEQ ID NO: 2) consisting of amino acids (AA) 1 to 168 of PD-1 was PCR-amplified and fused to either the γFc domain of the human IgG4 heavy chain or the His ₆ tag. Subcloned with a cDNA 3.1 system expression vector (Invitrogen, Carlsbad, California, USA) with, two recombinant fusion protein expression plasmids, PD-1-EC / His and PD-1-EC / FC. (Abbreviated as PD-1 / His and PD-1 / Fc) were obtained. Immunogenicity / antigen proteins are schematically shown in FIG. For the production of recombinant fusion proteins, PD-1 / His and PD-1 / Fc plasmids were transiently transfected into 293-F cells in 1-3 liters of medium (Invitrogen) and rotated. Incubated for 5-7 days in a CO ₂ incubator equipped with a formula shaker. The supernatant containing the recombinant protein was collected and removed by centrifugation at 15000 g for 30 minutes. PD-1 / His, HiRoad 16/60 Superdex 200 column (Cat. No. 17106901, GE Life Sciences) following immobilized metal affinity chromatography using Ni-Sepharose Fast Flow (Cat. No. 17531801, GE Life Sciences, Shanghai, China). Purified by size exclusion chromatography using (Shanghai, China). PD-1 / Fc was purified using a Protein G Sepharose Fast Flow Column (Cat. No. 17531801, GE Life Sciences). Both PD-1 / His and PD-1 / Fc proteins were dialyzed against phosphate buffered saline (PBS) and stored in a small aliquot in a -80 ° C freezer.

The cDNA encoding human PD-L1 was chemically synthesized by Genescript (Nanjing, China) based on the published sequence (NCBI Accession No. NM_014143). The PD-L2 expression plasmid was purchased from Origine, Inc. (Catalog No. SC108873, NCBI Accession No. NM_02539.2, Beijing, China). Both cDNAs were cloned with pcDNA3.1 / hygromycin (catalog number V870-20, Invitrogen) and cDNA 3.1 / V5-His (catalog number V810-20, Invitrogen), respectively.

Stable expression cell line

Stable cell lines expressing human PD-1, PD-L1 or PD-L2 are HuT78 (ATCC, Manasus, Virginia, USA) of the pcDNA3.1 plasmid containing PD-1, PD-L1 and PD-L2. ) And HEK293 (ATCC), respectively, and then selected on a medium containing 200 μg of hyglomycin (Cat. No. 10678-010, Invitrogen) or 1 mg of G418 (Sigma) per ml. Single clones were isolated from the medium-well surface by either picking up a single colony or limiting dilution by conventional methods. Western blot and FACS with all colonies using anti-PD-1, PD-L1, and PD-L2 antibodies (Cat. Nos. 12-9696, 17-5983, 12-5888, Ebioscience, San Diego, USA) Each was screened by analysis and top expression clones were selected for FACS binding assay to screen hybridoma monoclonal antibodies and used for functional analysis.

Immunization, hybridoma fusion and cloning

8-12 week old Balb / c mice (BEIJING HFK BIOCSIENCE CO., LTD, Beijing, China) were charged with 100 μl of adjuvant (catalog number KX0210041, KangBiQuant, Beijing, China) containing 5 μg of PD-1 / Fc. Subcutaneous immunization. Immunization was performed by injecting the immunogen twice every 3 weeks. Two weeks after immunization in the second round, mouse sera were evaluated for PD-1 binding by FACS (see below). Mice with high serum titers of anti-PD-1 antibody were selected and boosted intraperitoneally with 50 μg of PD-1 / Fc in the absence of adjuvant. Three days later, splenocytes were isolated using standard techniques (see literature [Geffer, ML et al., 1977 Somat Cell Genet, 3: 231-236]) and mouse myeloma cell lines. , SP2 / 0 cells (ATCC).

Evaluation of PD-1 binding activity of antibody by ELISA and FACS

Literature [Flanagan, M. et al. L. et al. The supernatant of hybridoma clones was first screened by the enzyme-linked immunosorbent assay (ELISA) (with some modifications) described in 2007 Methods in Molecular Biology 378: 33-52]. Simply put, 50-200 ng of PD-1 / His or PD-1 / Fc protein in 50 μl of phosphate buffered saline (PBS) on a 96-well plate (Shenzhen JinCanHua Industry Co., Ltd., Shenzhen, China). Coated in well units. ELISA signals were detected and developed using HRP-linked anti-mouse IgG antibodies (Cat. No. 7076S, Cell Signaling Technology, Shanghai, USA) and chemiluminescent reagents (Cat. No. PA107-01, TIANGEN, China) to a wavelength of 450 nm. It was read by a plate reader (PHREAstar FS, BMG LABTECH, Germany). ELISA-positive antibody-producing clones were further identified by fluorescence activated cell sorting (FACS) using conventional methods. Aforementioned PD-1 cell line stably expressing, HuT 78 / PD-1 (10 ⁵ cells / well), V-bottom 96-well plates (Cat # 3897, Corning, USA and Shanghai, China) with the anti-PD-1 hybridoma Stained with supernatant. To block human Fc receptors, cells were preincubated with human IgG (20 μg / ml) (Catalog No. H11296, Lifeholder, USA and Shanghai, China). The PD-1 antibody was detected with a DYlight® 649-labeled goat anti-mouse IgG antibody (Cat. No. 405312, BioLegend, San Diego, USA) and cell fluorescence was detected by a flow cytometer (Guava easeCyte 8HT, Merck-Millipore). , USA and Shanghai, China).

Hybridoma cells that showed positive signals in both ELISA and FACS assays were subjected to functional analysis to obtain antibodies with good functional activity in human immune cell line assays (in the specification). Identified. Antibodies with positive functional activity were further subcloned and characterized.

Subcloning and adaptation to serum-free or low-serum medium

Positive hybridoma clones from primary screening via ELISA, FACS and functional analysis were subcloned by conventional limiting dilution methods. RPMI1640 medium (catalog number SH30809.01B,) with each positive clone plated on a 96-well plate and containing 10% fetal bovine serum (FBS, catalog number SH30084.03, HYCLONE, Beijing, China) in a CO ₂ incubator, Cultured in Hyclone (Shanghai, China). Three subclones from each limiting dilution plate were selected and characterized by FACS and functional analysis. Subclones selected via functional analysis were defined as monoclonal antibodies. The top subclone was suitable for growth in CDM4 monoclonal antibody medium with 1-3% FBS (Cat. No. SH30801.02, Hycrone).

Expression and purification of monoclonal antibody

Either hybridoma cells producing mouse monoclonal antibodies or 293-F cells transfected with recombinant antibody plasmids (Cat. No. R79007, Invitrogen) were placed in CDM4 monoclonal antibody medium (catalog) in a CO ₂ incubator at 37 ° C., respectively. The cells were cultured in Freestyle 293 expression medium (Cat. No. 12338018, Invitrogen) for 5 to 7 days (No. SH30801.02, Hybridoma) or Freestyle293 expression medium. The conditioned medium was collected by centrifugation at 10000 g for 30 minutes to remove all cells and cell debris and filtered through a 0.22 μm membrane prior to purification. Mouse or recombinant antibody is applied to a Protein A column (Cat. No. 17127901, GE Life Sciences) according to the manufacturer's guide, bound to it, washed with PBS and containing 20 mM citric acid, 150 mM sodium chloride. It was eluted in buffer (pH 3.5). The eluted material was neutralized at 1 M Tris pH 8.0 and usually contained an antibody with a purity greater than 90%. Protein A affinity purified antibodies were dialyzed against PBS or further purified using a HiLoad 16/60 Superdex 200 column (Cat. No. 17531801, GE Life Sciences) to remove aggregates. Bradford assay (Cat. No. 1856210, Thermo Scientific, Rockford, Illinois, USA) by measuring absorbance at 280 nm or using a concentration defined as a standard bovine IgG (Cat. No. 23212, Thermo Scientific). ), The protein concentration was measured. The purified antibody was aliquoted in a freezer at −80 ° C.

