Antibody variants with improved pharmacokinetic properties

Abstract

The present disclosure provides a variant of an antibody having modified net charge characteristics compared to a corresponding unmodified antibody. A specific variant has improved pharmacokinetic characteristics compared to a corresponding unmodified antibody. A specific antibody variant binds to CD40. A composition and a method of using the same are also provided.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 026,499 filed on May 18, 2020, the entire contents of which are incorporated herein for all purposes.

[0003] Sequence list

[0004] The present application contains a list of sequences submitted electronically in ASCII format, the full text of which is incorporated herein by reference. The said ASCII copy, created on May 16, 2021, has the filename 200896_0016_WO-_SL.txt and is 281,538 bytes in size.

[0005] field

[0006] The present disclosure provides a variant antibody having improved pharmacokinetic properties compared to a corresponding unmodified antibody. In some cases, the antibody polypeptide binds to CD40 and does not exhibit CD40 agonist activity. A composition comprising the antibody, a method of use for treating a disease involving CD40 activity, and a use in the manufacture of a medicine for treating a disease involving CD40 activity are provided. Background Technology

[0007] Biotherapeutic molecules are often the subject of modification experiments intended to increase therapeutic efficacy, disease exposure, and / or safety profiles. In the case of antibody therapeutic molecules, modifications may include humanization, PEGylation, glycosylation, and conjugation to molecules such as albumin.

[0008] Pharmacokinetics (PK) refers to the movement of a drug into, through, and out of the body. Pharmacokinetics evaluates the onset, duration, and intensity of a drug's effect.

[0009] The pharmacokinetics of antibody therapeutics can be influenced by a wide range of properties, including molecular size, folding stability, solubility, target interactions, neonatal Fc binding ability, and charge (e.g., see literature [Warnders et al., 2018, Med. Res. Rev. 38: 1837-1873; Leipold and Prabhu, 2019, Clin. Transl. Sci. 12: 130-139]). Modification of the charge of antibodies can affect charge-dependent interactions. For example, an increase in basic / positive charge on a protein (cationization) can increase off-target interactions with membranes and the extracellular matrix and tend to decrease pharmacokinetics, whereas anionization of proteins with basic charge patches generally improves PK. However, protein modifications intended to intentionally tune in vivo behavior can also lead to unintended and undesirable effects due to the interdependence of many of the protein's properties. Therefore, pursuing charge modifications of antibodies to modulate PK is not simple and requires intelligent protein design / manipulation and experiments to identify effective mutations.

[0010] CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor (TNF) receptor superfamily present on antigen-presenting cells (APCs), including dendritic cells, B cells, and macrophages. APCs [are] where CD40 is T H It is activated upon binding to its ligand, CD154 (CD40L), on the cell. CD40-mediated APC activation is associated with various immune responses, including cytokine production, upregulation of co-stimulatory molecules (e.g., CD86), and enhanced antigen presentation and B cell proliferation. CD40 can also be expressed by endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells.

[0011] CD40 activation is also associated with various undesirable T cell responses, for example, autoimmune, transplant rejection, or allergic reactions. One strategy for controlling undesirable T cell responses is to target CD40 with antagonistic antibodies, thereby inducing the development of several monoclonal anti-CD40 antibodies, such as the monoclonal antibody HCD122 (lucatumumab), previously known as Chiron 1212, and the fully human domain antibody BMS-986090 (U.S. Patent No. 9,475 879). Also, refer, for example, to WO 2018 / 217976 and WO 2018 / 217988.

[0012] The present disclosure provides a variant antibody having improved pharmacokinetic properties compared to a corresponding unmodified antibody. A method for increasing at least one pharmacokinetic property is also provided. The present disclosure further provides an anti-CD40 monoclonal antibody variant having similar or improved pharmacokinetic properties compared to a corresponding unmodified parent antibody. The present disclosure also provides a method for intelligently designing an antibody variant having similar or improved pharmacokinetics compared to a corresponding unmodified antibody.

[0013] An isolated antibody or its antigen-binding portion that specifically binds to human CD40 is provided, wherein the antibody comprises a heavy chain variable region (V H A first polypeptide portion comprising ) and a light chain variable region (V L It comprises a second polypeptide portion including ), wherein the heavy chain variable region and the light chain variable region are selected from the following:

[0014] (i) The above heavy chain variable region is HC1

[0015] Includes; and the light chain variable region is LC4

[0016] Includes or;

[0017] (ii) The above heavy chain variable region is HC1

[0018] Includes; and the light chain variable region is LC3

[0019] Includes or;

[0020] (iii) The above heavy chain variable region is HC15

[0021] Includes; and the light chain variable region is LC3

[0022] Includes or;

[0023] (iv) The above heavy chain variable region is HC4

[0024] Includes; and the light chain variable region is LC1

[0025] Includes or;

[0026] (v) The above heavy chain variable region is HC4

[0027] Includes; and the light chain variable region is LC3

[0028] Includes or;

[0029] or

[0030] (vi) The above heavy chain variable region is HC5

[0031] Includes; and the light chain variable region is LC4

[0032] Includes

[0033] The isolated antibody or its antigen-binding portion may comprise a first polypeptide portion comprising a human heavy chain constant region; and a second polypeptide portion comprising a human light chain constant region. The isolated antibody or its antigen-binding portion described herein may comprise (1) a mutation at kavat position 238 that reduces binding to the Fc-gamma-receptor (FcγR) (wherein proline 238 (P238) is mutated to one residue selected from the group consisting of lysine, serine, alanine, arginine, and tryptophan, so that the antibody or its antigen-binding portion has reduced FcγR binding); or (2) a human IgG1 Fc domain comprising alanine substituted at kavat position 297.

[0034] In some embodiments of the isolated antibody or its antigen-binding portion described herein, the first polypeptide portion comprises a heavy chain variable region and a heavy chain constant region, and the second polypeptide portion comprises a light chain variable region and a light chain constant region, wherein

[0035] (i) The above heavy chain variable region is HC1

[0036] Includes; and the light chain variable region is LC4

[0037] Includes or;

[0038] (ii) The above heavy chain variable region is HC1

[0039] Includes; and the light chain variable region is LC3

[0040] Includes or;

[0041] (iii) The above heavy chain variable region is HC15

[0042] Includes; and the light chain variable region is LC3

[0043] Includes or;

[0044] (iv) The above heavy chain variable region is HC4

[0045] Includes; and the light chain variable region is LC1

[0046] Includes or;

[0047] (v) The above heavy chain variable region is HC4

[0048] Includes; and the light chain variable region is LC3

[0049] Includes or;

[0050] or

[0051] (vi) The above heavy chain variable region is HC5

[0052] Includes; and the light chain variable region is LC4

[0053] Includes

[0054] The isolated antibody or its antigen-binding portion described herein may comprise a human IgG1 Fc domain comprising a mutation at kavat position 238 that reduces binding to the Fc-gamma-receptor (FcγR), wherein proline 238 (P238) is mutated to one of a residue selected from the group consisting of lysine, serine, alanine, arginine, and tryptophan, so that the antibody or antigen-binding portion has reduced FcγR binding. An exemplary antibody may have P238 mutated to lysine.

[0055] The isolated antibody or its antigen-binding portion described herein may comprise an Fc domain comprising an amino acid sequence selected from the following:

[0056]

[0057]

[0058] The isolated antibody or its antigen-binding portion may include a human IgG1 Fc domain comprising the amino acid sequence of sequence identification number: 22 or sequence identification number: 23.

[0059] The isolated antibody or its antigen-binding portion described herein may comprise a human IgG1 Fc domain comprising an alanine substituted at kavat position 297.

[0060] An isolated antibody or its antigen-binding portion as described herein may antagonize the activity of CD40. An isolated antibody or its antigen-binding portion as described herein may be a chimeric antibody. An isolated antibody or its antigen-binding portion as described herein may be a humanized antibody. An isolated antibody or its antigen-binding portion as described herein may include a human heavy chain constant region and a human light chain constant region.

[0061] The antibody or its antigen-binding portion disclosed herein may comprise an antigen-binding portion selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, diabody, and scFv-Fc. An exemplary isolated antibody or its antigen-binding portion as described herein is scFv-Fc.

[0062] The antibody or its antigen-binding portion disclosed herein may be linked to a therapeutic agent.

[0063] The antibody or its antigen-binding portion disclosed herein may be connected to a second functional moiety having a binding specificity different from that of the antibody or its antigen-binding portion.

[0064] The antibody or its antigen-binding portion disclosed herein may additionally include additional moiety.

[0065] A nucleic acid molecule encoding an isolated antibody or its antigen-binding portion is disclosed herein. An expression vector comprising the nucleic acid molecule is disclosed herein. Additionally, a cell transformed with an expression vector capable of expressing an isolated antibody or its antigen-binding portion as disclosed herein is disclosed. Additionally, a method for preparing an anti-human CD40 antibody or its antigen-binding portion is also disclosed, comprising the following:

[0066] a) expressing the antibody or its antigen-binding portion in cells transformed with an expression vector comprising a nucleic acid molecule encoding the isolated antibody or its antigen-binding portion disclosed herein; and

[0067] b) A step of isolating an antibody or its antigen-binding portion from a cell.

[0068] Additionally, a pharmaceutical composition is provided comprising a) the antibody or its antigen-binding portion disclosed herein; and b) a pharmaceutically acceptable carrier.

[0069] A method for treating or preventing an immune response in a subject is provided, comprising administering to the subject the antibody disclosed herein or its antigen-binding portion thereof. A method for treating or preventing an autoimmune or inflammatory disease in a subject is further provided, comprising administering to the subject the antibody or its antigen-binding portion thereof. Optionally, the antibody or its antigen-binding portion thereof may be administered together with an immunosuppressant / immunomodulator and / or anti-inflammatory agent. Administration may be simultaneous or sequential. An exemplary agent for co-administration is a CTLA4 mutant molecule, e.g., L104EA29Y-Ig (velatasept).

[0070] In this method for treating or preventing an immune response in a subject, and in this method for treating or preventing an autoimmune or inflammatory disease in a subject, preferably the subject has Addison's disease, allergy, anaphylaxis, ankylosing spondylitis, asthma, atherosclerotic arteriosclerosis, atopic allergy, autoimmune disease of the ear, autoimmune disease of the eye, autoimmune hepatitis, autoimmune mumps, bronchial asthma, coronary heart disease, Crohn's disease, diabetes mellitus, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, an immune response to a recombinant drug product (e.g., Factor VII in hemophilia), lupus nephritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, He has a disease selected from the group consisting of sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, transplant rejection, vasculitis, and ulcerative colitis.

[0071] Additionally, antibodies or their antigen-binding portions disclosed herein for use as medicine are considered. Furthermore, antibodies or their antigen-binding portions disclosed herein or medicines containing the same are considered for use in treating subjects requiring treatment. Additionally, a therapeutically effective amount of antibodies or their antigen-binding portions disclosed herein for use in treating or preventing an immune response is considered for administration to a patient requiring treatment or prevention of an immune response. Brief explanation of the drawing

[0072] Figure 1 shows a graph of the on-rate versus off-rate (isoaffinity) plot for hCD40 binding to a protein A-captured antibody. The x-axis is off-rate (kd), the y-axis is on-rate (ka); the graph is on a logarithmic scale. Wild type = Data for HC1 / LC1 antibodies. HC13 basic variants = Data for antibodies HC13 / LC1, HC13 / LC2, HC13 / LC3, HC13 / LC4, HC13 / LC5, and HC13 / LC6. HC11 patch 1 = Data for antibodies HC11 / LC1, HC11 / LC2, HC11 / LC3, HC11 / LC4, HC11 / LC5, and HC11 / LC6. HC12 Patch 1 = Data for antibodies HC12 / LC1, HC12 / LC2, HC12 / LC3, HC12 / LC4, HC12 / LC5 and HC12 / LC6. Figure 2 shows the presence of soluble human IL-4 (+ IL-4 20 ng / ml) or the absence of IL-4 (medium). 3 Data regarding the efficacy of BMS-986325 and its variants on human B cell proliferation, measured by H thymidine incorporation, are presented. These data were obtained using human B cells from donor NABVHJ-OC2PVS. Figure 3 shows the presence of IL-4 (+ IL-4 20 ng / ml) or the absence of IL-4 (medium). 3 Data regarding the efficacy of BMS-986325 and its variants on human B cell proliferation, measured by H thymidine incorporation, are presented. These data were obtained using human B cells from donor NABZWC-06906T. Figure 4 shows the presence of IL-4 (+ IL-4 20 ng / ml) or the absence of IL-4 (medium). 3 Data regarding the efficacy of BMS-986325 and its variants on human B cell proliferation, measured by H. thymidine incorporation, are presented. These data were obtained using human B cells from donor NABZWC-069062. Figure 5 shows data regarding BMS-986325 and its variants for human B cell IL-6 secretion under medium or + IL-4 (+ IL-4 20 ng / ml) using human B cells from donor NABVHJ-OC2PVS. Figure 6 shows data regarding BMS-986325 and its variants on human B cell IL-6 secretion under medium or + IL-4 (+ IL-4 20 ng / ml) using human B cells from donor NABZWC-06906T. Figure 7 shows data regarding BMS-986325 and its variants on human B cell IL-6 secretion under medium or + IL-4 (+ IL-4 20 ng / ml) using human B cells from donor NABZWC-069062. Figure 8 shows data on the single-dose pharmacokinetics (PK) of BMS-986325 and its variants upon intravenous administration of 1 mg / kg in C57 / BL6 mice. Specific details for implementing the invention

[0073] The present disclosure provides a variant antibody having improved pharmacokinetic properties compared to a corresponding unmodified antibody. As presented herein, specific sites or locations of mutations for modifying surface charge patches have been found to be important for improving antibody PK. This finding is unexpected, as prior art suggested that merely modifying the total antibody charge is necessary to achieve PK modification. Advantageously, in some cases, a variant having mutations at only one or two strategic locations with a small change in total charge, e.g. -2 or -3, has an equivalent or improved PK compared to a variant with multiple mutations and a larger change in charge, e.g. -8.

