Anti-CD103 antibody

Abstract

This invention relates to anti-CD103 antibodies and their use in the diagnosis, prognosis, monitoring, and treatment of diseases. An imaging agent comprising the anti-CD103 antibody and a detectable label is also disclosed, wherein the antibody does not block CD103 binding to E-cadherin or at least partially blocks CD103 binding to E-cadherin. Treatment methods involve administering the anti-CD103 antibody, optionally conjugated to a cytotoxic agent. Diseases that need to be treated include, for example, hairy cell leukemia, HCLv, intestinal and extraintestinal lymphoma, enteropathy-associated T-cell lymphoma (EATL), T-lymphoblastic leukemia / lymphoma (T-ALL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell leukemia / lymphoma (ATLL), mycosis fungoides (ME), anaplastic large cell lymphoma ALCL, cutaneous T-cell lymphoma (CTCL), Cezari syndrome (SS), Alzheimer's disease, Parkinson's disease, or multiple sclerosis.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 704,258, filed April 30, 2020, which is incorporated herein by reference in its entirety. Invention Field

[0003] This invention relates to anti-CD103 antibodies and their use in the diagnosis, prognosis, monitoring, and treatment of diseases. Background of the Invention

[0005] CD103 (integrin αE) is a type I membrane protein expressed on a subset of lamina propria T cells, epithelial dendritic cells, lamina propria-derived dendritic cells, and a small subset of peripheral lymphocytes. reg Cells express high levels of CD103. Mature CD103 protein cleaves into two chains: a 150 kD (C-terminal) chain and a 25 kD (N-terminal) chain, which are linked by disulfide bonds. CD103 combines with β7 integrin to form the αE / β7 heterodimer, which represents the E-cadherin-binding integrin known as the human mucosal lymphocyte-1 antigen.

[0006] The identification and understanding of immune checkpoint proteins and their roles in immune responses represent a breakthrough in cancer therapy. With this discovery, work has focused on blocking immune checkpoint pathways to activate T cells directed towards cancer cells that are considered ineffective in stimulating useful anti-tumor responses. In 2011, the U.S. Food and Drug Administration (FDA) approved ipilimumab for the treatment of metastatic melanoma; ipilimumab is an antibody that binds to and functionally blocks CTLA-4. Following ipilimumab, antibodies targeting the programmed cell death-1 (PD-1) receptor and its ligand programmed death-ligand 1 (PD-L1), present on many cancer cells, were also approved. These checkpoint inhibitors have revolutionized cancer therapy.

[0007] While significant clinical benefits are attributed to immunotherapies targeting the immune checkpoint pathway, most cancer patients fail to respond to checkpoint inhibitors. Specifically, studies have shown that checkpoint inhibition may be insufficient in patients, resulting in limited infiltration of functional T cells into the tumor environment. Furthermore, although T cells can accumulate in tissues surrounding tumor masses, they may not interact with tumor cells on their own.

[0008] Although they can produce T cell-specific chemokines and present antigens as well as co-stimulatory or inhibitory signals, it is the tumor-associated antigen-presenting cells that are optimally positioned to form anti-tumor effector immunity. Tissue-resident dendritic cells consist of two functionally specialized subsets: those involved in CD8... + T cell activation and cross-presentation of cell-associated antigens to CD103 on the T cells + -CD8 + DC, and more effectively drive CD4 + CD11b, which helps T cell response + DC. CD103 + -CD8 + Type I interferon production in the DC lineage controls the initiation of spontaneous T cell responses to tumor antigens. Therefore, the composition of tumor-associated myelosimilar compartments may play a major role in tumor responses to checkpoint blockers.

[0009] CD103 reported in cancer patients + The association between tumor-infiltrating T lymphocytes and improved clinical outcomes underscores the need for a clear and comprehensive understanding of the expression profiles of cancer-specific lymphocytes and their implications for future immunotherapies. Summary of the Invention

[0010] In a first aspect, the present invention provides an anti-CD103 antibody and its antigen-binding fragment comprising the structural and functional features specified below.