Example 2 Comparison of binding activity between anti-PD-1 antibodies

We screened thousands of hybridoma clones and identified several top monoclonal antibodies (mAbs) that bind to human PD-1 with high specificity and strength. As shown in the ELISA assay (FIG. 2), the three top antibodies elicited such binding strength and specificity. From the results of FACS analysis, it was found that the selected monoclonal antibody binds to the native PD-1 protein expressed on the cell surface. Mouse monoclonal antibody 317 (mu317), the mu326 and Mu150, showing concentration dependent binding activity, their binding _EC 50 (effective concentration at 50% active) was significantly lower than the control Mu55 (Figure 3).

The binding affinity of the monoclonal antibody is evaluated by surface plasmon resonance (SPR).

Monoclonal antibodies with high binding activity in ELISA and FACS, as well as strong functional activity in cell line assays (in the specification), were examined for binding rate constants in real-time binding reactions. Mouse anti-PD-1 monoclonal antibody on a hybridoma by exclusion chromatography using a Protein A flow column (Cat. No. 17531801, GE Life Sciences) followed by a HiLoad 16/60 Superdex 200 column (Cat. No. 17106901, GE Life Sciences). Purified from Qing. The purified anti-PD-1 antibody was concentrated to 0.5 mg / ml in PBS and aliquoted in a -80 ° C freezer.

To measure the binding affinity of PD-1 monoclonal antibodies, HBS-N buffer (10 mM HEPES pH 7.4, 0.15 M) was used using a BIAcore® T-200 device (GE Lifescience). SPR measurements were performed in NaCl, 3 mM HEPES, 0.005% v / v surfactant P20, GE Healthcare). The anti-mouse Fc CM5 biosensor chip (GE Healthcare) was made using standard primary amine coupling protocols. 0.3 μg / ml PD-1 monoclonal antibody was captured on the anti-mouse Fc surface at 10 μl / min for 1 minute. The dissociated phase continues for 10 minutes after injecting PD-1 / Fc in a serial dilution of 3.3 nM to 120 nM at 30 μl / min onto the antibody-bound surface for 3 minutes. Association rate _{(K a} or _{k on)} and dissociation rates _{(K d} or _{k off)} one-to-one Langmuir binding model (BIA evaluation software, GE Life Sciences) was calculated using the. The equilibrium dissociation constant _{(K D)} was calculated as the ratio _k off _{/ k on.}

As shown in Table 1, both mu326 and Mu517, the homologous sequence family member related to Mu317, have _{K D} sub-nanomolar corresponding to significantly better, respectively 0.324nM and 0.289nM than mu134 .. k _on rate was similar between the three monoclonal antibodies shown in Table 1, K _off rate varies significantly faster dissociation rate was observed in Mu134.

Affinity measurement of anti-PD-1FAb by SPR

The anti-PD-1 monoclonal antibody was converted to the FAb version by PCR, and the variable regions of the heavy and light chains were fused with the N-terminal and κ chain constant regions of human IgG2-CH1, respectively, and the pcDNA3.1 vector (Invitrogen). ) Was subcloned. Both expression vectors were co-expressed in 293-F cells using a transient transfection protocol similar to the transient expression of whole antibody. Simply put, the Fab kappa strand was PCR amplified and subcloned with a pcDNA3.1 lineage expression vector (Invitrogen, Carlsbad, CA, USA). In another plasmid, PCR was performed to fuse with the CH1 coding sequence from human IgG2 along with the heavy chain variable region (VH) C-terminal c-myc-His8 tag, which was then subcloned into the expression vector. C232S and C233S (literature [KAbat residue numbering, KAbat et al . Sequence of proteins of immunologic interest, 5 th ed Bethesda, MD, NIH 1991] Referring) mutation was introduced into IgG2 heavy chain, disulfide in IgG2-A structure It prevented bond exchange and stabilized human IgG2. References [Lightle et al. 2010 Protein Sci 19 (4): 753-762]. Both structures contained a signal peptide upstream of the FAb mature sequence. Secretory expression of FAb was obtained by co-transfection of the above two plasmids into 293-F cells, and the cell culture supernatant was recovered 6-7 days after the transfer. Fabs tagged with His8 were subjected to size exclusion chromatography using a nickel-cepharose fast flow column (catalog number 17531801, GE Life Sciences) followed by a HiLoad 16/60 Superdex 200 column (catalog number 17106901, GE Life Sciences). Purified from cell culture supernatant. The purified Fab was concentrated in PBS to 0.5-5 mg / mL and aliquoted in a -80 ° C freezer.

An SPR assay was performed using a BIAcore® T-200 instrument (GE Life Sciences) to measure the affinity of anti-PD-1Fab. Simply put, human PD-1 / His or cynomolgus monkey PD-1 / His is bound to an activated CM5 biosensor chip (catalog number BR100530, GE Life Sciences) to produce approximately 100-200 response units (RU). Once obtained, unreacted groups were blocked with 1M ethanolamine. Fab samples with concentrations increasing from 0.12 nM to 90 nM were injected into SPR running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4) at 30 μL / min and human PD-1. Binding reactions on / His or monkey PD-1 / His were calculated by subtracting RU from the blank flow-cell. The association rate _{(k on)} and dissociation rates _{(k off)} is a one-to-one Langmuir binding model (BIA evaluation software, GE Life Sciences, Inc.) was calculated using the. The equilibrium dissociation constant _{(K D)} was calculated as the ratio _k off _{/ k on.}

The binding affinity of anti-PD-1Fab measured by SPR is shown in Table 18. Each anti-PD-1Fab bound to human PD-1 with high affinity (K _d = 0.15-1 nM). All Fabs, with the exception of 326-3G1, bound with an affinity slightly lower than that for cynomolgus monkey PD-1, but comparable (within K _d 5-fold).

Example 3 Functional activity of anti-PD-1 antibody in human T cells

Creation of stable cell lines

The retrovirus packaging cell line PT67, human T cell line HuT78 and HEK293 were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland). The HuT78 subline HuT78 / PD-1 expressing PD-1 is a pFB-neo vector containing the PD-1 gene (Transategene /) according to the protocol described above (see literature [Zhang et al. 2005 Blood 106: 1544-1551]). Genent Tech, Santa Clara, Calif.) By retroviral transduction. The T cell engager, membrane anchor chimeric antibody (OS8) is an anti-human CD3 monoclonal antibody, OKT3 (see [Kipriyanov et al. 1997, PEDS 10: 445-453]), a single-stranded variable fragment (see scFV) was constructed by fusing to the C-terminal domain (113-220) of mouse CD8α (NCBI Accession No .: NP_0010747591) containing the hinge, transmembrane domain, and cytoplasmic domain. By doing so, the anti-CD3 scFv is anchored on the cell surface as a T cell activator. Human PD-L1, PD-L2 and OS8 cDNAs were subcloned into the cDNA 3.1 vector. The stable cell lines HEK293 / OS8 / PD-L1, Hep3B / OS8 / PD-L1 and HEK293 / OS8 / PD-L2, which co-express both OS8 and PD-L1 or PD-L2 cDNA, are paired with HEK293 and HEK293. It was prepared by co-transfecting Hep3B cells (ATCC) and then selecting hyglomycin or G418 for 10-14 days. The cell line was then cloned by limiting dilution as described above (see Document [Fuller SA, et al. Curr Protocol Mol Biol. Chapter 11: Unit 11.8., 2001]). The chimeric PD-1 receptor, named P3Z, was constructed by fusing the extracellular and transmembrane domains of human PD-1 with the cytoplasmic domain of the human CD3ζ chain (NCBI Accession No. NP_932170.1). The cDNA sequence encoding P3Z was cloned into PFB-neo and delivered to HuT78 cells via retroviral transduction to generate HUT78 / P3Z cells.