[0074] The present disclosure further provides a variant antibody having improved pharmacokinetic properties compared to a corresponding unmodified antibody as a variant of an antibody that binds to CD40. The antibody polypeptide binds to CD40 and does not exhibit CD40 agonist activity. A composition comprising the antibody, a method of use for treating a disease involving CD40 activity, and a use in the manufacture of a medicine for treating a disease involving CD40 activity are provided.

[0075] The variant antibody of the present disclosure was identified by the method described in Example 1.

[0076] Definitions & Abbreviations

[0077] Additional abbreviations and definitions are provided below.

[0078] APC antigen-presenting cells

[0079] Area under the AUC curve

[0080] BSA bovine serum albumin

[0081] CD54 is also referred to as ICAM-1.

[0082] CDR Complementarity Determination Region

[0083] C H or CH invariant heavy chain

[0084] C L or CL invariant light chain

[0085] CHO cells Chinese hamster ovary cells

[0086] DC dendritic cells

[0087] FcgR is interchangeable with FcγR

[0088] FcγR Fc-gamma-receptor

[0089] FR Framework Area

[0090] GM-CSF Granulocyte Macrophage Colony Stimulating Factor

[0091] HC heavy chain

[0092] ICAM-1 intracellular adhesion molecule 1

[0093] iDC immature dendritic cells

[0094] IFN interferon

[0095] IgG Immunoglobulin G

[0096] IL-6 Interleukin-6

[0097] LC light chain

[0098] mAb monoclonal antibody

[0099] mg milligrams

[0100] ml or mL milliliters

[0101] ng nanogram

[0102] nM nanomoles

[0103] pI isoelectric point

[0104] SPR Surface Plasmon Resonance

[0105] TNF Tumor Necrosis Factor

[0106] μg micrograms

[0107] μM micromolar

[0108] V L or VL or Vl variable light chain domain

[0109] Vκ or Vk or VK kappa variable light chain domain

[0110] Vλ lambda variable light chain domain

[0111] V H or VH or Vh variable heavy chain domain

[0112] In accordance with this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms used herein include plural subjects unless the context clearly indicates otherwise. Thus, for example, a reference to "antibody" includes plural such antibodies, and a reference to "dosage" includes one or more doses and equivalents thereof known to a person skilled in the art.

[0113] The term “about” as used herein is understood by a person skilled in the art and may vary to some extent depending on the context in which it is used. Generally, “about” encompasses a range of values ​​plus or minus 10% of a reference value unless otherwise indicated in the specification.

[0114] Any and all whole or partial integers between the presented ranges are understood to be included herein.

[0115] CD40 is also known as B-cell surface antigen CD40, Bp50, CD40L receptor, CDw40, CDW40, MGC9013, p50, TNFRSF5, and tumor necrosis factor (TNF) receptor superfamily member 5. "Human CD40" refers to CD40 containing the following amino acid sequence:

[0116]

[0117] As used herein, the term "variable domain" refers to the immunoglobulin variable domain defined in the literature [Kabat et al., Sequences of Immunological Interest, 5th ed., US Dept. Health & Human Services, Washington, DC (1991)]. The numbering and positioning of CDR amino acid residues within the variable domain follow the widely known Kabat numbering convention. VH, "variable heavy chain," and "variable heavy chain domain" refer to the variable domain of the heavy chain. VL, "variable light chain," and "variable light chain domain" refer to the variable domain of the light chain.

[0118] The term “human” means that, when applied to an antibody, the antibody has a sequence derived from human immunoglobulin, e.g., FR and / or CH domains. The sequence is derived from “human immunoglobulin coding sequences” where the sequence is (a) isolated from a human individual or from a cell or cell line from a human individual; (b) isolated from a library of cloned human antibody gene sequences or human antibody variable domain sequences; or (c) diversified by mutation and selection from one or more of the polypeptides.

[0119] The term "isolated" compound as used herein means that the compound is isolated from at least one component that is naturally associated with the compound in nature.

[0120] The antibody of the present disclosure, e.g., an anti-CD40 antibody, comprises a variable heavy chain and a variable light chain, each of which contains three complementary determining regions (CDRs) and four framework regions (FRs) arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs contain most of the residues that form specific interactions with the antigen and are primarily responsible for antigen recognition.

[0121] Pharmacokinetics (PK) refers to the movement of a drug into, through, and out of the body; PK evaluates the absorption, distribution, metabolism, and elimination of the drug from the body. Parameters for evaluating pharmacokinetics include AUC 0-inf (μM·h), T-half (h), MRT (h), CL (mL / h / kg), and Vss (L / kg).

[0122]

[0123] PK parameters can be evaluated by the method described herein.

[0124] As used herein, “improved pharmacokinetic properties” means that at least one PK parameter for the antibody variant is increased (in the case of AUC, T-half, and MRT) or decreased (in the case of CL and Vss) compared to the same PK parameter measured in the corresponding non-modified antibody. In the embodiments, the antibody variant has improved pharmacokinetic properties in at least two, at least three, at least four, or at least five PK parameters compared to the same PK parameter in the corresponding non-modified antibody. As used herein, improved pharmacokinetic properties refer to pharmacokinetic properties of the variant antibody that are at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 100% greater than the same pharmacokinetic properties of the corresponding non-modified antibody.

[0125] An exemplary anti-CD40 antibody of the present disclosure is a variant of the humanized antibody BMS-986325 (also referred to as Y12XX-hz28). An overview of the amino acid sequences of the heavy chain variable region and the light chain variable region of BMS-986325 is provided in Table 1.

[0126] Table 1

[0127]

[0128] Details of the amino acid sequence of BMS-986325 are provided in Table 2.

[0129] Table 2: BMS-986325: Y12XX-hz28 sequence

[0130]

[0131]

[0132] The anti-CD40 variant antibody of the present disclosure has at least one specific anionizing mutation within the variable domain relative to at least the corresponding framework region within BMS-986325. The anionizing mutation is a mutation of a lysine (Lys; K) or arginine (Arg; R) residue located in the framework region of the variable chain, and in some variants, located in the CDR. Generally, the specific lysine and arginine residues may be mutated into uncharged residues, such as glutamine (Gln; Q) or asparagine (Asn; N), or into negatively charged (acidic) residues, such as glutamate (Glu; E) or aspartate (Asp; D). To avoid potential deamidation or isomerization, mutations to Gln and Glu are prioritized over mutations to Asn and Asp, respectively, thereby avoiding potential deamidation (Asn) or isomerization (Asp) problems common to the shorter Asn and Asp side chains. The disclosed variant has an improved PK associated with BMS-986325.

[0133] Combinations of heavy chain variable region and light chain variable region sequences of variants of BMS-986325 disclosed herein are provided in Tables 3-8. These combinations each have a change in the net charge of the variable region compared to BMS-986325. Specifically, each combination has a decrease in the net positive charge, except for the combination containing HC13. The variants bind to human CD40 with a KD value similar to that of BMS-986325 or up to about 4 times higher (as measured by hCD40 binding to BMS-986325 and BMS-986325 variant antibodies captured from the supernatant).

[0134] Table 3 includes various combinations of heavy chain variable region sequences and light chain variable region LC1.

[0135] Table 3

[0136]

[0137]

[0138] Table 4 includes various combinations of heavy chain variable region sequences and light chain variable region LC2.

[0139] Table 4

[0140]

[0141]

[0142] Table 5 includes various combinations of heavy chain variable region sequences and light chain variable region LC3.

[0143] Table 5

[0144]

[0145]

[0146] Table 6 includes various combinations of heavy chain variable region sequences and light chain variable region LC4.

[0147] Table 6

[0148]

[0149]

[0150] Table 7 includes various combinations of heavy chain variable region sequences and light chain variable region LC5.

[0151] Table 7

[0152]

[0153]

[0154] Table 8 includes various combinations of heavy chain variable region sequences and light chain variable region LC6.

[0155] Table 8

[0156]

[0157]

[0158] The heavy chain variable region and light chain variable region sequences of an exemplary variant of BMS-986325 with an improved PK are provided in Table 9.

[0159] Table 9

[0160]

[0161]

[0162] An exemplary CD40 antibody of the present disclosure may comprise an isolated antibody or its antigen-binding portion that specifically binds to human CD40, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, and wherein

[0163] (i) The above heavy chain variable region is HC1

[0164] Includes; and the light chain variable region is LC4

[0165] Includes or;

[0166] (ii) The above heavy chain variable region is HC1

[0167] Includes; and the light chain variable region is LC3

[0168] Includes or;

[0169] (iii) The above heavy chain variable region is HC15

[0170] Includes; and the light chain variable region is LC3

[0171] Includes or;

[0172] (iv) The above heavy chain variable region is HC4

[0173] Includes; and the light chain variable region is LC1

[0174] Includes or;

[0175] (v) The above heavy chain variable region is HC4

[0176] Includes; and the light chain variable region is LC3

[0177] Includes or;

[0178] or

[0179] (vi) The above heavy chain variable region is HC5

[0180] Includes; and the light chain variable region is LC4

[0181] Includes

[0182] An exemplary CD40 antibody of the present disclosure may comprise an isolated antibody or its antigen-binding portion that specifically binds to human CD40, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, and wherein

[0183] (i) The above heavy chain variable region is HC1

[0184] Includes; and the light chain variable region is LC3

[0185] Includes or;

[0186] or

[0187] (ii) The above heavy chain variable region is HC15

[0188] Includes; and the light chain variable region is LC3

[0189] These two exemplary antibodies have the fewest mutations and also have a particularly advantageous combination of characteristics, including at least one improved PK parameter.

[0190] The “antibody” (Ab) will, without limitation, comprise an immunoglobulin or its antigen-binding portion comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, which specifically bind to an antigen. Each H chain comprises a heavy chain variable region (V herein). H It includes the heavy chain invariant region (abbreviated as ). The heavy chain invariant region consists of three invariant domains C H1 , C H2 and C H3 Each light chain includes a light chain variable region (V in this application). L It includes (abbreviated as ) and a light chain invariant region. The light chain invariant region consists of one invariant domain C L Includes. V H and V L The region can be further subdivided into a hypervariable region called the Complementarity Decision Region (CDR), which is interposed with a more conserved region called the Framework Region (FR). Each V H and V L It contains three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0191] The “antigen-binding portion” of Ab (also referred to as an “antigen-binding fragment”) or its antigen-binding portion refers to one or more sequences of Ab (the full length or fragment of a full-length antibody) that possess the ability to specifically bind to the antigen to which the whole Ab binds. Examples of antigen-binding fragments include Fab, F(ab')2, scFv (single-chain variable fragment), Fab', dsFv, sc(Fv)2, and scFv-Fc.

[0192] "Humanized" antibodies refer to antibodies in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulin. In one embodiment of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids from human immunoglobulin, whereas some, most, or all of the amino acids within one or more of the CDR domains remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies retain antigen specificity similar to that of the original antibody.

[0193] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0194] The "specific binding" as used herein refers to a dissociation constant (K) of about 1 μM or less when measured, for example, by surface plasmon resonance (SPR). d It refers to the binding of an antibody to an antigen. Suitable assay systems include the BIAcore™ (GE Healthcare Life Sciences, Marlborough, Massachusetts) surface plasmon resonance system and BIAcore™ kinetics evaluation software (e.g., version 2.1).

[0195] The binding of the antibody of the present invention to CD40 antagonizes at least one type of CD40 activity. "CD40 activity" includes, but is not limited to, T cell activation (e.g., induction of T cell proliferation or cytokine secretion), macrophage activation (e.g., induction of reactive oxygen species and nitric oxide in macrophages), and B cell activation (e.g., B cell proliferation, antibody isomorphism conversion, or differentiation into plasma cells). CD40 activity may be mediated by interactions with other molecules. "CD40 activity" includes functional interactions between CD40 and the following molecules (identified by the Uniprot accession number in parentheses).

[0196] CALR (P27797);

[0197] ERP44 (Q9BS26);

[0198] FBL (P22087);

[0199] POLR2H (P52434);

[0200] RFC5 (P40937);

[0201] SGK1 (O00141);

[0202] SLC30A7 (Q8NEW0);

[0203] SLC39A7 (Q92504);

[0204] TRAF2 (Q5T1L5);

[0205] TRAF3 (Q13114);

[0206] TRAF6 (Q9Y4K3);

[0207] TXN (Q5T937);

[0208] UGGT1 (Q9NYU2); and

[0209] USP15 (Q9Y4E8).

[0210] For example, CD40 "activation" involves interaction with TRAF2. CD40 / TRAF2 interaction activates NF-κB and JNK. See literature [Davies et al., Mol. Cell Biol. 25: 9806-19 (2005)]. Therefore, this CD40 activity can be determined by CD40-dependent cellular NF-κB and JNK activation compared to the reference.

[0211] As used herein, the terms “activate,” “activate,” and “activated” refer to a given measurable CD40 activity increased by at least 10% compared to the reference, e.g., at least 10%, 25%, 50%, 75%, or even at least 100% or more. CD40 activity is “antagonized” when CD40 activity is reduced by at least 10% compared to the absence of an antagonist, or in exemplary embodiments, by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or even 100% (i.e., no detectable activity). For example, the antibody may antagonize some or all of CD40 activity without activating CD40. For example, the antibody may not activate B cell proliferation. The antibody may not activate cytokine secretion by T cells, where the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-6, IL-10, IL-13, TNF-α and IFN-γ.

[0212] The isolated antibody or its antigen-binding portion may antagonize one or more activities of CD40. The isolated antibody or its antigen-binding portion may be a chimeric antibody. The isolated antibody or its antigen-binding portion may be a humanized antibody. The isolated antibody or its antigen-binding portion may include a human heavy chain constant region and a human light chain constant region.