[0011] In various embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to human CD103, said antibody or antigen-binding fragment comprising:

[0012] a. Heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence different from SEQ ID NO: 1 due to 1, 2 or 3 conserved substitutions,

[0013] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 2 or differs from the amino acid sequence of SEQ ID NO: 2 due to 1, 2 or 3 conserved substitutions.

[0014] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 3 or differs from the amino acid sequence of SEQ ID NO: 3 due to 1, 2 or 3 conserved substitutions.

[0015] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 4 or differs from the amino acid sequence of SEQ ID NO: 4 due to 1, 2 or 3 conserved substitutions.

[0016] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence different from SEQ ID NO: 5 due to 1, 2 or 3 conserved substitutions, and

[0017] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 6 or differs from the amino acid sequence of SEQ ID NO: 6 due to 1, 2 or 3 conserved substitutions;

[0018] or

[0019] g. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 9 or differs from the amino acid sequence of SEQ ID NO: 9 due to 1, 2 or 3 conserved substitutions.

[0020] h. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 10 or differs from the amino acid sequence of SEQ ID NO: 10 due to 1, 2 or 3 conserved substitutions.

[0021] i. Heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or differing from the amino acid sequence of SEQ ID NO: 11 due to 1, 2 or 3 conserved substitutions,

[0022] j. Light chain variable region CDR1, said light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or differing from the amino acid sequence of SEQ ID NO: 12 due to 1, 2 or 3 conserved substitutions,

[0023] k. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence different from SEQ ID NO: 13 due to 1, 2 or 3 conserved substitutions, and

[0024] 1. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 14 or differs from the amino acid sequence of SEQ ID NO: 14 due to 1, 2 or 3 conserved substitutions;

[0025] or

[0026] m. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 17 or differs from the amino acid sequence of SEQ ID NO: 17 due to 1, 2 or 3 conserved substitutions.

[0027] n. Heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 18 or differing from the amino acid sequence of SEQ ID NO: 18 due to 1, 2 or 3 conserved substitutions.

[0028] o. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 19 or differs from the amino acid sequence of SEQ ID NO: 19 due to 1, 2 or 3 conserved substitutions.

[0029] p. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or differs from the amino acid sequence of SEQ ID NO: 20 due to 1, 2 or 3 conserved substitutions.

[0030] q. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence different from SEQ ID NO: 21 due to 1, 2 or 3 conserved substitutions, and

[0031] r. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or differs from the amino acid sequence of SEQ ID NO: 22 due to 1, 2 or 3 conserved substitutions;

[0032] or

[0033] s. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 25 or differs from the amino acid sequence of SEQ ID NO: 25 due to 1, 2 or 3 conserved substitutions.

[0034] t. Heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 26 or differing from the amino acid sequence of SEQ ID NO: 26 due to 1, 2 or 3 conserved substitutions,

[0035] u. Heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 27 or differing from the amino acid sequence of SEQ ID NO: 27 due to 1, 2 or 3 conserved substitutions,

[0036] v. Light chain variable region CDR1, said light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 28 or differing from the amino acid sequence of SEQ ID NO: 28 due to 1, 2 or 3 conserved substitutions.

[0037] w. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence different from SEQ ID NO: 29 due to 1, 2 or 3 conserved substitutions, and

[0038] x. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 30 or differs from the amino acid sequence of SEQ ID NO: 30 due to 1, 2 or 3 conserved substitutions;

[0039] or

[0040] y. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 33 or differs from the amino acid sequence of SEQ ID NO: 33 due to 1, 2 or 3 conserved substitutions.

[0041] z. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 34 or differs from the amino acid sequence of SEQ ID NO: 34 due to 1, 2 or 3 conserved substitutions.

[0042] aa. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 35 or differs from the amino acid sequence of SEQ ID NO: 35 due to 1, 2 or 3 conserved substitutions.

[0043] bb. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 36 or differs from the amino acid sequence of SEQ ID NO: 36 due to 1, 2 or 3 conserved substitutions.