Determination of PD-1 antibody function by release of IL-2 in HuT78 / PD-1 cells

HuT78 / PD-1 cells (to determine if anti-PD-1 antibodies can block PD-L1-induced (PD-L1-induced) PD-1 signaling interactions. HEK293 / OS8 / PD-L1 or HEK293 / OS8 / on a flat-bottomed plate supplied with 200 μl RPMI1640 growth medium per well at 37 ° C. after preincubation with 1.5 × 10 ⁴ cells per well in a 96-well plate for 15 minutes. It was co-cultured with PD-L2 cells (4 × 10 ⁴ per well). After 16-18 hours, the supernatant of the co-culture was collected. IL-2 is human IL-2 Ready-Set-Go! Assayed by ELISA using an ELISA kit (Cat. No. 88-7025, eBiosciences, San Diego, CA). In this assay, blocking PD-1 signaling by anti-PD-1 antibody resulted in enhanced TCR signaling and IL-2 production (FIG. 4).

As shown in FIG. 5 and Table 2, the mouse anti-PD-1 monoclonal antibodies mu317 and mu326 induce significantly higher functional activity than mu30, leading to increased IL-2 secretion PD-L1-inducible PD- 1 Inhibited signal transduction. Both a higher IL-2 secretion (top line, Table 2), the effective concentration of monoclonal antibody in conjunction with a low _EC 50 (50% levels of IL-2 secretion-inducing than mu30 antibodies each with 675 and 634pg / ml ) Was.

Engagement of anti-PD-1 monoclonal antibody to HuT78 / PD-1 cells not only blocked PD-L1-induced T cell activation, but also blocked PD-L2-induced IL-2 release. .. Table 3 shows that mu317 and mu326 are more potent than mu476 in the T cell activity indicated by the IL-2 secretion parameter (EC ₅₀ ).

Determination of PD-1 antibody function by reverse signaling of IL-2 release in HUT78 / P3Z cells

At the chimeric receptor P3Z, the PD-1 signaling domain was exchanged for the cytoplasmic domain of CD3ζ. Therefore, P3Z mediates activation upon binding to PD-L1 rather than inhibition as the original PD-1 receptor. In this assay, HuT78 / P3Z cells (3 × 10 ⁴ / well) were pre-incubated with hybridoma supernatant or PD-1 antibody for 1 minute and then HEK293 / OS8 / PD-L1 or on a 96-well flat bottom plate at 37 ° C. It was co-cultured with HEK293 / OS8 / PD-L2 cells (4 × 10 ⁴ per well) (total volume 200 μl per well). After 16-18 hours, the supernatant of the co-culture was collected and assayed by ELISA as described above.

The functional activity of the mouse anti-PD-1 monoclonal antibody was further confirmed by direct readout of T cell activation in the reverse signaling assay described above. Consistent with the above results, mu317 and mu326 had the highest functional activity among the monoclonal antibodies we screened. Table 4 and Table 5, Mu317 and mu326, both in terms of _{IC 50} and maximum inhibition, one low active monoclonal antibodies, were much more potent than Mu37.

Example 4 Activation of IFN-γ secretion by anti-PD-1 antibody in primary human PBMC co-cultured with HEK293 / OS8 / PD-L1 cells

To determine if the top mAbs selected for PD-1 also exert a functional effect on primary human immune cells, we predominantly T cells (50-70%), B cells and Antibody function was assayed using freshly isolated peripheral blood mononuclear cells (PBMCs) composed of NK cells (15-30%) and monocytes (2-10%). According to the manufacturer's instructions, human PBMCs were isolated from healthy donors by density gradient centrifugation using Ficoll lymphocyte isolation medium (Histopaque-1077; Sigma-Aldrich, Misori). All human blood draws followed Humane's internal procedures. The PBMC was then stimulated with anti-CD3 monoclonal antibody (40 ng / ml) OKT3 (Cat. No. 16-0037, Ebioscience, CA) for 3 days prior to the assay. FACS analysis (Example 1) showed that PD-1 expression in activated PBMCs (mainly T cells) was variably increased depending on the individual donor (Table 6). After binding of the TCR / CD3 complex, PBMC (1 × 10 ⁴ ) was HEK293 in a 96-well flat bottom plate to determine the response of pre-activated T cells to PD-1 ligand-positive tumor cells. It was co-cultured with either / OS8 / PD-L1 or HEK293 / OS8 / PD-L2 cells (3 × 10 ⁴ ) for 15-18 hours. Cell-free supernatants are the most prominent indicator of T cell activation and other immunized cell activation, Ready-Set-Go! Assayed for IFN-γ levels by ELISA using an ELISA kit. Literature [Thakur A. et al. 2012 Vaccine, 30: 4907-4920].

FIG. 6 demonstrated that the presence of the monoclonal antibodies mu317 and mu326 in the co-culture of pre-activated PBMC and HEK293 / OS8 / PD-L1 cells resulted in a dose-dependent increase in IFN-γ accumulation. Although the base level of IFN-γ by IgG treatment in control mice varies between donors, the increase in IFN-γ secretion in PBMC treated with mu317 or mu326 is statistical in antibody treatment in the 0.1-10 μg / ml range. Significantly. IFN-γ secretion at 0.1-10 μg / ml concentration levels induced by mu317 and mu326 was 2.5-3.2 in PBMCs from donor 19 when compared to the corresponding levels of mIgG-treated PBMCs. It increased by a factor of 1.4 to 2.3 in PMBC of donor 20.

Example 5 Activation of human NK cells by anti-PD-1 monoclonal antibody

Stable cell line for functional analysis in NK cells

It has been previously reported that primary human NK cells express PD-1 protein in response to IL-2 treatment and inhibit the cytotoxicity of NK cells enhanced by PD-1 mediated signaling. See reference [2010 Blood, 116: 2286]. For quantitative analysis of the functional effects exerted by anti-PD-1 monoclonal antibodies in NK cells, human NK cell line NK92MI (ATCC) and lung cancer cell line SK-Mes-1 (ATCC) were added to the aforementioned protocol. By introducing retroviral traits based on, human PD-1 and PD-L1 were engineered for stable expression. References [Zhang et al. 2005, Blood 106: 1544-1551], literature [Zhang et al. 2006, Cancer Res, 66: 5927]. The two stable cell lines were named NK92MI / PD-1 and SK-Mes-1 / PD-L1.