[0213] In certain aspects, the present disclosure describes variant framework regions (FRs) of variable domains and, in some cases, CDRs, wherein specific positions having basic amino acids are mutated to neutral or acidic amino acids. The disclosed variant FRs may be variants of the framework region encoded by human wiring antibody gene fragments, such as the VH1 heavy chain wiring and VK1 light chain wiring, or variants of modified FRs of human wiring antibody gene fragments, such as variants arising from mutagenic affinity maturation of antibody libraries. A preferred framework sequence for use with the antibodies described herein is structurally similar to the framework sequence used by the anti-CD40 antibody described herein. V of any antibody H CDR1, 2, and 3 sequences and V L The CDR1, 2, and 3 sequences are considered to be graftable onto the framework region disclosed herein to improve one or more PK parameters. Additionally, as described herein, specific positions within the CDR having basic amino acids are also considered to be modified.

[0214] Accordingly, the present disclosure considers monoclonal antibody variants having similar or improved pharmacokinetic properties compared to the corresponding unmodified parent antibody. The parent antibody is an amino acid sequence

[0215] A first polypeptide portion comprising a heavy chain variable region having; and an amino acid sequence

[0216] It comprises a second polypeptide portion comprising a light chain variable region having. In sequence identification number: 75, position 108 is the first amino acid of the constant region (CL). Position 108 may be a basic amino acid, e.g., arginine as presented in sequence identification number: 75.

[0217] The antibody variant comprises at least one anionization mutation in a basic residue. The heavy chain variable region of the variant may comprise a mutation at at least one position having a basic residue within the parent antibody, which is at least one position selected from the group consisting of at least one position of SEQ ID No. 73, 12, 13, 19, 23, 38, 57, 63, 67, and 74 and combinations thereof. The heavy chain variable region of the variant may comprise at least one mutation as a position selected from the group consisting of K12, K13, K19, K23, R38, R57, K63, R67, and R74 and combinations thereof. The mutation may replace a basic amino acid with a neutral amino acid or an acidic amino acid. Exemplary neutral amino acids include glutamine, asparagine, valine, serine, alanine, and threonine. Exemplary acidic amino acids include glutamate and aspartate. Combinations of mutations may occur at two or more of positions 12, 13, 19, 23, 38, 57, 63, 67, and 74 of sequence identification number: 73. Examples include, but are not limited to, mutations at positions 12 and 13; mutations at positions 12, 13, and 23; mutations at positions 38, 63, and 67; mutations at positions 63 and 67; and mutations at positions 57 and 74. In some variant antibodies, examples of combinations include, but are not limited to, mutations at K12 and K13Q; mutations at K12, K13, and K23; mutations at R38, K63, and R67; mutations at K63 and R67; and mutations at R57 and K74. Exemplary combinations of mutations include K12Q and K13Q; K12Q, K13Q, and K23Q; Includes K12E, K13Q and K23E; K12V, K19S and K23A; R38Q, K63Q and R67Q; K63Q and R67E; and R57E and K74Q.

[0218] Heavy chain variable region - Vh wiring

[0219]

[0220] The light chain variable region of the variant may comprise a mutation at at least one position having a basic residue in the parent antibody, which is at least one position selected from the group consisting of 45, 54, 61, and 107 of sequence identification number: 74 and combinations thereof, or at least one position selected from the group consisting of 45, 54, 61, 107, and 108 of sequence identification number: 75 and combinations thereof. The light chain variable region of the variant may comprise a mutation at at least one position selected from the group consisting of K45, R54, R61, K107, and R108 if present, and combinations thereof. The mutation may replace a basic amino acid with a neutral amino acid or an acidic amino acid. Exemplary neutral amino acids include glutamine (E), asparagine (N), valine (V), serine (S), alanine (A), and threonine (T). In some cases, the neutral amino acid is glutamine. Exemplary acidic amino acids include glutamate (E) and aspartate (E). In some cases, the acidic amino acid is glutamate. Combinations of mutations may occur at two or more of positions 45, 54, 61, and 107 of SEQ ID NO: 74 or positions 45, 54, 61, 107, and 108 of SEQ ID NO: 75. Examples include, but are not limited to, mutations at positions 45, 54, and 61; or mutations at positions 107 and 108. In some variant antibodies, examples of combinations include, but are not limited to, K45, R54, and R61; and K107 and K108. Exemplary combinations of mutations include K45Q, R54Q, and R61Q; and K107Q and K108Q.

[0221] Light chain variable area - Vk wiring

[0222]

[0223] The present disclosure further provides a method for improving at least one pharmacokinetic property of a parent antibody. The method comprises mutating a residue at at least one position selected from 12, 13, 19, 23, 38, 57, 63, 67 and 74 of SEQ ID NO: 73 and / or 45, 54, 61 and 107 of SEQ ID NO: 74 to produce a variant having at least one improved pharmacokinetic property compared to an unmodified parent antibody. In some cases, the method comprises mutating a residue at at least one position selected from 12, 13, 19, 23, 38, 57, 63, 67 and 74 of sequence identification number: 73 and / or 45, 54, 61, 107 and 108 of sequence identification number: 75 to produce a variant having at least one improved pharmacokinetic property compared to the non-modified parent antibody.

[0224] In the practice of the method, the mutation may be a neutral amino acid or an acidic amino acid. Exemplary neutral amino acids include glutamine, asparagine, valine, serine, alanine, and threonine. Exemplary acidic amino acids include glutamate and aspartate.

[0225] Combinations of mutations may occur at two or more residues of positions 45, 54, 61, and 107 of sequence identification number: 74 and combinations thereof, or 45, 54, 61, 107, and 108 of sequence identification number: 75 and combinations thereof. Examples include, but are not limited to, mutations at positions 45, 54, and 61 of sequence identification number: 74; or mutations at 107 and 108 of sequence identification number: 75.

[0226] Combinations of mutations may occur at two or more of positions 12, 13, 19, 23, 38, 57, 63, 67 and 74 of sequence identification number: 73 and combinations thereof. Examples include, but are not limited to, mutations at positions 12 and 13; positions 12, 13 and 23; positions 38, 63 and 67; positions 63 and 67 and positions 57 and 74.

[0227] The improved pharmacokinetic properties obtained by the method may be the area under the concentration-time curve from time 0 to infinity (AUC 0-inf (uM.h)), half-life (T-half (h)), mean retention time (MRT (h)), clearance (CL (mL / h / kg)), and volume of distribution at steady state (Vss (L / kg)).

[0228] Exemplary combinations of variant framework regions are provided in Table 10.

[0229] Table 10

[0230]

[0231]

[0232] The antibody considered in the present disclosure may comprise an isolated antibody that specifically binds to an antigen or its antigen-binding portion, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, and wherein

[0233] (i) The above heavy chain variable region is the HC1 framework

[0234] Includes; and the light chain variable region is an LC4 framework

[0235] Includes or;

[0236] (ii) The above heavy chain variable region is the HC1 framework

[0237] Includes; and the light chain variable region is an LC3 framework

[0238] Includes or;

[0239] (iii) The above heavy chain variable region is the HC15 framework

[0240] Includes; and the light chain variable region is an LC3 framework

[0241] Includes or;

[0242] (iv) The above heavy chain variable region is the HC4 framework

[0243] Includes; and the light chain variable region is an LC1 framework

[0244] Includes or;

[0245] (v) The above heavy chain variable region is the HC4 framework

[0246] Includes; and the light chain variable region is an LC3 framework

[0247] Includes or;

[0248] or

[0249] (vi) The above heavy chain variable region is the HC5 framework

[0250] Includes; and the light chain variable region is an LC4 framework

[0251] Includes

[0252] The antibody considered in the present disclosure may comprise an isolated antibody that specifically binds to an antigen or its antigen-binding portion, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, wherein the heavy chain variable region is an HC1 framework

[0253] Includes; and the light chain variable region is an LC3 framework

[0254] Includes. The antibody considered in the present disclosure may include an isolated antibody that specifically binds to an antigen or its antigen-binding portion, wherein the antibody includes a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, wherein the heavy chain variable region is the HC15 framework

[0255] Includes; and the light chain variable region is an LC3 framework

[0256] Includes

[0257] Fc domain and invariant region

[0258] The carboxyl-terminal "half" of the heavy chain defines an invariant region (Fc), which primarily functions as an effector. As used herein, the term "Fc domain" refers to the literature [Kabat et al., Sequences of Immunological Interest, 5 th[Refers to an antibody sequence comprising CH2 and CH3 constant domains defined according to [ed., US Dept. Health & Human Services, Washington, DC (1991)]. The Fc domain may be derived from human IgG. For example, the Fc domain may be derived from human IgG1 or human IgG4 Fc domains. A heavy chain variable domain may be fused to the Fc domain. The carboxyl terminus of the variable domain may be connected or fused to the amino terminus of the Fc CH2 domain. Alternatively, the carboxyl terminus of the variable domain may be connected or fused to the amino terminus of a linker amino acid sequence that is fused to the amino terminus of the Fc domain. Alternatively, the carboxyl terminus of the variable domain may be connected or fused to the amino terminus of a CH1 domain that is fused to the Fc CH2 domain. Optionally, the protein may include a hinge region after the CH1 domain wholly or partially. Optionally, an amino acid linker sequence exists between the variable domain and the Fc domain. The carboxyl terminus of the light chain variable domain can be connected to or fused to the amino terminus of the CL domain.

[0259] An exemplary sequence for heavy chain CH1 is sequence identification number: 82

[0260] These are amino acids 118–215. An exemplary sequence for the light chain CL is sequence identification number: 83

[0261] The amino acids are 108-214.

[0262] Exemplary heavy chain variable region and light chain variable region sequences of an exemplary variant of BMS-986325 having an improved PK are provided in Table 11. In these sequences, the heavy chain contains an exemplary CH1 domain, and the light chain contains an exemplary CL domain.

[0263] Table 11

[0264]

[0265]

[0266] The antibody may be a fusion antibody comprising a first variable domain that specifically binds to human CD40 and a second domain including an Fc domain.

[0267] An exemplary Fc domain used in the fusion protein may include a human IgG domain. An exemplary human IgG Fc domain includes an IgG4 Fc domain and an IgG1 Fc domain. Human IgG heavy chain genes code for C-terminal lysine, but lysine is often absent from endogenous antibodies as a result of cleavage during blood circulation. When antibodies having an IgG heavy chain containing C-terminal lysine are expressed in mammalian cell cultures, variable levels of C-terminal lysine may also be present (Cai et al., 2011, Biotechnol. Bioeng. 108(2): 404-12). Therefore, the C-terminal lysine of any IgG heavy chain Fc domain disclosed herein may be omitted.

[0268] The isolated antibody or its antigen-binding portion described herein may comprise an Fc domain comprising the following amino acid sequence:

[0269] Parentheses indicate the possible amino acid identity at that position. For example, kavat position 238 can be proline (P) or lysine (K), which is denoted as (P / K). Additional exemplary non-restrictive consensus sequences are sequence identification numbers: 118-120:

[0270]

[0271] The isolated antibody or its antigen-binding portion described herein may comprise a human IgG1 Fc domain comprising a mutation at kavat position 238 that reduces binding to the Fc-gamma-receptor (FcγR), wherein proline 238 (P238) is mutated to one of a residue selected from the group consisting of lysine (K), serine (S), alanine (A), arginine (R), and tryptophan (W), so that the antibody or its antigen-binding portion has reduced FcγR binding. The isolated antibody or its antigen-binding portion described herein may have P238 mutated to lysine in the human IgG1 Fc domain.

[0272] The isolated antibody or its antigen-binding portion comprises an Fc domain comprising an amino acid sequence selected from sequence identification number: 22-29.

[0273]

[0274]

[0275] The exemplary sequence containing the IgG1 Fc domain above includes four different VH chain sequences presented in Table 12.

[0276] The isolated antibody or its antigen-binding portion described herein may comprise a human IgG1 Fc domain comprising alanine substituted at kavat position 297. For example, the isolated antibody or its antigen-binding portion comprises an Fc domain comprising an amino acid sequence selected from sequence identification numbers: 141-148.

[0277] Exemplary heavy chain variable region and light chain variable region sequences of an exemplary variant of BMS-986325 having improved PK are provided in Table 12. In these sequences, the heavy chain comprises an exemplary CH1 domain and a human IgG1 C domain comprising a mutation at kavat position 238 that reduces binding to the Fc-gamma-receptor (FcγR), wherein proline 238 (P238) is mutated to one residue selected from the group consisting of lysine (K). The light chain comprises an exemplary CL domain.

[0278] Table 12

[0279]

[0280]

[0281]

[0282] The antigen-binding portion thereof of any antibody disclosed herein may be selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, diabody, and scFv-Fc.

[0283] The antibody or its antigen-binding portion disclosed herein may be an immunoconjugate, wherein the antibody or its antigen-binding portion is linked to a therapeutic agent.

[0284] The antibody or its antigen-binding portion disclosed herein may be a bispecific antibody, wherein the antibody or its antigen-binding portion is connected to a second functional moiety having a binding specificity different from that of the antibody or its antigen-binding portion.

[0285] The antibody or its antigen-binding portion disclosed herein may additionally include additional moiety.

[0286] The variable region of the antibody of the present invention may optionally be connected to the Fc domain by an "amino acid linker" or a "linker." For example, the C-terminus of the variable heavy chain domain may be fused to the N-terminus of the amino acid linker, and the Fc domain may be fused to the C-terminus of the linker. The amino acid linker may be of any length and may consist of any combination of amino acids, but the linker length may be relatively short to reduce interactions between the linked domains (e.g., 5 or fewer amino acids). The amino acid composition of the linker may also be adjusted to reduce the number of amino acids having bulky side chains or amino acids that may introduce a secondary structure. Suitable amino acid linkers include, but are not limited to, having a length of 3, 4, 5, 6, 7, 10, 15, 20, or 25 or fewer amino acids. Representative amino acid linker sequences include GGGGS (sequence identification number: 92) and linkers containing 2, 3, 4, or 5 copies of GGGGS (sequence identification numbers: 93 to 96, respectively). Table 13 lists linker sequences suitable for use in the present disclosure.