Anti-PD-1Ab promotes IFN-γ production and secretion in NK92MI / PD-1 cells

The functional activity of the anti-PD-1 monoclonal antibody against NK cells was 1: 2 with the lung cancer cell line SK-MES-1 / PD-L1 on a 96-well flat-bottomed plate with a total of 6 × 10 ⁴ cells per well. The assay was performed by quantitative measurement of IFN-γ production and secretion in NK92MI / PD-1 cells co-cultured in. The anti-PD-1 monoclonal antibody was added to NK92MI / PD-1 15 minutes before the start of co-culture, after which the cells were co-cultured overnight in a CO ₂ incubator. Cell-free supernatants were assayed for IFN-γ levels by ELISA as described in Example 4.

All anti-PD-1 monoclonal antibodies significantly increased IFN-γ production from the baseline with low antibody therapeutic concentrations to the top line with high antibody therapeutic concentrations. Two top Antibodies, Mu317 and mu326 has a lower _{EC 50} than the comparative antibody 5C, it indicates that it has a strong activating effect against NK cells (Table 7).

Anti-PD-1 antibody enhances cancer cell death mediated by NK92MI / PD-1 cells

Lactate dehydrogenation of NK92MI / PD-1 cell cytotoxicity against SK-MES-1 / PD-L1 cells using the CytoTox96 non-radioactive cytotoxicity assay kit (Promega, Madison, Wisconsin). Determined by elementary enzyme (LDH) release assay. Briefly, preincubated with NK92MI / PD-1 cells (10 ⁵⁾ the 0.004-10μg / ml anti-PD-1 monoclonal antibody at a final concentration ranging from effector in 96-well V-bottom plates SK-MES-1 / PD-L1 cells (2 × 10 ⁴ ) were added to the immunized cell culture at a ratio of tumor cells (E: T) of 5: 1 and then co-cultured for 5 hours. Complete tumor cell lysis was defined as maximal cell killing, and the LDH release assay reading of each sample was calculated as the rate of maximal cell killing (%). Cell killing (%) of all samples was standardized through plates (cross) using 10% of baseline as a common criterion.

In the particular cytotoxicity assay set as described above, the selected anti-PD-1 monoclonal antibody killed final tumor cells in the range of 19% to 20.2% at high concentrations of monoclonal antibody input (value). Top line-base line). mu317 and mu326 has had a lower _{EC 50} than Mu336, it indicates that the result in NK92MI / PD-1 cell-mediated tumor cell killing (Table 8).

Example 6 Cloning and sequence analysis of PD-1 monoclonal antibody

Mouse hybridoma clones secreting a particular monoclonal antibody are cultured in a 100 mm-tissue culture dish to a density of 3-10 x 10 ⁶ (cells) and the cells are centrifuged in a swing bucket rotor at 1500 rpm. Collected. Whole cell RNA was isolated using an ultrapure RNA kit (Ultrafine RNA Kit) (Cat. No. CW0581, CWBIOTECH, Beijing, China) according to the manufacturer's protocol. RNA was resuspended in double deionized water and its concentration was measured by Nanodrop (Thermo Fisher, Shanghai, China).

Monoclonal Antibodies PCR primers for cDNA cloning were synthesized by Invitrogen (Beijing, China) based on previously reported sequences (see literature [Blocks et al. 2001 Mol Med 7: 461-469]). First-strand cDNA was synthesized using reverse transcriptase (Cat. No. AH301-02, Transgene Biotech, Beijing, China). PCR reagent kits (Cat. No. Ap221-12, Transgene Biotech, Beijing, China) were used to perform PCR amplification of specific monoclonal antibody cDNAs according to the manufacturer's protocol. The PCR product was sequenced directly by a service provider (GeneWiz, Beijing, China) or subcloned into a pCR vector (Invitrogen) and then sequenced (GeneWiz).

The protein sequences of mouse monoclonal antibodies were analyzed by sequence homology alignment. Monoclonal antibodies were classified based on sequence homology and epitope mapping results (Example 13). Kabat by sequence analysis and internet sequence analysis (http://www.imgt.org/IMGT_vquest/share/textes/index.html and http: //www.ncbi.nlm.nih.GOV/igblast/). See literature [Wu, TT and Kabat, EA, 1970 J. Exp. Med. 132: 211-250]) and the IMGT system (literature [Lefranc MP et al., 1999 National Center for Biotechnology Information]. The complementarity determination region (CDR) was identified based on (see Research, 27, 209-212]). As shown in Table 9, the CDRs of mu317 and mu326 are very different in sequence length and identity.

Example 7 Humanization of mouse monoclonal antibody

Simulation of antibody 3D structure

Three-dimensional structure was simulated for the mu317 and mu326 variable domains to identify framework residues that may be important to support the CDR loop structure. Potentially important framework residues were maintained as original mouse residues in the first round of antibody humanization. Adopting a previously established structural modeling method for an antibody (see [Morea et al. Methods 2000 20: 267-279]), a known standard structure of an antibody (see [Al-Lazikani et al. 1997] JouRNAl of The 3D structure of the anti-PD-1 monoclonal antibody was simulated based on (see Molecular Literature 273: 927-948)). Simply put, in order to identify antibody sequences that are most homologous to known high resolution structures (less than 2.5 angstroms), the sequences of each variable domain (Vk and Vh) of mouse antibodies are sequenced in a PDB database (Protein Data bank). , Htp: //blast.ncbi.nlm.nih.gov/). Templates selected for mu317 and mu326 modeling (shown in Table 10) are similar standard in the modeled target antibodies L-CDR1, L-CDR2, L-CDR3, H-CDR1, and H-CDR2. Had a loop structure. When the Vk and Vh templates are derived from different immunoglobulins, Vk-Vh is used as a template for structural homology modeling by the Swiss model program (see literature [Kiefer et al. 2009 Nuclear Acids Research 37, D387-D392]). They are packed together by a least-square fit of the main chain atoms to form a hybrid structure of interface reimmunity. Specific side chain structures were adjusted while retaining the main chain structure (conformation). The side chain structure was retained at sites where the parent structure and the modeled structure had the same residue. At sites with different residues, the side chain structure was modeled based on template structure, rotational isomer library and packing considerations. After homology modeling, a PLOP program (see literature [Jacobson et al. 2002 JouRNAl of Physical Chemistry 106: 11673-11680]) is used to minimize total nuclear energy (all-atom energy) and Vh and Vh. The homology model was purified (improved) to optimize the interface of. This step is performed to improve stereochemistry, especially in regions where structural segments from different antibodies are bound.

The structures of CDR transplants 317-1 and 326-1 were simulated to guide further steps in antibody engineering to increase the degree of humanization and / or enhance antibody stability. The selected structural molds are also shown in Table 10. Structural simulations were taken from PDB template 1AY1 for 317-1 and PDB template 3CXD for 326-1, respectively, where the possible structure of H-CDR3 contains H-CDR3 of similar size and torso region. The procedure was the same as above, except for the above points. Energy minimization for the transplanted H-CDR3 residues was performed using PLOP.

Humanization

For humanization of anti-PD-1 monoclonal antibodies, we have IMGT (http://www.imgt.org/IMGT_vquest/share/textes/index.html) and the NCBI website (http://www.ncbi.). Human germline IgG genes homologous to the cDNA sequences of the mu317 and mu326 variable regions were examined by blasting a database of human immunoglobulin genes at nlm.nih.gov/igblast/). Human IVGH and IGVγ, which have high homology with PD-1 monoclonal antibody, were selected as templates for humanization.

Humanization was performed by CDR transplantation in principle. In the first round of humanization, mouse-to-human mutations of amino acid residues in the variable region framework sequence are guided by a simulated 3D structure, so that the mouse's overall antibody is retained. Only the CDR loop structure was mutated to the human sequence described above. Early versions of humanized monoclonal antibodies are heavy chains with humanized variable heavy chains (Vh) fused to the human IgG2 constant region (NCBI Accession No. P01859) and humanization fused to the human IgκC-region (NCBI Accession No. P01834). Hu317-1 (SEQ ID NO: 47-50) and hu326-1 (SEQ ID NO: 55-58) containing a light chain having a variable light chain κ (Vκ). Similarly, we generated chimeric antibodies from mu317 and mu326 composed of mouse VH fused to the human IgG2 constant region and mouse Vκ fused to the human IgκC-region. The full chimeric antibodies were named ch317 and ch326, respectively. As described in Example 1, all recombinant monoclonal antibodies were expressed and purified.

FACS and functional analysis demonstrated that the monoclonal antibody hu317-1 retains approximately the same binding and functional activity as mu317 and ch317. difference _{EC 50} of the FACS analysis between mu317 pair ch317 and hu317-1 can be interpreted by the fact that using two different detection antibodies, goat anti-mouse IgG and goat anti-human IgG by FACS. In the two functional analyzes, all three versions of 317 were treated more equally, with results close to each other (Table 11).

As a result of the first round of humanization of mu326, the monoclonal antibody hu326-1 retains functional features similar to the parental ch326 and mu326, but with the FACS binding assay and the HuT78 / PD-1 cell line IL- Functional activity in the two-release assay may be slightly weaker than ch326 (Table 12).

Based on the first round of humanization, we mutated other mouse amino acid (AA) residues within the framework (FR) of hu317-1_Vh and _Vκ to further assess the effect on antibody function. I let you. As shown in Table 13, all seven individual mutations (forms) in Vh and one mutation (form) in Vκ of hu317-1 have similar functional activity. Among the mutations (body), very slight changes were observed in some Vh mutations (body) such as hu317-2_K71V, which has a slightly weaker inhibitory function. However, when all mouse amino acid residues were mutated to humans (hu317-3A), their function was clearly weaker in FACS and IL-2 release assays than in other mutations (forms).

In the initial studies described above, hu326-1 reached significant humanization levels in FR, except that it left some mouse AA residues. However, it has a weaker function than mu326. Therefore, we made either additional individual mutations (forms), such as back to mouse residues or to human residues, and examined the contribution of individual AAs to monoclonal antibody 326 function. Table 14 shows all single AA mutations (forms) made based on the hu326-1_Vh template (SEQ ID NO: 56, SEQ ID NO: 57) and the results of their functional analysis. Most of the mutations were comparable to the original mu326 monoclonal antibody and showed better functional activity than hu326-1. The two mutations (forms) (E46K and F95Y) resulted in slightly less potential in the EC ₅₀ or IC ₅₀ , indicating the role of their residues in antibody structure and function.

To investigate the best possible Vh and Vκ sequence composition for monoclonal antibodies 317 and 326 that can be used as therapeutic agents in humans, we discuss humanization levels, functional activity, and physicochemical in FR. Make various combinations of mutations (including some mutations in the CDR sequence), taking into account antibody characteristics such as properties, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (DCD). It was. Most mutations were considered not to pass the accreditation criteria. Through the manipulation process, six humanized recombinant monoclonal antibodies were selected for their potential therapeutic utility: hu317-4B2 (SEQ ID NO: 43-44), hu317-4B5 (SEQ ID NO: 45-46), hu317-4B6. (SEQ ID NO: 23-26), hu326-3B1 (SEQ ID NO: 51-52), hu326-3G1 (SEQ ID NO: 53-54) and hu326-4A3 (SEQ ID NO: 27-30). Tables 15 and 16 show a comparison of the CDRs of the monoclonal antibody with the CDRs of the original mouse antibody.

Of the six monoclonal antibodies, hu317-4B2, hu317-4B5, and hu317-4B6 are closely related to each other in sequence and very similar in their functional activity and intensity. On the other hand, hu326-3B1, hu326-3G1 and hu326-4A3 are very close to each other in sequence and function (Table 17-18). Monoclonal antibodies belonging to each of these two groups, with some minor differences, other than sequence and function, such as physicochemical properties and binding epitopes (described in Examples 10 and 11). It also shared many features.

Affinity determination of humanized anti-PD-1Fab by SPR

The anti-PD-1 monoclonal antibody was converted to the Fab version by PCR, and the variable regions of the heavy and light chains were fused to the N-terminal and κ chain constant regions of human IgG2-CH1, respectively, and the pcDNA3.1 vector (Invitrogen). ) Was subcloned. Both expression vectors were expressed in 293-F cells using the complete antibody transient transfection protocol. Simply put, the Fab kappa strand was PCR amplified and subcloned into a pcDNA3.1 lineage expression vector (Invitrogen, Carlsbad, CA, USA). By overlaying PCR with another plasmid, the heavy chain variable region (VH) was fused with the C-terminal c-Myc-His8 tag along with the CH1 coding sequence from human IgG2 and then subcloned into an expression vector. C232S and C233S (literature [KAbat residue numbering, KAbat et al . Sequence of proteins of immunologic interest, 5 th ed Bethesda, MD, NIH 1991] Referring) by introducing a mutation into IgG2 heavy chain disulfide bond in the IgG2-A structure Exchange was prevented and human IgG2 was stabilized (see [Lightlet et al. 2010 Protein Sci 19 (4): 753-762]). Both structures contained a signal peptide upstream of the Fab mature sequence. Secretory expression of Fab was obtained by co-transfection of the above two plasmids into 293-F cells, and the cell culture supernatant was collected 6-7 days after the transfer. Fabs tagged with His8 are subjected to size exclusion chromatography (Cat. No. 17106901, GE Life Sciences) using a Ni-Sepharose Fast Flow Column (Cat. No. 17531801, GE Life Sciences) and a HiLoad 16/60 Superdex 200 column from cell culture supernatants. Purification was carried out in sequence. The purified Fab was concentrated in PBS to 0.5-5 mg / ml and aliquoted in a -80 ° C freezer.

To measure the affinity of anti-PD-1Fab, the SPR assay was performed on a BIAcore® T-200 instrument (GE Life Sciences). Simply put, human PD-1 / His or cynomolgus monkey PD-1 / His is combined with an activated CM5 biosensor chip (catalog number BR100530, GE Life Sciences) to produce approximately 100-200 response units (RU). Once obtained, unreacted groups were blocked with 1M ethanolamine. Fab samples with concentrations increasing from 0.12 nM to 90 nM were injected into SPR running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4) at 30 μL / min and human PD-1. Binding reaction on / His or monkey PD-1 / His was calculated by subtracting RU from the blank flow-cell. The association rate _{(k on)} and dissociation rates _{(k off)} is a one-to-one Langmuir binding model (BIA evaluation software, GE Life Sciences, Inc.) was calculated using the. The equilibrium dissociation constant _{(K D)} was calculated as the ratio _k off _{/ k on.}

The binding affinity of Fab of anti-PD-1 measured by SPR is shown in Table 18. Each anti-PD-1Fab bound to human PD-1 with high affinity (K _d = 0.15-1 nM). All Fabs, with the exception of 326-3G1, bound with a slightly lower but comparable affinity (within K _d 5-fold) for cynomolgus monkey PD-1.