[0287] Table 13

[0288] Representative linker sequences

[0289]

[0290] Antibody production

[0291] Antibodies are produced and purified using conventional techniques in suitable mammalian host cell lines, such as CHO, 293, COS, NSO, etc., and then purified using one or a combination of methods including protein A affinity chromatography, ion exchange, reverse phase techniques, etc.

[0292] As is widely known in the relevant art, multiple codons can code for the same amino acid. Therefore, nucleic acids coding for protein sequences include nucleic acids having codon axiom. The polypeptide sequence disclosed herein can be coded by various nucleic acids. Genetic codes are universal and widely known. Nucleic acids coding for any polypeptide sequence disclosed herein can be easily devised and optimized for production based on ordinary knowledge in the relevant art. While there are many possible nucleic acid sequences coding for a given polypeptide when considering standard genetic code tables and using a computer as an aid, a person skilled in the art can easily generate all possible combinations of nucleic acid sequences coding for a given polypeptide.

[0293] Representative nucleic acid sequences encoding four of the heavy chain variable domains are provided below. In these sequences, nucleotides 1-351 code for the heavy chain variable region, where nucleotides 91-105 code for CDR1 of the heavy chain variable domain, nucleotides 148-195 code for CDR2, and nucleotides 295-318 code for CDR3. Nucleotides 352-645 code for the CH1 domain, and nucleotides 646-1341 code for IgG1-P238K. Nucleotides 1342-1344 are stop codons.

[0294] Representative nucleic acid sequences encoding the heavy chain variable domains (HC1, i.e., HC-wt) of M49 and M33 (CDR is underlined), including the constant region CH1 (italicized) and the Fc domain IgG1-P238K, are as follows:

[0295]

[0296] Representative nucleic acid sequences encoding the heavy chain variable domain (HC-15) of M47, including the constant region CH1 and Fc domain IgG1-P238K, are as follows:

[0297]

[0298] Representative nucleic acid sequences encoding the constant region CH1 and Fc domain IgG1-P238K, as well as the heavy chain variable domain (HC-4) of M4 and M36, are as follows:

[0299]

[0300] Representative nucleic acid sequences encoding the heavy chain variable domain (HC-5) of M53, including the constant region CH1 and Fc domain IgG1-P238K, are as follows:

[0301]

[0302] Representative nucleic acid sequences encoding three of the light chain variable domains are provided below. In these sequences, nucleotides 1-321 code for the light chain variable region, where nucleotides 70-102 code for CDR1, nucleotides 148-168 code for CDR2, and nucleotides 265-291 code for CDR3. Nucleotides 322-642 code for CL. Nucleotides 643-645 are stop codons.

[0303] Representative nucleic acid sequences encoding the light chain variable domain (LC4) of M49 and M53 (CDR is underlined), including the invariant region CL (italicized), are as follows:

[0304]

[0305] Representative nucleic acid sequences encoding the light chain variable domain (LC3) of M33, M47, and M36, including the invariant region CL, are as follows:

[0306]

[0307] Representative nucleic acid sequences encoding the M4 light chain variable domain (LC1, i.e., LC-wt), including the invariant region CL, are as follows:

[0308]

[0309] Exemplary coding sequences are summarized in Table 14 below. The sequences are provided in the sequence list.

[0310] Table 14

[0311]

[0312] The coding sequence for the heavy chain and / or light chain may optionally code for a signal peptide, e.g., MRAWIFFLLCLAGRALA (Sequence ID: 51), at the 5' end of the coding sequence. An exemplary nucleic acid coding sequence for such a signal peptide is

[0313] am.

[0314] Accordingly, nucleic acids encoding the antibodies disclosed herein are also considered. Such nucleic acids may be inserted into a vector, e.g., a suitable expression vector, e.g., pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137). Isolated host cells containing the vector and / or nucleic acids are further provided.

[0315] The antibodies of the present disclosure may be produced and purified using only ordinary techniques in any suitable mammalian host cell line, such as CHO (Chinese hamster ovary cells), 293 (human embryonic kidney 293 cells), COS cells, NSO cells, etc., and then purified using one or a combination of methods including protein A affinity chromatography, ion exchange, reverse phase techniques, etc.

[0316] Pharmaceutical composition and treatment method

[0317] The pharmaceutical composition comprises a therapeutically effective amount of one or more antibodies of the present disclosure and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, saline solution, phosphate-buffered saline solution, dextrose, glycerol, ethanol, etc., as well as combinations thereof. The pharmaceutically acceptable carrier may further comprise trace amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, to enhance the shelf life or efficacy of the fusion protein. The composition may be formulated to provide rapid, sustained, or delayed release of the active ingredient(s) after administration. Suitable pharmaceutical compositions and methods for preparing the same are known in the art. For example, refer to the literature [Remington, The Science and Practice of Pharmacy, A. Gennaro, et al., eds., 21st ed., Mack Publishing Co. (2005)].

[0318] In certain embodiments, the pharmaceutical composition may be administered alone or in combination therapy with an immunosuppressant / immunomodulator and / or anti-inflammatory agent (i.e., simultaneously or sequentially). An exemplary type of agent is a cytotoxic T lymphocyte-associated protein 4 (CTLA4) mutant molecule. An exemplary CTLA4 mutant molecule is L104EA29Y-Ig (velatacept), which is a modified CTLA4-Ig. Different immune diseases may require the use of specific adjuvants useful for treating the immune disease, which may be determined on a patient-to-patient basis. For example, the pharmaceutical composition may be administered in combination with one or more suitable ajuvants, e.g., cytokines (e.g., IL-10 and IL-13), or other immunostimulators, e.g., chemokines, tumor-associated antigens, and peptides. Suitable ajuvants are known in the art.

[0319] In certain embodiments, a method for treating an immune disease in a patient requiring treatment for an immune disease may include administering to the patient a therapeutically effective amount of an antibody or its antigen-binding portion as described herein. A method for treating or preventing an autoimmune or inflammatory disease in said patient is further provided, which may include administering to a patient requiring treatment for an autoimmune or inflammatory disease a therapeutically effective amount of an antibody or its antigen-binding portion as described herein. Additionally, a use of the antibody or its antigen-binding portion or a pharmaceutically acceptable salt thereof of the present disclosure is provided for treating an immune disease in a patient requiring such treatment and / or treating or preventing an autoimmune or inflammatory disease in a patient requiring such treatment, which may include administering to a patient requiring treatment for an immune disease and / or an autoimmune or inflammatory disease a therapeutically effective amount of an antibody or its antigen-binding portion. Antagonizing CD40-mediated T cell activation may suppress undesirable T cell responses occurring, for example, during autoimmune, transplant rejection, or allergic reactions. Suppressing CD40-mediated T cell activation can alleviate the progression and / or severity of these diseases.

[0320] In certain embodiments, the use of the antibody of the present disclosure or its antigen-binding portion or its pharmaceutically acceptable salt is also provided in the manufacture of a medicine for the treatment of an immune disease and / or the treatment or prevention of an autoimmune or inflammatory disease in a patient requiring treatment of an immune disease and / or an autoimmune or inflammatory disease. The medicine may be administered, for example, in combination with an immunosuppressant / immunomodulator and / or anti-inflammatory agent.

[0321] As used herein, “patient” means an animal, a mammal, including, for example, humans. For example, a patient may be diagnosed with an immune disease. “Treatment,” “to treat,” or “treating” means a process involving the progression or remission of the severity of a symptom, disorder, condition, or disease. “Immune disease” refers to any disease associated with the occurrence of an immune response, including cellular and / or humoral immune responses, in an individual. Examples of immune diseases include, but are not limited to, inflammation, allergies, autoimmune diseases, or graft-related diseases. Thus, a patient may be diagnosed with an autoimmune disease or an inflammatory disease. “Autoimmune disease” refers to any disease associated with the occurrence of an autoimmune response, including cellular and / or humoral immune responses, in an individual. Examples of autoimmune diseases include, but are not limited to, ulcerative colitis and Crohn's disease, inflammatory bowel disease (IBD). Other autoimmune diseases include systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, diabetes mellitus, psoriasis, scleroderma, and atherosclerosis. Graft-related diseases include graft-versus-host disease (GVHD), acute transplant rejection, and chronic transplant rejection.

[0322] In certain embodiments, diseases that may be treated by administering the antibodies of the present disclosure include Addison's disease, allergy, anaphylaxis, ankylosing spondylitis, asthma, atherosclerosis, atopic allergy, autoimmune disease of the ear, autoimmune disease of the eye, autoimmune hepatitis, autoimmune parotitis, bronchial asthma, coronary heart disease, Crohn's disease, diabetes mellitus, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant drug products (e.g., factor VII in hemophilia), lupus nephritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, transplant rejection, and vasculitis. It can be selected from the group consisting of ulcerative colitis.

[0323] Any suitable method or route may be used to administer the antibody or its antigen-binding portion or pharmaceutical composition. Routes of administration include, for example, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The therapeutically effective dose of the administered antibody depends on numerous factors, for example, the type and severity of the disease to be treated, the use of combination therapy, the route of administration of the antibody or its antigen-binding portion or pharmaceutical composition, and the patient's body weight. The non-limiting range for the therapeutically effective dose of the antibody is 0.1–20 milligrams / kilogram (mg / kg) and, on one side, 1–10 mg / kg, based on the patient's body weight.

[0324] Kit

[0325] A kit useful for treating immune diseases in human patients is provided. A kit useful for treating or preventing autoimmune or inflammatory diseases in human patients is also provided. The kit may comprise (a) a predetermined dose of the antibody of the present disclosure or its antigen-binding portion, and (b) instructions for using the antibody or its antigen-binding portion in a method for treating an immune disease in a patient or using the antibody or its antigen-binding portion in a method for treating or preventing an autoimmune or inflammatory disease in a patient.

[0326] As used herein, the term “instructions” includes publications, records, diagrams, or any other media of expression that may be used to inform the usefulness of the compositions and / or compounds of the present invention within the kit. The instructions of the kit may, for example, be attached to a container containing the compounds and / or compositions of the present invention, or delivered together with the container containing the compounds and / or compositions. Alternatively, the instructions may be delivered separately from the container if the recipient intends to use the instructions and the compounds together. Delivery of the instructions may be achieved, for example, by physical delivery of a publication or other media of expression informing the usefulness of the kit, or alternatively by electronic transmission, for example by a computer, such as by email or download from a website.

[0327] Exemplary embodiment

[0328] Embodiment 1. An isolated antibody or its antigen-binding portion that specifically binds to human CD40, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, and wherein

[0329] (i) The heavy chain variable region comprises an amino acid sequence selected from HC2, HC3, HC4, HC5, HC6, HC7, HC8, HC9, HC16, HC10, HC15, HC11, HC12, and HC14; and the light chain variable region comprises LC1 as presented in Table 3;

[0330] Table 3

[0331]

[0332]

[0333] (ii) The heavy chain variable region comprises an amino acid sequence selected from HC1, HC2, HC3, HC4, HC5, HC6, HC7, HC8, HC9, HC16, HC10, HC15, HC11, HC12 and HC14 and H13; and the light chain variable region comprises LC2 as presented in Table 4;

[0334] Table 4

[0335]

[0336]

[0337] (iii) The heavy chain variable region comprises an amino acid sequence selected from HC1, HC2, HC3, HC4, HC5, HC6, HC7, HC8, HC9, HC16, HC10, HC15, HC11, HC12 and HC14 and H13; and the light chain variable region comprises LC3 as presented in Table 5;

[0338] Table 5

[0339]

[0340]

[0341] (iv) The heavy chain variable region comprises an amino acid sequence selected from HC1, HC2, HC3, HC4, HC5, HC6, HC7, HC8, HC9, HC16, HC10, HC15, HC11, HC12 and HC14 and H13; and the light chain variable region comprises LC4 as presented in Table 6;

[0342] Table 6

[0343]

[0344]

[0345] (v) The heavy chain variable region comprises an amino acid sequence selected from HC1, HC2, HC3, HC4, HC5, HC6, HC7, HC8, HC9, HC16, HC10, HC15, HC11, HC12 and HC14 and H13; and the light chain variable region comprises LC5 as presented in Table 7;

[0346] Table 7

[0347]

[0348]

[0349] or

[0350] (vi) The heavy chain variable region comprises an amino acid sequence selected from HC1, HC2, HC3, HC4, HC5, HC6, HC7, HC8, HC9, HC16, HC10, HC15, HC11, HC12 and HC14 and H13; and the light chain variable region comprises LC6 as presented in Table 8.

[0351] Isolated antibody or its antigen-binding portion.

[0352] Table 8

[0353]

[0354]

[0355] Embodiment 2. An isolated antibody or its antigen-binding portion that specifically binds to human CD40, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, and wherein

[0356] (i) The above heavy chain variable region is HC1

[0357] Includes; and the light chain variable region is LC4

[0358] Includes or;

[0359] (ii) The above heavy chain variable region is HC1

[0360] Includes; and the light chain variable region is LC3

[0361] Includes or;

[0362] (iii) The above heavy chain variable region is HC15

[0363] Includes; and the light chain variable region is LC3

[0364] Includes or;

[0365] (iv) The above heavy chain variable region is HC4

[0366] Includes; and the light chain variable region is LC1

[0367] Includes or;

[0368] (v) The above heavy chain variable region is HC4

[0369] Includes; and the light chain variable region is LC3

[0370] Includes or;

[0371] or

[0372] (vi) The above heavy chain variable region is HC5

[0373] Includes; and the light chain variable region is LC4

[0374] including

[0375] Isolated antibody or its antigen-binding portion.

[0376] Embodiment 3. In Embodiment 2, the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, wherein

[0377] (i) The above heavy chain variable region is HC1

[0378] Includes; and the light chain variable region is LC4

[0379] Includes or;

[0380] (ii) The above heavy chain variable region is HC1

[0381] Includes; and the light chain variable region is LC3

[0382] Includes or;

[0383] (iii) The above heavy chain variable region is HC15

[0384] Includes; and the light chain variable region is LC3

[0385] Includes or;

[0386] (iv) The above heavy chain variable region is HC4

[0387] Includes; and the light chain variable region is LC1

[0388] Includes or;

[0389] (v) The above heavy chain variable region is HC4

[0390] Includes; and the light chain variable region is LC3

[0391] Includes or;

[0392] or

[0393] (vi) The above heavy chain variable region is HC5

[0394] Includes; and the light chain variable region is LC4

[0395] including

[0396] Isolated antibody or its antigen-binding portion.