Example 8 Preparation and expression of a recombinant anti-PD-1 monoclonal antibody having a mutant human IgG4 constant region.

Since PD-1 is predominantly expressed in activated T cells, PD-1 blocking antibodies linked to the Fc portion of native IgG differ in Fc-mediated effector functions such as ADCC and DCD depending on the IgG subclass. It is expected to induce to some extent and, as a result, eliminate activated T cells. References [Natsume A, et al, 2009 Drug Des Devel Ther. 3: See 7-16]. It has been widely reported that the human antibody subclass IgG4 has a small amount of ADCC and has almost no CDC effector function. References [Moore GL, et al. 2010 mAbs, 2: 181-189]. On the other hand, natural IgG4 has been known to be inferior in stability under stress conditions such as acidic buffers or elevated temperatures. References [Angal, S.A. 1993 Mol Immunol, 30: 105-108]; Reference [Dall'Acqua, W. et al. et al, 1998 Biochemistry, 37: 9266-9273]; literature [Aalberse et al. See 2002 Immunol, 105: 9-19]. Humanized monoclonal antibodies were bound to IgG4 engineered by a combination of mutations to evade PD-1 ⁺ cells from death and improve the physicochemical properties of anti-PD-1 antibody, resulting in reduced or null. ) FcγR binding or C1q binding activity was imparted, thereby weakening or eliminating ADCC and CDC effector function. Given the physicochemical properties of the antibody as a biological agent, one of the less desirable inherent properties of IgG4 is that the two heavy chains dynamically separate in solution to form a half-antibody (half antibody). Is to form. This produces bispecific antibodies in vivo through a process called "Fab arm exchange". References [Van der Neut Kolfschoten M, et al. See 2007 Science, 317-1554-157]. Mutation of serin to proline at position 228 (EU numbering system) appeared to suppress the heavy chain separation of IgG4. References [Angal, S.A. 1993 Mol Immunol, 30: 105-108]; and the literature [Aalberse et al. See 2002 Immunol, 105: 9-19]. Several amino acid residues in the hinge and γFc regions have been reported to affect antibody interactions with Fcγ receptors. References [Chappel SM, et al. 1991 Proc. Natl. Acad. Sci. USA, 88: 9036-9040]; literature [Mukherjee, J. et al. et al. , 1995 FASEB J, 9: 115-119]; Ref. [Armor, K. et al. L. et al. , 1999 Eur J Immunol, 29: 2613-2624]; Reference [Clynes, R. et al. A. et al. , 2000 Nature Medicine, 6: 443-446]; and literature [Arnold J. et al. N. , 2007 Annu Rev Immunol, 25: 21-50]. In addition, some IgG4 isoforms that appear rarely in the human population can induce different physicochemical properties. Literature [Brusco, A. et al. 1998 Eur J Immunogenet, 25: 349-55]; and the literature [Aalberse et al. See 2002 Immunol, 105: 9-19]. Collectively treating all previously reported mutations and isoforms as specific antibodies does not guarantee that the ideal antibody molecule will share all of the therapeutic agent characteristics as described above. It is based on the effect of variable regions on the effector function and physicochemical properties of the antibody and the contradictory effects of the combined mutations. References [Igawa T. et al. , 2010 Prot Eng Design Select, 23: 385-392]; and the literature [Perchiacca J. et al. M. and Tessier P. M. , 2012 Ann Rev Biomol Eng 3: 263-286].

To produce anti-PD-1 monoclonal antibodies with minimal ADCC, CDC and instability, we introduced a number of mutation combinations into the hinge and γFc regions of human IgG4 to yield IgG4mt1 and IgG4mt12. I let you. Some mutated IgG4 variants were found to be less desirable, as shown in our analysis. Table 19 summarizes several related IgG4 variants and mutant sequences. Evaluations of these antibodies are described herein.

Example 9 IgGmt10 has no FcγR binding and has the lowest ADCC and CDC effector function.

ATCC is initiated when the antibody binds to a cell surface target protein and is linked to the Fcγ receptor (FcγR) expressed on effector cells. Human IgG1 has a higher binding affinity for FcgγR, especially FcγR-I and FcγR-IIIA, than IgG2 and IgG4, which is well documented to be related to the strength of IgG1 that activates ADCC. It was. CDC, which resembles ADCC, is activated when the antibody cross-binds to the cell surface target and C1q protein, followed by cascade reactions such as complement complex formation and artillery cell lysis. As a proxy for ADCC and CDC, the assay for antibodies that bind to FcγR and C1q can serve as a basic indicator of ADCC and CDC. Therefore, we systematically evaluated monoclonal antibody binding to all major FcγRs.

FcγR binding

The binding of various IgG4 variants to FcγR was determined by flow cytometry. Simply put, a series of HEK293 transfectants expressing human FcγRs have been established. These transfectants expressed FcγRI, FcγRIIA, FcγRIIB, or FcγRIIIA. The multi-subunit FcγR (ie, FcγRI and FcγRIIIA) was co-expressed with FcRγ. Polymorphic variants (ie, FcγRIIA H131 and R131, FcγRIIIA F158 and V158) were also included. Mutant IgG4 variants against FcγR ⁺ HEK293 cells using secondary antibodies (goat anti-human IgG F (ab) '2-Alexafluoro488, Jackson Immunoresearch, West Grove, PA, USA) (Table) The binding of the anti-PD-1 monoclonal antibody having 9) was detected. As expected, IgG1 format anti-PD-1 monoclonal antibodies (hu317-1 / IgG1 and hu317-4B6 / IgG1) are capable of producing FcγRI, FcγRIIA (H131 and R131 alleles), FcγRIIB, and FcγRIIIA (V158 and F158 alleles). It binds strongly to all FcγRs, including (Table 20). Interestingly, two different versions of humanized monoclonal antibodies, hu317-1 and hu317-4B6 (different in both Vh and Vκ), were made in the same IgG4 mutant format (eg, either IgG4mt1 or IgG4mt6 format). Sometimes their bond strength (MFI) varies from 2x to nearly 100x (eg, 455.2 / 115.7 = 3.9x; 13.6 / 1.0 = 13.6). Times; 434.6 / 4.9 = 88.7 times, etc., see Table 20). This is consistent with previous findings by other researchers that the variable region of the antibody affects effector function, such as ADCC, as it significantly affects binding to FcR. References [Igawa T. et al. , 2010 Prot Eng Design Select, 23: 385-392]; and the literature [Perchiacca J. et al. M. and Tessier P. M. , 2012 Ann Rev Biomol Eng 3: 263-286].

As shown in Table 20, when hu317-4B6 and hu326-4A3 were prepared in the IgG4mt10 format, they were the PD-1 monoclonal antibody and IgG variant formats shown in the table and the many we tested in this study. It has the lowest binding activity to FcγR among other humanized monoclonal antibodies and IgG formats. Here, the uniqueness of the IgG4mt10 format hu317-4B6 and hu326-4A3 cannot be extended to humanized monoclonal antibodies of the same series, such as the hu317-1 described above, which have somewhat distant sequence homology.

ADCC

Classical ADCC is involved in the activation of NK cells by antibodies that bind to FcγRIIIA or CD16. NK92MI / generated from NK92MI cells by co-transfecting an expression plasmid containing CD16 (V158 allelic gene) and FcRγ gene to confirm whether the humanized anti-PD-1 monoclonal antibody induces ADCC. CD16V cells were used as effector cells and PD-1 expressing T cell lineage, HuT78 / PD-1, was used as target cells. NK92MI / CD16V cells (4 × 10 ⁴ ) were co-cultured with the same number of HuT78 / PD-1 cells on a 96-well V-bottom plate. Cytotoxicity was measured by the LDH release assay described in the previous section. From the results, it was confirmed that all of hu317-4B2 / IgG4mt6, hu317-4B6 / IgG4mt6, hu317-4B6 / IgG4mt10 and hu326-4A3 / IgG4mt10 had base level ADCC as compared with the positive control group (). FIG. 7). The slight difference in ADCC between these four monoclonal antibodies may be due to experimental error (see error bars in FIG. 7).

CDC

Human IgG4 antibodies generally do not induce any CDC via the classical pathway. Whether the IgG4mt10 format anti-PD-1 monoclonal antibody induces CDC was evaluated using PD-1 expressing T cell lines, HuT78 / PD-1, and fresh human serum from healthy donors. Cell lysis by CDC was measured with the CELLTITER Glo assay kit (Promega, Beijing, China). Simply put, HuT78 / PD-1 cells (2 x 10 ⁴ cells) were incubated with anti-PD-1Ab (10 μg / ml) in serum-free RPMI1640 (Invitrogen) at 37 ° C. for 15 minutes and then 96-well flat bottom. A total volume of 120 μl of normal human serum (NHS) was added to the plate to a final concentration of 15-50%. After incubating overnight at 37 ° C., cells were lysed and assayed for ATP concentration. Isolated from healthy donors prior to co-culture with anti-PD-1 to test whether humanized anti-PD-1 monoclonal antibody in IgG4mt10 can kill PD-1 ⁺ via CDC PBMC was pre-activated with anti-CD3 Ab OKT3 (40 ng / ml) for 3 days. The amount of ATP is directly proportional to the number of cells present in the culture. Fluorescence was read using a 96-well fluorometer (PHERA Star FS, BMG LABTECH). Results are shown in relative fluorescence units (RFU) proportional to the number of viable cells. % CDC activity was calculated as follows:% CDC activity = [(RFU test-RFU background) / (RFU-RFU background during total cell lysis)] x 100. In general, we were unable to detect any ADCC mediated by IgG4mt10 format anti-PD-1 that binds to activated PBMC. Under hypersensitive experimental conditions such as using highly PD-1 expressing cell lines, high serum and antibody concentrations, we detected very low levels of CDC in some situations, with different versions and anti-PD- There is not much difference between 1 monoclonal antibodies. It shows that the IgG4 mutant format anti-PD-1 monoclonal antibody retained the characteristic of low CDC activity or no CDC activity as a general form of IgG4.

Example 10 Humanized anti-PD-1 monoclonal antibody in IgG4mt10 format has enhanced stability under stress conditions.

Stability of anti-PD-1 antibody under high temperature and acidic conditions

All anti-PD-1 antibodies used in the stability study were purified by post-size exclusion chromatography (SEC) of the Protein A column as described in the previous section. After purification, the content of aggregates in the purified antibody sample was monitored by analytical size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). It was in the range of 0% to 0.5%.

For SEC-HPLC analysis, antibody samples were subjected to isocratic elution conditions (elution buffer 00.2 M sodium phosphate, pH 7.2) TSKgel G3000 SWXL column (7.8 x 300 mm, catalog number 08541, Tosoh Bioscience). , Shanghai, China), and subsequently analyzed by detection at UV-215 nm. In each experiment, 10 μl of antibody sample was packed in a column and eluted at a flow rate of 1 mL / min. Antibodies of dimer or larger aggregate species were separated from the monomer species and the proportions of dimer and aggregate were determined based on the integrated peak area from the UV trace.

In the accelerated storage stability study, anti-PD-1 antibody (10-40 mg / mL in PBS) was used in an incubator at 40-50 ° C for 4-7 days to test the stability of the antibody under high temperature conditions. Maintained. The antibody sample was then analyzed by SEC-HPLC for thermal dimer and aggregate formation. For each anti-PD-1 analyzed, less than 2% were high molecular weight species (dimers and aggregates), which means that the anti-PD-1 antibody has good stability under high temperature conditions. Is shown.

The stability of the antibody under acidic conditions has become an important issue in the downstream production process (see literature [Liu et al. 2010 Abs 2: 480-499]). Elution of antibody from protein A and inactivation of virus usually requires incubation of the antibody under low pH (2.5-4) conditions. However, such acidic conditions can potentially cause antibody denaturation and aggregation. Human IgG4 is known to be less stable than IgG1 and IgG2. See reference [2002 Immunology 105: 9]. Therefore, we assayed humanized monoclonal antibodies made with various IgG4 mutation formats. Simply put, the stability of the antibody under low pH conditions is a low pH buffer containing 50 mM sodium citrate and 100 mM NaCl at pH 3.6, 3.3, 3.0, or 2.7. Each antibody sample (10 mg / mL in PBS) mixed with 1: 1 volume was examined. After incubating for 1 hour at room temperature, antibody samples under low pH conditions were neutralized into SEC-HPLC elution buffer (containing 0.2 M sodium phosphate, pH 7.2) by 1: 5 dilution. SEC-HPLC analysis was performed as described above to quantify the percentage of dimers and aggregates induced by low pH conditions. The IgG1 form of the anti-PD-1 monoclonal antibody 317-4B6 was most stable under bioprocessing-related acidic conditions, even when the pH value was as low as 2.7. Of the anti-PD-1 monoclonal antibodies made as several IgG4 variants, acid-induced aggregates are significant to levels comparable to IgG1 format anti-PD-1 monoclonal antibodies 317-4B6 and 326-4A3. Hu317-4B6 / IgG4mt10 and hu326-4A3 / IgG4mt10 were the most stable under acid buffer conditions (Table 21).

Example 11 Mapping of binding epitopes of anti-PD-1 monoclonal antibody

Previous reports on the crystal structure of the PD-1 / PD-L1 and PD-1 / PD-L2 complexes shed light on the understanding of the important amino acids (AA) on PD-1 required for ligand binding. .. References [Zhang et al. 2004 Immunity, 20: 337-347]; Ref. [Lin D. Y. et al. 2008 PNAS 105: 3011-3016]; and the literature [Lazar-Molnar E. et al. et al. 2008 PNAS, 105: 10483-1488]. In fact, these six AA amino acid residues were identified on the receptor by point mutation analysis required for PD-L1 binding. Five of the above six AA residues were also required for PD-LS binding. Literature [Lin D. Y. et al. 2008 PNAS 105: 3011-3016]. Based on information from structure-induced mutation analysis, we find that the most effective way for functional monoclonal antibodies to block PD-1-mediated signaling is to bind to 6 important AA residues. So, we hypothesized that it would compete with PD-1 ligands by occupying the binding epitopes required for ligand binding. To study the hypothesis and understand the mechanism of action by functional PD-1 antibodies, we individually put each of the six key AAs (ie, K45A, I93A, L95A, P97A, I101A, and E103A) into Ala. By substituting, 6 variants of PD-1 were prepared. Literature [Lin D. Y. et al. 2008 PNAS 105: 3011-3016 based on AA residue numbering]. Variants PD-1 / Fc and PD-1 / His (Fig. 1) are rolling circles using a PCR-guided mutagenesis or fast mutagenesis system (Cat. No. FM111, Transgen Biotech, Beijing, China). Used as a template for mutagenesis. All mutants were subcloned in our pcDNA-based expression vector and confirmed by sequencing. Mutant PD-1 and wild-type PD-1 proteins were expressed by transient transfection (described in Example 1) and prepared after 4-6 days of culture. The conditioned medium (CM) was analyzed by Western blot to verify expression of PD-1 protein in terms of quality and quantity. The supernatant (CM) was used directly in an ELISA analysis or Western blot for epitope mapping after clearing the cell debris.