[0397] Embodiment 4. The isolated antibody or its antigen-binding portion in Embodiment 2, wherein the first polypeptide portion comprises or is composed of an amino acid sequence selected from the group consisting of:

[0398]

[0399] Embodiment 5. In Embodiment 2, the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, wherein

[0400] (i) The above heavy chain variable region is HC1

[0401] Includes; and the light chain variable region is LC4

[0402] Includes or;

[0403] (ii) The above heavy chain variable region is HC1

[0404] Includes; and the light chain variable region is LC3

[0405] Includes or;

[0406] (iii) The above heavy chain variable region is HC15

[0407] Includes; and the light chain variable region is LC3

[0408] Includes or;

[0409] (iv) The above heavy chain variable region is HC4

[0410] Includes; and the light chain variable region is LC1

[0411] Includes or;

[0412] (v) The above heavy chain variable region is HC4

[0413] Includes; and the light chain variable region is LC3

[0414] Includes or;

[0415] or

[0416] (vi) The above heavy chain variable region is HC5

[0417] Includes; and the light chain variable region is LC4

[0418] including

[0419] Isolated antibody or its antigen-binding portion.

[0420] Embodiment 6. In Embodiment 2, the heavy chain variable region is HC1

[0421] Includes; and the light chain variable region is LC4

[0422] An isolated antibody or its antigen-binding portion comprising

[0423] Embodiment 7. In Embodiment 2, the heavy chain variable region is HC1

[0424] Includes; and the light chain variable region is LC3

[0425] An isolated antibody or its antigen-binding portion comprising

[0426] Embodiment 8. In Embodiment 2, the heavy chain variable region is HC15

[0427] Includes; and the light chain variable region is LC3

[0428] An isolated antibody or its antigen-binding portion comprising

[0429] Embodiment 9. In Embodiment 2, the heavy chain variable region is HC4

[0430] Includes; and the light chain variable region is LC1

[0431] An isolated antibody or its antigen-binding portion comprising

[0432] Embodiment 10. In Embodiment 2, the heavy chain variable region is HC4

[0433] Includes; and the light chain variable region is LC3

[0434] An isolated antibody or its antigen-binding portion comprising

[0435] Embodiment 11. In Embodiment 2, the heavy chain variable region is HC5

[0436] Includes; and the light chain variable region is LC4

[0437] An isolated antibody or its antigen-binding portion comprising

[0438] Embodiment 12. An antibody in which the antigen-binding portion is scFv-Fc or the antigen-binding portion thereof, in any one of Embodiments 2-11.

[0439] Embodiment 13. In any one of Embodiments 2-12, an antibody or its antigen-binding portion connected to a therapeutic agent.

[0440] Embodiment 14. In any one of Embodiments 2-13, an antibody or its antigen-binding portion connected to a second functional moiety having a binding specificity different from that of the antibody or its antigen-binding portion.

[0441] Embodiment 15. An antibody or its antigen-binding portion further comprising an additional moiety in any one of embodiments 2-14.

[0442] Embodiment 16. A method for treating or preventing an immune response in a subject, comprising administering to the subject any one of the antibodies of Embodiments 2-15 or its antigen-binding portion.

[0443] Embodiment 17. A method for treating or preventing an autoimmune or inflammatory disease in a subject, comprising administering to the subject any one of the antibodies of Embodiments 2-15 or its antigen-binding portion.

[0444] Embodiment 18. The method of Embodiment 16 or 17, wherein the antibody or its antigen-binding portion is administered together with an immunosuppressant / immunomodulator and / or anti-inflammatory agent.

[0445] Embodiment 19. The method of Embodiment 18, wherein the immunosuppressant / immunomodulator and / or anti-inflammatory agent is a CTLA4 mutant molecule.

[0446] Embodiment 20. The method of Embodiment 19, wherein the CTLA4 mutant molecule is L104EA29Y-Ig (velatasept).

[0447] Embodiment 21. In Embodiment 16 or 17, the subject has Addison's disease, allergy, anaphylaxis, ankylosing spondylitis, asthma, atherosclerotic arteriosclerosis, atopic allergy, autoimmune disease of the ear, autoimmune disease of the eye, autoimmune hepatitis, autoimmune mumps, bronchial asthma, coronary heart disease, Crohn's disease, diabetes mellitus, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, an immune response to a recombinant drug product (e.g., Factor VII in hemophilia), lupus nephritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, A method having a disease selected from the group consisting of transplant rejection, vasculitis, and ulcerative colitis.

[0448] Embodiment 22. An isolated antibody or its antigen-binding portion, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, and wherein

[0449] (i) The above heavy chain variable region is the HC1 framework

[0450] or including its mutation; the light chain variable region is an LC1 framework

[0451] or including his mutation;

[0452] Herein, at least one of the heavy chain variable region and the light chain variable region comprises a mutation in a basic residue, wherein the heavy chain variable region mutation is selected from the group consisting of positions 12, 13, 19, 23, 38, 57, 63, 67 and 74 of sequence identification number: 73 and combinations thereof, or the light chain variable region mutation is selected from the group consisting of positions 45, 54, 61 and 107 of sequence identification number: 74 and combinations thereof;

[0453] Here, at least one mutation in the basic residue is a mutation to a neutral amino acid or an acidic amino acid.

[0454] Isolated antibody or its antigen-binding portion.

[0455] Embodiment 23. The isolated antibody or its antigen-binding portion in Embodiment 22, wherein the neutral amino acid is selected from glutamine, asparagine, valine, serine, alanine, and threonine.

[0456] Embodiment 24. The isolated antibody or its antigen-binding portion in Embodiment 22, wherein the acidic amino acid is selected from glutamate or aspartate.

[0457] Embodiment 25. An isolated antibody or its antigen-binding portion in Embodiment 22, wherein at least two mutations are present in a basic residue selected from the group consisting of 45, 54, 61 and 107 of sequence identification number: 74 and combinations thereof within the light chain variable region.

[0458] Embodiment 26. An isolated antibody or its antigen-binding portion in Embodiment 22, wherein at least two mutations are present in a basic residue selected from the group consisting of 12, 13, 19, 23, 38, 57, 63, 67 and 74 of sequence identification number: 73 within a heavy chain variable region.

[0459] Embodiment 27. In Embodiment 22, the light chain variable region is an LC1 framework

[0460] or an isolated antibody or its antigen-binding portion containing its mutation, wherein the positions of 45, 54, 61, 107 and 108 and combinations thereof are mutable.

[0461] Embodiment 28. The isolated antibody or its antigen-binding portion for specifically binding to human CD40 in Embodiment 22.

[0462] Embodiment 29. A method for improving at least one pharmacokinetic property of a first antibody, comprising mutating a residue at at least one position selected from 12, 13, 19, 23, 38, 57, 63, 67 and 74 of sequence identification number: 73 or a combination thereof and / or at least one position selected from 45, 54, 61 and 107 of sequence identification number: 74 or a combination thereof, to produce a variant of the first antibody having at least one mutated residue and at least one improved pharmacokinetic property compared to a non-modified first antibody.

[0463] Embodiment 30. The method of Embodiment 29, wherein the first antibody specifically binds to human CD40.

[0464] Embodiment 31. An isolated antibody or its antigen-binding portion, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, and wherein

[0465] (i) The above heavy chain variable region is the HC1 framework

[0466] Includes; and the light chain variable region is an LC4 framework

[0467] Includes or;

[0468] (ii) The above heavy chain variable region is the HC1 framework

[0469] Includes; and the light chain variable region is an LC3 framework

[0470] Includes or;

[0471] (iii) The above heavy chain variable region is the HC15 framework

[0472] Includes; and the light chain variable region is an LC3 framework

[0473] Includes or;

[0474] (iv) The above heavy chain variable region is the HC4 framework

[0475] Includes; and the light chain variable region is an LC1 framework

[0476] Includes or;

[0477] (v) The above heavy chain variable region is the HC4 framework

[0478] Includes; and the light chain variable region is an LC3 framework

[0479] Includes or;

[0480] or

[0481] (vi) The above heavy chain variable region is the HC5 framework

[0482] Includes; and the light chain variable region is an LC4 framework

[0483] including

[0484] Isolated antibody or its antigen-binding portion.

[0485] Embodiment 32. An isolated antibody or its antigen-binding portion in Embodiment 31, wherein the first polypeptide portion comprises a human heavy chain constant region; and the second polypeptide portion comprises a human light chain constant region.

[0486] Embodiment 33. An isolated antibody of any one of Embodiments 1 to 15, 22 to 28, 31 and 32, or a nucleic acid molecule encoding its antigen-binding portion.

[0487] Embodiment 34. An expression vector comprising the nucleic acid molecule of Embodiment 33.

[0488] Embodiment 35. A cell transformed with the expression vector of Embodiment 34 or the nucleic acid of Embodiment 33.

[0489] Embodiment 36. A method for producing an anti-human CD40 antibody or its antigen-binding portion, wherein

[0490] a) a step of expressing an antibody or its antigen-binding portion in a cell of embodiment 35; and

[0491] b) A step of isolating an antibody or its antigen-binding portion from a cell

[0492] A method including

[0493] Embodiment 37. a) an antibody or its antigen-binding portion of any one of Embodiments 1 to 15, 22 to 28, 31 and 32; and b) a pharmaceutically acceptable carrier comprising a pharmaceutical composition.

[0494] Embodiment 38. An antibody or its antigen-binding portion for use as a medicine in any one of Embodiments 1 to 15, 22 to 28, 31 and 32.

[0495] Embodiment 39. An antibody or its antigen-binding portion for use in treating a subject requiring treatment, in any one of embodiments 1 to 15, 22 to 28, 31 and 32.

[0496] Examples

[0497] Example 1: Manipulation of BMS-986325 variant for improved pharmacokinetic properties

[0498] The anti-CD40 monoclonal antibody BMS-986325 (PCT / US19 / 62011) was selected to develop a protein manipulation strategy for optimizing pharmacokinetic (PK) properties. The amino acid sequences of the heavy chain variable region and light chain variable region of BMS-986325 are presented in Table 15. The CDR for each variable region is underlined and in bold.

[0499] Table 15

[0500]

[0501] The protein manipulation strategy was to disrupt positively charged (basic) patches on the antibody surface that could accompany undesirable binding to negatively charged (acidic) intracellular surfaces, such as cell membranes or the extracellular matrix (ECM). As part of this strategy, it was also important to maintain the high-affinity interaction with CD40 and functional efficacy, as well as the favorable biophysical properties of the antibody.

[0502] To limit potential immunogenicity risks, initial optimization focused on variable regions of the heavy and light chains, which naturally tend to exhibit greater sequence variability. Both these variable heavy chain (Vh) and variable light chain (Vl) regions were analyzed at the primary amino acid sequence level as well as by generating a structural model of the BMS-986325 Fab domain. Homology models were generated based on available X-ray structures using the antibody modeler at the Molecular Operating Environment (MOE) (Chemical Computing Group). The amino acid sequences were loaded into the modeling GUI. The tool then searches for framework and CDR loop templates. The antibody backbone is constructed from the framework template most similar to the original, followed by the generation of the CDR loop. The final step of model construction is refinement performed by all-atom minimization using the Amber10EHT field in the MOE.

[0503] Sequence analysis first involved identifying all lysine (Lys) and arginine (Arg) residues within the heavy chain variable region (Vh) and light chain variable region (Vl), which serve as primary sources of positive charge at physiological pH and temperature. The positions of these amino acid residues within BMS-986325 were evaluated against the natural human wiring repertoire to identify residues capable of undergoing mutations to improve CD40 binding, as well as against a set of antibodies from the same sequence family identified from the same CD40 immunization that identified BMS-986325. Refer to Table 16. (The light chain variable region for BMS-986325 is the kappa light chain "Vk").

[0504] Table 16

[0505]

[0506]

[0507]

[0508] Sequence analysis was used to help bias protein manipulation efforts from residues that could potentially be associated with CD40 binding. Analysis of the structural model included (1) evaluating the positions of Lys and Arg residues in relation to charged patches and hydrophobic patches on the antibody surface, and (2) evaluating the potential impact that mutations to non-basic residues might have on these surface characteristics. Charged patches refer to one or more charged residues spatially close to each other on the surface of the folder protein structure. Hydrophobic patches refer to one or more non-charged residues spatially close to each other on the surface of the folder protein structure.

[0509] A total of 7 Lys and 5 Arg residues of BMS-986325 were identified in the Vh sequence, and 6 Lys and 3 Arg residues were identified in the Vl sequence. Additionally, since the last residue (K107) of Vl and the first residue (R108) of the light chain constant region are basic residues, the R108 residue was considered as part of the substrate for mutation. Based on the above sequence and structural model analysis, each Lys and Arg residue was annotated regarding (1) clustering into its charge patches, (2) the likelihood that the mutation will affect binding based on (i) CDR proximity and (ii) binding data for sequence family variants, (3) wiring analysis, and (4) other predicted characteristics based on structural modeling. See Table 17.

[0510] Table 17: Annotations of selected Lys and Arg residues in BMS-986325

[0511]

[0512]

[0513] Basic residues were mutated into (1) uncharged amino acids or (2) acidic residues. To select the amino acid residue to mutate the basic residue, Gln was prioritized as the amino acid to replace the basic side chain of Lys or Arg with an uncharged side chain of similar length. Glu was prioritized as the acidic residue to induce more dramatic destruction of the positively charged patch by reversing the positive charge of Lys or Arg into a negatively charged side chain of similar length. Additionally, Gln and Glu were prioritized over Asn and Asp residues, respectively, to avoid potential deamidation (Asn) or isomerization (Asp) problems common to the shorter Asn and Asp side chains. Glu and Gln were also prioritized because they have relatively low immunogenicity potential. Additionally, by comparing Lys and Arg positions across the human wiring repertoire, alternative natural wiring residues were identified in which the basic Lys or Arg side chains can be replaced with neutral or acidic residues known to be structurally acceptable in other human IgGs and to possess a low immunogenicity risk.