To examine the binding epitope of humanized anti-PD-1 monoclonal antibody, ELISA assay using wild-type (WT) and mutant (Mt) PD-1 was performed, hu317-4B5, hu317-4B6, hu326-3B1. And the binding activity of hu326-4A3 was evaluated. Two reference antibodies (reference Ab-1 and reference Ab-2 from US8008449B2 and US8168757B2, respectively) were included in this study for comparison confirming the uniqueness of the antibody binding characteristics. In the same ELISA assay, 96-wells were coated with the same volume of CM containing WT or MtPD-1 for all monoclonal antibodies. All Elisa results were normalized using mean Elisa measurements of WT PD-1 binding signals as standard. The ELISA binding signal for specific Mt PD-1 was further standardized for the highest antibody binding readout (set as 100%) for specific Mt PD-1. For convenience of data analysis, amino acid residues are defined as important binding epitopes when the Elisa binding signal of a particular mutant monoclonal antibody falls below 50% relative to WT PD-1. .. That is because the mutation significantly eliminates antibody binding. Similarly, it is defined as very important when the Elisa binding signal of a particular mutant monoclonal antibody drops below 25%. As shown in FIG. Two of the amino acid residues important to PD-1, K45 and I93, are important or very important epitopes for binding the monoclonal antibodies hu317-4B5 and hu317-4B6, and the three AA residues, I93, L95 and P97 are important or very important epitopes for hu326-3B1 and hu326-4A3. On the other hand, the two reference antibodies have their own binding epitopes, with P97 being important for reference Ab-1, while L95 and P97 are important for reference Ab-2.

Interestingly, when the PD-1 protein was denatured on Western blots, important binding epitopes (K45 and I93) were not close to each other (non-linear) but were still able to bind to WT PD-1. This indicates that the PD-1 protein was denatured in SDS-PAGE of the Western blot process and then regenerated to some extent. This allows the anti-PD-1 monoclonal antibody to recognize and bind to it. Using this observation, we performed Western blot analysis on all six antibodies used in the ELISA study above. Overall results from Western blots well supported the ELISA results. That is, mutation of a significant or very important epitope gave a low binding signal in ELISA, but showed the weakest Western blot band compared to binding to other mutant PD-1 (Figure). 8). There were also some slight differences between ELISA and Western blots (eg, the ELISA binding signals on I93A and E103A by reference Ab-2 were relatively stronger than those on Western blots). This may indicate that these AA residues may contribute to binding as they affected binding even under stress conditions (ie, denaturing or losing natural structure) when mutated. unknown. As summarized in Table 22, in the present invention, the anti-PD-1 monoclonal antibody has a distinguishable binding epitope that is different from other anti-PD-1 antibodies.

Example 12 Anti-PD-1 monoclonal antibody activates primary human PBMCs and inhibits tumor growth in xenograft mouse models

Humanized anti-PD-1 monoclonal antibody activates human PBMC

Throughout the humanization process, humanized anti-PD-1 monoclonal antibodies at various stages retained similar functional activity as assessed by ELISA, FACS, and immune cell line cytokine release assays. To confirm the function of the final version of the humanized monoclonal antibody, we assayed the activation function of hu317-4B5, hu317-4B6, hu326-3B1 and hu326-4A3 using primary human PBMCs. The results show that through humanization, the degree of activation of these monoclonal antibodies varies among the four donors due to differences in individual genetic backgrounds, but the original mouse monoclonal antibody function that activates primary PBMCs. Demonstrated that it was maintained.

Human anti-PD-1 monoclonal antibody enhances NK cell line cytotoxicity against cancer cells

Humanized anti-PD-1 monoclonal antibodies from the original mouse monoclonal antibody, hu317-4B5, hu317-4B6, hu326-3B1 and hu326-3G1, were NK92MI / PD against the target lung cancer cells, SK-MES-1 / PD-L1. -1 Dose-dependent enhancement of cell-mediated cytotoxicity (Fig. 10, Table 23). In principle, humanized anti-PD-1 monoclonal antibodies disrupt immune cell tolerance mediated by PD-1 signaling and enhance cancer-killing activity by immune cells such as NK cells and cytotoxic T lymphocytes. It became clear that it could work to do so.

Human anti-PD-1 monoclonal antibody activates human PBMC in vivo and inhibits tumor growth in vivo in a mouse xenograft model.

All experimental evidence shows that the anti-PD-1 monoclonal antibody is a mouse cancer in which human PBMC is transplanted into immunodeficient mice xenografted with human cancer cells and treated with a monoclonal antibody to inhibit cancer cell growth in vivo. It has been shown that it can act in the model. The experiment was designed as follows. 7-8 week old SCID-male mice (Vital River Laboratories, China) in 50% matrigels (BD Biosciences, NJ, USA) with 3 × 10 ⁶ Hep3B / OS8-PD-L1 cells on the right flank Was inoculated subcutaneously. Fifteen days after tumor inoculation, mice with tumors 100-250 mm ³ in size were randomized and divided into three treatment groups. The two pooled from healthy donors PBMC (5 × ¹⁰ 5) 100μl was injected into the tumor. Three days after transplantation of PBMC, anti-PD-1 antibody (Hu317-IgG4mt2) and human IgG were administered subcutaneously at a dose of 10 mg / kg, respectively. The antibody treatment was repeated once every 10 days for a total of 3 times. PBS was injected into the parallel group as a negative control group. Tumors were measured with calipers twice a week from day 7. Tumor volume was calculated using the following formula: [D × (d ² )] / 2 (D represents the large diameter of the tumor and d represents the small diameter). All animal studies were performed according to the Beijing Animal Care and Use Procedure.

In the in vivo study, 60% of the tumors in the control group returned automatically, but the rest of the in vivo experiments were still quite informative, which is shown in Figure 11. Each control group (vehicle-treated group or human IgG (huIgG) treated group) showed 40% tumor growth (proliferation) from the baseline at the starting point (2 out of 5 mice). Two tumors in the PBS-treated group grew larger (> 2000 mm ^{3 and} one tumor mouse that exceeded the tumor size limit by protocol was terminated prematurely). Two tumors huIgG treated group, although smaller than the size of the PBS treated group, significantly above the average baseline 164 mm ^3, was grown to 800~1370mm ^3. On the other hand, in the anti-PD-1 monoclonal antibody (hu317-1 / IgG4mt2) treatment group, the tumors were completely regressed or close to baseline size (one tumor = 200 mm ³ , which is from PBMC transplantation). After returning to 50% of the baseline at 2 weeks, it slowly returned to its original state). This result indicates that the anti-PD-1 antibody described above activates human immune cells that inhibit tumor cell growth in a mouse in vivo cancer model, which is consistent with the results of the in vitro experiment described above. Is.

Claims (2)

A monoclonal antibody that binds to human PD-1
An antibody comprising a PD-1 binding domain and an IgG4 heavy chain effector or IgG4Fc region in which the constant region comprises SEQ ID NO: 85 or 88 . In patients who have undergone determination of having cancer or viral infection, an antibody of claim 1 for use in the treatment of cancer or viral infection.