[0514] Based on the above analysis, relative mutagenic priorities were assigned to each Lys and Arg residue within the Vh and Vl regions, and then further incorporated into a short list of 14 mutants HC and 5 mutants LC, consisting of single mutants or combinations of mutants that may represent a subset of the highest priority mutants. See Tables 18 and 19.

[0515] Table 18: Antibody heavy chain and light chain sequences.

[0516]

[0517]

[0518]

[0519]

[0520]

[0521] Table 19: Change in net charge of the variable region for different combinations of heavy and light chains.

[0522]

[0523] Furthermore, one additional mutant, HC (HC13), was designed as a proof-of-concept control by replacing two acidic residues with two basic residues (E46K, E62K) and introducing a more positively charged surface patch with increased off-target binding and reduced PK, which is predicted to potentially demonstrate opposite characteristics to other engineered variants.

[0524] Collectively, these 15 mutant HC and 5 mutant LC, together with wild-type HC and wild-type LC, produced a total of 16 HC and 6 LC constructs (Table 18), which could be combined into all possible HC x LC combinations to generate 96 unique antibodies. The total net charge change for these different combinations of HC and LC ranges from minus 8 (-8) for the combination of HC5 (HC-K12E, K13Q, K23E) and LC4 (LC-K45Q, R54Q, R61Q) to plus 4 (+4) for the proof-of-concept antibody combination of HC13 (HC-E46K, E62K) and wild-type LC1 (LC-wt). See Table 19.

[0525] Example 2: Analysis of the potency of the BMS-986325 variant supernatant

[0526] Antibody mutant identification numbers (M#) from M1-M96 were assigned to 96 antibodies of 16 HC x 6 LC combinations (Table 20).

[0527] Table 20: Mutant identification numbers (M#) assigned to different combinations of heavy chain (HC) and light chain (LC).

[0528]

[0529] 96 types of antibodies generated from 16 types of HC x 6 types of LC combinations were produced by transient transfection and 3 mL scale, and the titers were analyzed using a ForteBio Octet RED96 instrument.

[0530] Antibody expression (titer) was detected for each HC x LC combination. Titers varied widely from as low as 5 μg / ml (M56) to as high as 322 μg / ml (M80), and the wild-type antibody (M1 / wt) had a titer of 134 μg / ml. Refer to Table 21.

[0531] Table 21: Antibody titer data (μg / ml) determined by octet BLI assay for different combinations of heavy chain (HC) and light chain (LC).

[0532]

[0533] Several trends were observed in the titer data associated with specific HC or LC mutations. For example, double or triple mutations for HC patch #2 (HC8 = HC-R38Q, K63Q, R67Q and HC9 = HC-K63Q, R67E) significantly reduced antibody titers when combined with any of the six LCs, and the triple mutant (HC8) exhibited particularly low titers (5–7 μg / ml) when paired with any of the six different LCs. Interestingly, LC mutations generally improved antibody titers, and 73 / 80 (91%) of the antibodies contained mutated LCs that had higher titers than when each HC was paired with a wild-type LC. See Table 21.

[0534] Example 3: CD40 binding SPR analysis of BMS-986325 and variant supernatants

[0535] Ninety-six mutant BMS-986325 antibodies were tested for CD40 binding by surface plasmon resonance (SPR). Using titer data (Table 21), the antibody concentration in each supernatant was normalized to 3 μg / ml, and these antibodies were captured from the supernatant on a Protein A CM5 Series S sensor chip (GE Healthcare) and tested for binding to the soluble hCD40 extracellular domain at two concentrations (5 nM and 50 nM). Purified wild-type BMS-986325 was included as a control at the start, middle, and end of the experiment for a total of n = 3 in each of the three flow cells, which demonstrated excellent reproducibility throughout the experimental process in each flow cell (Table 22).

[0536] Table 22: Kinetic and affinity values ​​for hCD40 binding to purified BMS-986325 determined by SPR. The triple data (n = 3) for each flow cell (Fc) were fitted overall to obtain the ka, kd, and KD values ​​for the binding interactions on each Fc.

[0537]

[0538] The KD values ​​for CD40 binding to 96 supernatant samples are summarized in Table 23, and a plot of kinetic on-rate (ka) and off-rate (kd) values ​​(isoaffinity plot) is provided in Fig. 1. In Fig. 1, it can be seen that the majority of variants (72 / 95 variants = 76%) possessed equivalent or even improved affinity for CD40 compared to the wild-type antibody M1. Some of the mutants that consistently improved binding affinity across multiple HC x LC combinations included HC mutants for Patch 2 (HC8, HC9, HC10, HC15, HC16), as well as the triple LC mutant LC4 (LC-K45Q, R54Q, R61Q). Variants with reduced affinity included all antibodies containing HC11, HC12, and HC13. Refer to Fig. 1. Of these, HC11 and HC12 both contain mutations in basic patch 1, which is the patch closest to the CDR region. HC13 is a proof-of-concept control sample engineered to increase the net positive charge (HC-E46K, E62K).

[0539] Table 23: KD values ​​for hCD40 binding to BMS-986325 and BMS-986325 variant antibodies captured from the supernatant as determined by SPR.

[0540]

[0541] Example 4: Selection of BMS-986325 variants for further purification and further characterization

[0542] To identify a subset of antibodies to be expressed and purified on a larger scale for further characterization, titer data from antibody sequence and structure models, hCD40 binding SPR data, and in silico analysis were considered comprehensively. For this analysis, characteristics such as lower titers or reduced affinity compared to wild-type antibodies were considered undesirable and more likely to be reduced in priority. However, rather than being biased toward producing only these specific HC x LC combinations with the highest affinity and titers, the goal was to have a purified set of antibodies exhibiting a wide range of different characteristics, including at least one mutation for each of the five basic patches. For example, all Patch 3 mutants were well accepted for their favorable titers and CD40 binding characteristics and appeared to combine favorably with any Patch 4 or Patch 5 LC mutant; however, antibodies containing HC4, HC5, and HC6 were preferred over antibodies containing HC2 or HC3 because the HC4, HC5, and HC6 mutants exhibited a larger change in net charge without any undesirable titer reduction or binding loss. Both M13 and M53 were included to ensure that the purified set exhibited the full range of net charge change from M13 (+4) to M53 (-8). For additional diversity in the purified set, the set included variants in which Lys or Arg were mutated to human wiring residues, including variants in which Lys and Arg were mutated to Glu or Gln, as well as M62 (HC-K74T) containing HC14 and M38 and M54 (HC-K12V, K19S, K23A) containing HC6. Furthermore, to keep the total mutation burden low and reduce the risk of instability or immunogenicity, multiple variants with only a single mutation for HC or LC were included.Considering all these factors, a final set of wild-type and 15 mutant antibodies was identified for larger-scale expression, purification, and characterization. The final set is presented in Table 24.

[0543] Table 24: 16 antibodies selected for larger-scale expression, purification, and characterization.

[0544]

[0545]

[0546] Example 5: Expression and Purification of BMS-986325 and Variants

[0547] The 16 antibodies from Table 24 were expressed in transient Expi293 cells (purchased from ThermoFisher Scientific) under the conditions indicated for these cells. The antibodies were purified for further analysis and biophysical characterization. Further characterization involved the production of two batches of M4 identified as M4 and M4-b to compare the materials produced from two distinct production runs; the two distinct production runs were found to have similar analytical and biophysical characteristics.

[0548] Example 6: aSEC analysis of BMS-986325 and variants

[0549] The purity and oligomer status of BMS-986325 and 15 variants were characterized by analytical size exclusion chromatography (aSEC). The data are presented in Table 25.

[0550] Table 25: aSEC data for purified BMS-986325 and variants

[0551]

[0552] All samples were found to have quality suitable for further study, containing more than 93% of monomers and less than 7% of high molecular weight (HMW) species, and no detectable low molecular weight (LMW) species.

[0553] Example 7: icIEF analysis of BMS-986325 and variants

[0554] The effects of various mutations on the charge characteristics of BMS-986325 were evaluated using imaging capillary isoelectric focusing (icIEF). These data are presented in Table 26.

[0555] Table 26: icIEF data for purified BMS-986325 and variants

[0556]

[0557] The wild-type BMS-986325 had a major peak isoelectric point (pI) of 9.21, and contained 76.1% major peaks, 22.2% acidic variants, and 1.7% basic variants. Refer to M1 in Table 26. The icIEF profiles for all other antibodies also consisted predominantly of major peaks (71.7–94.2%), some acidic variants (5.8–26.1%), and small amounts of basic variants or the absence of basic variants (0–2.4%).

[0558] As expected, M13, the only mutant designed to increase positive charge, was found to have a higher major peak pI (9.42) than the wild-type BMS-986325, whereas all other mutants designed to replace positively charged residues with neutral or acidic residues were found to have lower pIs than the wild-type BMS-986325.

[0559] Example 8: aHIC analysis of BMS-986325 and variants

[0560] The hydrophobicity of wild-type and mutant BMS-986325 molecules was evaluated by analytical hydrophobic interaction chromatography (aHIC). Data are provided in Table 27.

[0561] Table 27: aHIC data for purified BMS-986325 and variants

[0562]

[0563] In this analysis, wild-type BMS-986325 eluted as a single symmetric peak with a major peak retention time (RT) of 10.1 min. Refer to M1 in Table 27. Several mutants designed to disrupt positively charged patches, including M4, M10, M13, M33, M36, M47, M80, and M81, eluted in this manner while maintaining low hydrophobicity (RT = 10.1–10.3 min). Antibodies in this subset include variants in which one or two charged residues are mutated into uncharged residues, e.g., M47, M80, and M81. In contrast, all variants using LC4 (LC-K45Q, R54Q, R61Q), the most highly mutated light chain tested with three charged residues replaced by three uncharged residues, had increased hydrophobicity compared to the wild type (RT = 10.5 - 10.8 min).

[0564] The heterogeneity of the BMS-986325 variants also generally increased with increasing mutations. For example, all antibodies using HC or LC (HC5, HC6, LC4) containing three mutations eluted with a <80% major peak, along with correspondingly increased levels of post-peaks (eluted later) for the more hydrophobic species. Two variants (M53 and M54) with three mutations for each of the HC and LC, totaling six mutations, exhibited particularly high heterogeneity, with major peaks of 53.4–55.2% and post-peaks of 46.6–44.8%.

[0565] Example 9: Analysis of UNcle Thermal Stability of BMS-986325 and Variants

[0566] The structural and colloidal stability of the BMS-986325 variant was investigated by fluorescence spectroscopy and static light scattering (SLS), respectively, using an UNcle instrument (Unchained Labs, Pleasanton, California). Thermal denaturation of each antibody was accompanied by a distinct fluorescence change (which could be monitored using the center-of-gravity mean (BCM) method), which was fitted to the melting temperature 1 (T m1 The value of ) was determined. Subsequently, a significant increase in SLS, indicating the aggregation of denatured protein molecules at higher temperatures, followed, from which the aggregation initiation temperature (T agg ) at 266 nm (T agg 266) or 473 nm (T agg It can be determined by monitoring in 473).

[0567] The data is presented in Table 28.

[0568] Table 28: T for purified BMS-986325 and variants m and T agg Thermal stability data

[0569]

[0570] The value for *M1 (wt) is the mean ± standard deviation of three independent measurements.

[0571] T for wild-type BMS-986325 m and T agg The value (mean ± standard deviation for triple measurements) is T m1 = 65.8 ± 0.3℃, T agg 266 = 79.7 ± 0.1℃ and T agg473 = 79.8 ± 0.4℃. The Tm1 values ​​for all antibody variants ranged from 65.1 to 67.6℃, and all variants except M37 showed equivalent (within standard deviation) or slightly higher Tm1 values ​​compared to wild-type BMS-986325. m1 had. In contrast, T agg is T agg 266 (70.6-79.9℃) and T agg 473 (70.6-80.3℃) varied over a larger range for both, and all variants except M33 had lower T than the wild type. agg It had a value.

[0572] The number of mutations for this set of antibodies and T agg No direct correlation was observed between the values, suggesting that the location of mutations and the nature of amino acid changes are scientifically important for maintaining the thermal stability of antibodies. For example, T for M62 (4 total mutations) agg While it is higher for multiple mutants (M4, M10, M13, M36, M47, M49, M80, M81) with only 1, 2, or 3 mutations, the single mutation in M10 is T agg 266 = 72.6℃ and T agg 473 = 72.2℃ caused significant destabilization.

[0573] Example 10: ECM ELISA Analysis of BMS-986325 and Variants

[0574] To evaluate the potential for nonspecific binding of positively charged BMS-986325 surface patches to acidic surfaces and the effect of mutations on these interactions, an extracellular matrix (ECM) binding ELISA assay was used. Data are presented in Table 29.

[0575] Table 29: ECM scores for purified BMS-986325 and variants.

[0576]

[0577] Wild-type BMS-986325 (M1) produced a strong ECM binding response with an ECM score of 23.5 at a concentration of 1 μM. As expected, the proof-of-concept control molecule M13, which introduced an additional positively charged patch via the E46K-E62K double mutation, was found to exhibit a much stronger ECM binding response than wild-type BMS-986325, with an ECM score of 72.0 at a concentration of 1 μM. The single HC-R67E mutation (M10) had the smallest effect on the ECM score compared to the wild-type antibody, with an ECM score of 21.7 at a concentration of 1 μM. M4, M80, and M81 showed some reduction in ECM binding but maintained measurable ECM binding (ECM scores at 1 μM = 8.1–14.0). All other variants (M33, M36, M37, M38, M47, M49, M53, M54, M62, M63) showed significantly reduced ECM binding responses with ECM scores of 1.4–3.1 at 1 μM.

[0578] Example 11: CD40 binding SPR analysis of BMS-986325 and variants

[0579] The CD40 target binding kinetics and affinities of BMS-986325 and 15 purified variants were evaluated using an SPR method similar to the one previously used to screen 96 small-scale expression supernatants, with the exception of testing the full set of six CD40 analyte concentrations for the purified antibodies rather than just two analyte concentrations in the supernatant screening experiment. Additionally, to more accurately define the dissociation rate (kd), a longer dissociation time of 360 seconds was used in contrast to the shorter dissociation time of 180 seconds used in the supernatant screening experiment.

[0580] The data is presented in Table 30.

[0581] Table 30: Kinetics and affinity values ​​for hCD40 binding to purified BMS-986325 and BMS-986325 variants determined by SPR.

[0582]

[0583] The value for *M1 (wt) is the mean ± standard deviation of three independent measurements.

[0584] The observed effect of the mutation on CD40 binding to the purified antibody was similar to that observed in the supernatant, with M13 having a significantly lower affinity than the wild-type BMS-986325, and other variants having similar or slightly higher affinities.

[0585] Example 12: Analysis of Functional Efficacy of BMS-986325 Variant

[0586] The effect of mutations on the functional efficacy of BMS-986325 in inhibiting CD40L-stimulated activity in primary human amygdala B cells was tested. Data are provided in Table 31.

[0587] Table 31: Activity of BMS-986325 on human amygdala B cells. Inhibition of human amygdala B cell proliferation stimulated by IZ-hCD40L trimers or human CD40L-expressing CHO cells. The assay was performed in triplicate. B cells from n-donors were tested.

[0588]

[0589] All mutants exhibited potent inhibition of soluble CD40L trimer (IZ-hCD40L)-stimulated B cell proliferation, most mutants showed efficacy similar to BMS-986325, and IC50 values ​​were within 2–3 fold (typically within the range of donor variability in these tests, as exemplified by two lots of mutant M4). Exceptions included several mutants M62, M809, M10, and M38, which exhibited slightly lower efficacy.

[0590] Similarly, all mutants inhibited B cell proliferation stimulated by cell surface CD40L (CHO-CD40L), which is typically more difficult to inhibit. In these experiments, several mutants were slightly less potent but exhibited high variability between the two donors tested (M62, M80, M10, M63, M37, M38, M54); the remaining mutants showed efficacy within three times that observed for BMS-986325. Collectively, these data suggest that most mutants had minimal effect on CD40 efficacy, with the selected number of mutants showing only a slight shift in efficacy.

[0591] The prior art CD40 antibody is described as having the potential to possess agonist activity. In contrast, BMS-986325 is a pure antagonist that does not exhibit agonist activity against B cells, either alone or in combination with IL-4, and sensitizes B cells to proliferation and activation signals. Additionally, the potential of mutations affecting potential agonist activity was tested by monitoring B cell stimulation through the assessment of proliferation and cytokine production. Figures 2-7 illustrate the data for the evaluation of the potential agonist activity of BMS-986325 by IL-4-stimulated human B cells, measuring proliferation (Figure 2-4) and cytokine secretion (Figure 5-7). With the exception of one variant (M81), no other mutants induced agonist activity when tested in B cells, and each molecule was tested in a total of two donors. The M81 mutation showed a weak increase in proliferation in only one of the two donors tested in the presence of IL-4, and IL-6 production in only the presence of IL-4 in both donors tested. These data suggest that these mutations may alter the conformation of the generated antibodies, thereby enabling some degree of efficacy.

[0592] Example 13: Intrinsic pharmacokinetics of BMS-986325 and variants

[0593] The "inherent" PK of BMS-986325 and its variants is presented in FIG. 8, and the calculated "inherent" PK parameters are provided in Table 32.

[0594] Table 32: Single-dose PK parameters of BMS-986325 and its variants at 1 mg / kg IV in C57 / BL6 mice calculated by non-compartmental analysis (NCA).

[0595]

[0596] Following intravenous (IV) administration of BMS-986325 (a single 1 mg / kg dose) to mice, BMS-986325 exhibited a mean low total serum clearance "CL" of 0.56 mL / h / kg, a limited steady-state volume of distribution "Vss" of 0.14 L / kg, and an apparent elimination half-life "T-half" of 168 hours (~7 days). Within the variability range, all variants except M13 (M49, M33, M47, M4, M36, and M53) exhibited PKs comparable to or superior to WT (area under the concentration-time curve "AUC" and CL within twofold). In contrast, at this dose and within the variability range, the PK of the M13 variant was worse than that of WT (AUC as much lower and CL as much higher). Each of M49, M33, M47, M4, M36, and M53 had improved values ​​for at least one of these PK parameters, and most variants had improved values ​​for at least two of these PK parameters.

[0597] Therefore, at this dose, within the variability, the overall PK of all variants except M13 is similar to or slightly better than that of wild-type BMS-986325. That is, at this dose, within the variability, M13 has a worse PK (lower AUC and higher clearance) than wild-type BMS-986325, whereas the PK of all other variants is similar to or improved compared to wild-type BMS-986325.

[0598] Materials and methods for Examples 1 to 13

[0599] Cloning of BMS-986325 variants: Coding sequences for the CD40 mAb heavy chains BMS-986325-IgG1a-P238K-K12Q-K13Q and BMS-986325-IgG1a-P238K-R63Q were codon-optimized for Chinese hamster ovary (CHO) expression, and synthetic DNA fragments were cloned into a modified pTT5 mammalian expression vector. The remaining CD40 mAb heavy chains were generated by mutagenesis using one of the two constructs as a template.

[0600] The coding sequence for the CD40 mAb light chain BMS-986325-Vk-K45Q-hLC was also codon-optimized for CHO expression, and the synthetic DNA fragment was cloned into the same pTT5 vector. The remaining CD40 mAb light chains were generated by mutagenesis using the light chain construct as a template.

[0601] Expression of BMS-986325 and BMS-986325 variants: For initial screening experiments, antibodies were expressed at a 3 ml scale using the Thermo Fisher Scientific Expi293™ expression system (Thermo Fisher Scientific, Waltham, Massachusetts). The DNA / Expifectamine™ ratio was 1:2.7; and the DNA amount was 0.5 mg / L. The cell seeding density was 2.7 x 10⁶ 6 It was a dog (cell density after transfection was 2.5 x 10 6(It was a dog). 24 hours after transfection, cells were supplied with 0.5 M valproic acid from Gibco® (Thermo Fisher Scientific, Waltham, Massachusetts; cat# A10240-02) at a final concentration of 2 mM and CHO CD EfficientFeed™ B at a final volume of 5%. Culture growth conditions were 37°C and 8% CO2 under humidification. On the 5th day, the supernatant was collected by centrifugation. Larger-scale expression was performed at a 0.5 L scale.

[0602] Purification of BMS-986325 and BMS-986325 variants: Purified antibody-rich supernatant was conjugated to a 5 mL MabSelect SuRe™ (Cytiva, Marlborough, Massachusetts) column and washed with 5 column volumes of 1X phosphate-buffered saline (PBS) pH 7.2 until baseline was reached. The antibodies were eluted with 50 mM acetic acid pH 3.0 and run on a Superdex 26 / 10 desalting column, with the buffer exchanged to PBS pH 7.2. All samples contained more than 5% impurities, and these impurities were removed by running them on a Superdex® 200 26 / 600 purification grade (pg) purification SEC (pSEC) column (GE Healthcare, Chicago, Illinois). Next, the sample was concentrated to at least 1 mg / mL and filtered through a 0.2 μm filter before freezing.

[0603] Octet BLI Titer Analysis: Antibody titers were determined using ForteBio’s Octet RED384 and Protein A sensor tip. An 8-point standard curve was constructed using human IgG1f isotype standard antibodies in PBS-T buffer at concentrations ranging from 150 to 1.17 μg / mL. The standard curve was run in triplicate. Sample supernatants were diluted 1:2 in PBS-T buffer (10 mM NaPO4, 130 mM NaCl, 0.05% Tween 30 (PBS-T) pH 7.2). The standard curves and samples were placed in black flat-bottom 96-well plates (Corning), with a final volume of 100 μL in the wells. The Protein A sensor tip was hydrated in PBS-T buffer for approximately 10 minutes before the run was initiated. Assembly was performed at 30 μL / min for 180 seconds, and the protein A sensor tip was regenerated using 10 mM glycine pH 1.5. Data was obtained using Octet software data acquisition and data analysis.

[0604] CD40 Binding SPR of Antibody Supernatants: Surface Plasmon Resonance (SPR) studies were performed on a ViaCore™ T200 instrument (GE Healthcare, Chicago, Illinois). A Series S Protein A sensor chip (GE Healthcare, Chicago, Illinois) was equilibrated at 25°C with SPR run buffer consisting of 10 mM NaPO4, 130 mM NaCl, 0.05% Tween 20, (PBS-T) pH 7.2. Supernatants of 96 antibodies were normalized to a concentration of 3 μg / ml by diluting with PBS-T using a PerkinElmer Janus® G3 system (PerkinElmer, Akron, Ohio). After priming the system, 3 μg / ml antibody samples were captured on the surface of Protein A at 10 μl / min for 30 seconds. The binding of 50 nM and 500 nM human CD40 extracellular domains (in-house produced) was evaluated using an associative time of 180 seconds at 30 μl / min, followed by a dissociative time of 180 seconds at 30 μl / min. Regeneration between cycles was achieved using two 15-second injections of 10 mM glycine pH 1.5. Wild-type BMS-986325 was tested three times separately for each of three independent flow cells for a total of nine measurements. Data were analyzed by fitting a 1:1 Langmuir model using Viacore™ T200 evaluation software.

[0605] CD40 Binding SPR: SPR studies of purified antibodies were performed on a ViaCore™ T200 instrument (GE Healthcare, Chicago, Illinois). A Series S Protein A sensor chip (GE Healthcare, Chicago, Illinois) was equilibrated at 25°C with SPR run buffer consisting of 10 mM NaPO4, 130 mM NaCl, 0.05% Tween 20, (PBS-T) pH 7.2. Purified antibody samples were diluted to 3 μg / ml in PBS-T and captured on the surface of Protein A at 10 μl / min for 30 seconds. Binding to 3.91, 7.81, 15.6, 31.3, 62.5, and 125 nM human CD40 extracellular domains (in-house produced) was evaluated using an association time of 180 seconds at 30 μl / min, followed by a dissociation time of 360 seconds at 30 μl / min. Regeneration between cycles was achieved using two 15-second injections of 10 mM glycine pH 1.5. Wild-type BMS-986325 was tested once on each of three independent flow cells. Data were analyzed by fitting a 1:1 Langmuir model using Viacore™ T200 evaluation software.

[0606] aSEC Analysis: Isosolvent separation was performed on a Shodex™ K403-4F column (Showa Denko America, Inc., New York, NY) connected to an Agilent 1260 Series HPLC system in a buffer containing 100 mM sodium phosphate and 150 mM sodium chloride pH 7.3 (0.1 μm filtered) running at 0.3 mL / min. 20 μg of antibody was injected using an Agilent AutoSampler, and data were obtained using an Agilent diode array detector by reading at 280 nm, 260 nm, 214 nm, and 254 nm and subtracting a reference at 360 nm. Data were analyzed using ChemStation (Agilent) software.

[0607] icIEF Analysis: Imaging Capillary Isoelectric Focusing (icIEF) experiments were performed on a Maurice instrument (ProteinSimple, San Jose, California). The instrument setup included pre-focusing at 1500 V for 1 minute and focusing at 3000 V for 10 minutes. The antibody samples were first diluted in double distilled water (ddH2O) to a final concentration of 2 mg / mL. In the final plate, 20 μL of the sample was mixed with 180 μL of Master Mix (MM) for final concentrations of 0.35% methylcellulose (MC), 2.0 M urea, 1% v / v% Pharmalyte® 5-8, and 3% v / v% Pharmalyte® 8-10.5. MM contained 1.0% MC solution (70 μL), PharmaRite® 5-8 (2 μL), PharmaRite® 8-10.5 (6 μL), 8 M urea (50 μL), arginine (100x), dd water (50 μL), pI marker 5.85 (1 μL) and pI marker 10.10 (1 μL) per sample (PharmaRite®, Citiba, Marlborough, Massachusetts). Data were obtained and analyzed using Compass for iCE of ProteinSimple.

[0608] aHIC Analysis: High-performance analytical hydrophobic interaction chromatography (aHIC) was performed on an Agilent 1260 series HPLC. Data were collected at 280 nm and reference subtracted at 360 nm. A Tosoh TSK gel butyl NPR column with dimensions of 4.6 mm × 3.5 cm and a particle size of 2.5 μm was used for separation at a flow rate of 1 mL / min. A 20-minute linear gradient ranging from 1.8 M to 0.0 M ammonium sulfate in 0.1 M sodium phosphate buffer (pH 7.0) was used. The column and autosampler temperatures were set to 25°C and 4°C, respectively. The column loading was 10 μg. Data were analyzed using ChemStation (Agilent) software.

[0609] Thermal Stability Analysis: The melting and aggregation initiation temperatures were determined using a UNcle instrument (Unchained Labs). Briefly, 9 μL of the 1 mg / mL sample was loaded into a Sample Uni cuvette, sealed, and placed into the instrument. A temperature gradient from 25°C to 90°C was applied to the sample at 0.5°C / min. Full-spectral UV absorbance (250 nm–725 nm) as well as static light scattering emissions at 266 nm and 473 nm were specifically obtained at each time point. The generated T m / T agg The fitting of was performed by UNCLE analysis software (Unchained Labs).

[0610] ECM Binding ELISA Analysis: Extracellular Matrix (ECM) binding ELISA assays were performed using 96-well Corning® Thin-Layer Matrigel® matrix pre-coated ECM plates (Corning Incorporations Life Sciences, Tewkesbury, Massachusetts). The plates were incubated with 300 μl of blocking buffer (10% fetal bovine serum (FCS) in TBS) at room temperature for 1 hour. After incubation, 100 μl of fresh blocking buffer was added along with antibody samples of 1 μM, 0.33 μM, and 0.11 μM. Six wells were left blank for background and ECM score calculation. After 1 hour of sample incubation, the samples were removed, and the plates were washed 3X with PBS-T wash buffer. 100 μl of 10 ng / ml goat anti-human IgG-HRP (horsetail peroxidase) conjugated detection antibody was added to all wells. After an additional 1 hour of incubation at room temperature, the wells were washed 3X with PBS-T wash buffer. After washing, 100 μl of TMB (3,3',5,5'-tetramethylbenzidine) substrate was added to each well and allowed to react for 15 minutes, after which 100 μl of 1 M phosphate stop solution was added. Subsequently, absorbance was read at 450 nm with reference at 620 nm on a microplate reader.

[0611] Evaluation of the in vitro activity of BMS-986325 and its variants in human B cell assays: Briefly, human tonsil B cells were obtained from pediatric patients during commercial tonsillectomy. After slowly crushing the tissue, mononuclear cells were separated by density gradient separation using Lympholyte®-H separation medium (Cedarlane Labs, Burlington, Ontario, Canada), washed, rosetteed with sheep erythrocytes (SRBC, Colorado Serum Company; Denver, Colorado), and then T cells were removed by density gradient separation. Cells were washed and resuspended in complete medium consisting of RPMI-1640 containing 2 mM L-glutamine (Cat. #11875) supplemented with 10% fetal bovine serum (cat. #26140), 50 μg / ml gentamicin (cat. #15750), and 1% antibiotic-antifungal agent (cat. #15240) (all purchased from Gibco, Carlsbad, California).

[0612] Inhibition of soluble or membrane-bound CD40L-stimulated human B cell proliferation: Isoleucine zipper human CD40L trimer (IZ-hCD40L) or Chinese hamster ovary cells stably transfected with human CD40L (CHO-hCD40L) were used as stimulants for CD40 activation and as a positive control. The CD40 agonist mAb2141-hHCD-IgG2-puCOEgate-SP5 was used as a positive control for CD40 pathway activation induced by the agonist antibody. The BMS-986325 antibody was titrated in complete medium and triple-piptetted into 96-well round-bottom plates. 1 x 10⁶ 5 Renal tonsil B cells were added and stimulated with soluble IZ-hCD40L (3 μg / ml) or CHO-hCD40L irradiated at 10,000 rad, at a final volume of 200 μl per well, 2 x 10 3Cells were plated at a rate of 1 cell / well. Plates were incubated at 37°C for 72 hours in a humidified incubator with 5% CO2. During the last 7 hours of incubation, cells were treated with 0.5 μCi of [methyl- 3 Samples were labeled with [H]-thymidine, collected on glass fiber filter plates, and counted by liquid scintillation on a Packard Topcount® NXT™ counter (PerkinElmer Life and Analytical Sciences, Shelton, Connecticut). B cell proliferation was quantified based on thymidine incorporation. For analysis, a 4-parameter curve was generated from the triplicate values ​​and fitted using GraphPad PRISM software (ver. 7, GraphPad Software, San Diego, California).

[0613] Evaluation of potential efficacy of BMS-986325 and BMS-986325 variants on human B cells: BMS-986325 and variant antibodies were titrated in complete medium and double-piptetted into 96-well round-bottom plates. 2x10 5 Reticulobacteria B cells were added and stimulated with soluble hIL-4 (20 ng / ml, PeproTech, Inc.), antibody alone or antibody plus IL-4 and soluble IZ-hCD40L (3 μg / ml). Plates were incubated at 37°C for 72 hours in a humidified incubator with 5% CO2.

[0614] The medium was sampled at 48 hours by AlphaLisa® (cat. #AL3025C, PerkinElmer; Waltham, Massachusetts) according to the manufacturer's instructions to measure IL-6 and read on an EnVision® 2105 multimode plate reader (PerkinElmer; Waltham, Massachusetts). For IL-6 production, an induction of more than twofold compared to the control group was considered a positive indicator of efficacy.

[0615] During the last 7 hours of incubation, cells were treated with 0.5 μCi of [methyl- 3 The cells were labeled with [H]-thymidine (PerkinElmer, Waltham, Massachusetts), collected on glass fiber filter plates, and counted by liquid scintillation on a Packard TopCount® NXT™ counter (PerkinElmer, Waltham, Massachusetts). B cell proliferation was quantified based on thymidine incorporation. For analysis, double values ​​were averaged and quantified using GraphPad PRISM software (ver. 7, GraphPad Software, San Diego, California). Induction of more than twofold compared to the control, non-stimulated, or IL-4 alone was considered positive for agonist activity.

[0616] Single-dose pharmacokinetic study in C57BL / 6 mice following 1 mg / kg intravenous administration of BMS-986325 and its variants: Since neither BMS-986325 nor its variants cross-react with murine CD40, the PKs thus collected were the "inherent PKs" of the antibodies, unaffected by any typical target-mediated drug batch (TMDD) for anti-CD40 antibodies. Briefly, 1 mg / kg of the antibody in phosphate-buffered saline (PBS) was administered intravenously (IV) to C57 / BL6 mice, and 10 μL of whole blood was collected over a period of 6 weeks into tubes containing 90 μL of Rexxip® A buffer (Gyros Protein Technologies, Tucson, Arizona). After thoroughly mixing the tubes, they were frozen until bioanalysis.

[0617] Analysis of BMS-986325 and its variants in mouse micro-samples by Gyrolab immunoassay: After thawing, blood samples were centrifuged, and the supernatant was analyzed for antibodies using a validated automated microfluidic fluorescence immunoassay on a Gyrolab® xP workstation (Gyrolab Protein Technologies, Tucson, Arizona). Briefly, 100 μg / ml of biotinylated huCD40-mouse IgG2b was used as the capture molecule for "active / free" antibodies. Samples were analyzed at a minimum required dilution of 10% in 1% BSA / PBS / 0.05% Tween®20 (PTB; Croda International, Edison, NJ). Standard, QC, and study samples were prepared to a final matrix concentration of 10% mouse blood in Rexship® A buffer and loaded onto the Gyrolab. The 3-step Wizard method using the Gyrolab® Bioaffy 200 CD was used. After the final wash step, captured "active / free" antibodies were detected using Alexa 64-labeled monkey anti-human IgG Fc mAb clone 1628.3379.10C7.D12. The concentration of antibodies ("active / free") in the blood samples was calculated based on the corresponding fluorescence intensity measured by the Gyrolab using a 4PL (parameter logistic) regression standard calibration curve. The assay performance was within the acceptable range, with the % CV of the standard and QC being less than 20%, and the QC recovery rate within ±20% of the nominal value.

[0618] These data are consistent with the hypothesis that specific sites or locations of mutations that modify surface charge patches, rather than merely modifying the total antibody charge, are important for improving antibody PK. For example, a variant with 1 or 2 strategically placed mutations and a smaller change in total charge of -2 (M33) or -3 (M47) has equivalent or superior PK compared to a mutant with 6 mutations and a larger change in charge of -8 (M53).

[0619] Although the embodiments described herein have been described in detail with reference to the above embodiments, it is understood that various modifications may be made without departing from the spirit of these embodiments, and that this will be readily known to those skilled in the art.

[0620] The exemplary specific treatment methods, medicines, and uses listed herein, as well as other aspects disclosed herein, will be apparent from the teachings contained herein.

Claims

Claim 1 An isolated antibody or its antigen-binding portion that specifically binds to human CD40, wherein the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, wherein (i) the heavy chain variable region comprises HC1 (SEQ ID: 40); and the light chain variable region comprises LC4 (SEQ ID: 41); or (ii) the heavy chain variable region comprises HC1 (SEQ ID: 40); and the light chain variable region comprises LC3 (SEQ ID: 42); or (iii) the heavy chain variable region comprises HC15 (SEQ ID: 43); and the light chain variable region comprises LC3 (SEQ ID: 42); or (iv) the heavy chain variable region comprises HC4 (SEQ ID: 44); An isolated antibody or its antigen-binding portion wherein the light chain variable region comprises LC1 (sequence identification number: 45); (v) the heavy chain variable region comprises HC4 (sequence identification number: 44); the light chain variable region comprises LC3 (sequence identification number: 42); or (vi) the heavy chain variable region comprises HC5 (sequence identification number: 46); and the light chain variable region comprises LC4 (sequence identification number: 41). Claim 2 In claim 1, the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, wherein (i) the heavy chain variable region comprises HC1 (Sequence Identification No.: 47); and the light chain variable region comprises LC4 (Sequence Identification No.: 20); (ii) the heavy chain variable region comprises HC1 (Sequence Identification No.: 47); and the light chain variable region comprises LC3 (Sequence Identification No.: 19); (iii) the heavy chain variable region comprises HC15 (Sequence Identification No.: 86); and the light chain variable region comprises LC3 (Sequence Identification No.: 19); and (iv) the heavy chain variable region comprises HC4 (Sequence Identification No.: 49); An isolated antibody or its antigen-binding portion wherein the light chain variable region comprises LC1 (sequence identification number: 17); (v) the heavy chain variable region comprises HC4 (sequence identification number: 49); the light chain variable region comprises LC3 (sequence identification number: 19); or (vi) the heavy chain variable region comprises HC5 (sequence identification number: 50); and the light chain variable region comprises LC4 (sequence identification number: 20). Claim 3 In claim 1, the isolated antibody or its antigen-binding portion wherein the first polypeptide portion comprises or is composed of an amino acid sequence selected from the group consisting of (i) sequence identification number: 91; (ii) sequence identification number: 84; and (iii) sequence identification number:

85. Claim 4 In claim 1, the antibody comprises a first polypeptide portion comprising a heavy chain variable region and a second polypeptide portion comprising a light chain variable region, wherein (i) the heavy chain variable region comprises HC1 (Sequence Identification No.: 56); and the light chain variable region comprises LC4 (Sequence Identification No.: 20); (ii) the heavy chain variable region comprises HC1 (Sequence Identification No.: 56); and the light chain variable region comprises LC3 (Sequence Identification No.: 19); (iii) the heavy chain variable region comprises HC15 (Sequence Identification No.: 35); and the light chain variable region comprises LC3 (Sequence Identification No.: 19); and (iv) the heavy chain variable region comprises HC4 (Sequence Identification No.: 84); An isolated antibody or its antigen-binding portion wherein the light chain variable region comprises LC1 (sequence identification number: 17); (v) the heavy chain variable region comprises HC4 (sequence identification number: 84); the light chain variable region comprises LC3 (sequence identification number: 19); or (vi) the heavy chain variable region comprises HC5 (sequence identification number: 85); and the light chain variable region comprises LC4 (sequence identification number: 20). Claim 5 An isolated antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region comprises HC1 (sequence identification number: 40); and the light chain variable region comprises LC4 (sequence identification number: 41). Claim 6 An isolated antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region comprises HC1 (sequence identification number: 40); and the light chain variable region comprises LC3 (sequence identification number: 42). Claim 7 An isolated antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region comprises HC15 (sequence identification number: 43); and the light chain variable region comprises LC3 (sequence identification number: 42). Claim 8 An isolated antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region comprises HC4 (sequence identification number: 44); and the light chain variable region comprises LC1 (sequence identification number: 45). Claim 9 An isolated antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region comprises HC4 (sequence identification number: 44); and the light chain variable region comprises LC3 (sequence identification number: 42). Claim 10 An isolated antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region comprises HC5 (sequence identification number: 46); and the light chain variable region comprises LC4 (sequence identification number: 41). Claim 11 An antibody or its antigen-binding portion having scFv-Fc in any one of claims 1 to 10. Claim 12 In any one of paragraphs 1 to 10, an antibody or its antigen-binding portion connected to a therapeutic agent. Claim 13 In any one of claims 1 to 10, an antibody or its antigen-binding portion connected to a second functional moiety having a binding specificity different from that of the antibody or its antigen-binding portion. Claim 14 An antibody or its antigen-binding portion further comprising an additional moiety in any one of claims 1 to 10. Claim 15 A pharmaceutical composition for treating or preventing an immune response to a recombinant drug product, an autoimmune disease, or an inflammatory disease in a subject, comprising an antibody of any one of claims 1 to 10 or its antigen-binding portion. Claim 16 A pharmaceutical composition according to claim 15, wherein the antibody or its antigen-binding portion is administered together with an immunosuppressant / immunomodulator and / or anti-inflammatory agent. Claim 17 A pharmaceutical composition according to claim 16, wherein the immunosuppressant / immunomodulator and / or anti-inflammatory agent is L104EA29Y-Ig (velatacept). Claim 18 In claim 15, a pharmaceutical product in which the subject has a disease selected from the group consisting of Addison's disease, allergy, anaphylaxis, ankylosing spondylitis, asthma, atherosclerosis, atopic allergy, autoimmune disease of the ear, autoimmune disease of the eye, autoimmune hepatitis, autoimmune parotitis, bronchial asthma, coronary heart disease, Crohn's disease, diabetes mellitus, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant drug factor VII, lupus nephritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, transplant rejection, vasculitis, and ulcerative colitis Composition. Claim 19 A nucleic acid molecule encoding an isolated antibody or its antigen-binding portion according to any one of claims 1 to 10. Claim 20 An expression vector comprising the nucleic acid molecule of claim 19. Claim 21 Isolated cells transformed with the expression vector of claim 20. Claim 22 A method for producing an anti-human CD40 antibody or its antigen-binding portion, comprising: a) expressing the antibody or its antigen-binding portion in the isolated cells of claim 21; and b) isolating the antibody or its antigen-binding portion from the cells. Claim 23 a) an antibody or its antigen-binding portion according to any one of claims 1 to 10; and b) a pharmaceutically acceptable carrier, comprising a pharmaceutical composition for treating or preventing an immune response to a recombinant drug product, an autoimmune disease, or an inflammatory disease in a subject. Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete

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