[0044] cc. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence different from SEQ ID NO: 37 due to 1, 2 or 3 conserved substitutions, and

[0045] dd. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 38 or differs from the amino acid sequence of SEQ ID NO: 38 due to 1, 2 or 3 conserved substitutions.

[0046] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0047] a. The heavy chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 1,

[0048] b. The heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 2,

[0049] c. The heavy chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 3,

[0050] d. The light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 4,

[0051] e. The light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 5, and

[0052] f. The light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0053] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0054] a. The heavy chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 9,

[0055] b. The heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 10,

[0056] c. The heavy chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 11,

[0057] d. The light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 12,

[0058] e. The light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 13, and

[0059] f. The light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 14.

[0060] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0061] a. The heavy chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 17,

[0062] b. The heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 18,

[0063] c. The heavy chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 19,

[0064] d. The light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 20,

[0065] e. The light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 21, and

[0066] f. The light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22.

[0067] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0068] a. The heavy chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 25,

[0069] b. The heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 26,

[0070] c. The heavy chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 27,

[0071] d. The light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 28,

[0072] e. The light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 29, and

[0073] f. The light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 30.

[0074] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0075] a. The heavy chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 33,

[0076] b. The heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 34,

[0077] c. The heavy chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 35,

[0078] d. The light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 36,

[0079] e. The light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 37, and

[0080] f. The light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 38.

[0081] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0082] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 1 due to one conserved substitution.

[0083] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 2 due to one conserved substitution.

[0084] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 3 due to one conserved substitution.

[0085] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 4 due to one conserved substitution.

[0086] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 5 due to one conserved substitution, and

[0087] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 6 due to one conserved substitution.

[0088] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0089] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 9 due to one conserved substitution.

[0090] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises an amino acid sequence different from SEQ ID NO: 10 due to one conserved substitution.

[0091] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises an amino acid sequence different from SEQ ID NO: 11 due to one conserved substitution.

[0092] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 12 due to one conserved substitution.

[0093] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 13 due to one conserved substitution, and

[0094] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 14 due to one conserved substitution.

[0095] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0096] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 17 due to one conserved substitution.

[0097] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises an amino acid sequence different from SEQ ID NO: 18 due to one conserved substitution.

[0098] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises an amino acid sequence different from SEQ ID NO: 19 due to one conserved substitution.

[0099] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 20 due to one conserved substitution.

[0100] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 21 due to one conserved substitution, and

[0101] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 22 due to one conserved substitution.

[0102] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0103] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 25 due to one conserved substitution.

[0104] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises an amino acid sequence different from SEQ ID NO: 26 due to one conserved substitution.

[0105] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises an amino acid sequence different from SEQ ID NO: 27 due to one conserved substitution.

[0106] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 28 due to one conserved substitution.

[0107] e. Light chain variable region CDR2, wherein the light chain variable region CDR2 comprises an amino acid sequence different from SEQ ID NO: 29 due to one conserved substitution, and

[0108] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 30 due to one conserved substitution.

[0109] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0110] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 33 due to one conserved substitution.

[0111] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 34 due to one conserved substitution.

[0112] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises an amino acid sequence different from SEQ ID NO: 35 due to one conserved substitution.

[0113] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 36 due to one conserved substitution.

[0114] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 37 due to one conserved substitution, and

[0115] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 38 due to one conserved substitution.

[0116] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0117] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 1 due to two conserved substitutions.

[0118] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 2 due to two conserved substitutions.

[0119] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 3 due to two conserved substitutions.

[0120] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 4 due to two conserved substitutions.

[0121] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 5 due to two conserved substitutions, and

[0122] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 6 due to two conserved substitutions.

[0123] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0124] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 9 due to two conserved substitutions.

[0125] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises an amino acid sequence different from SEQ ID NO: 10 due to two conserved substitutions.

[0126] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 11 due to two conserved substitutions.

[0127] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 12 due to two conserved substitutions.

[0128] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 13 due to two conserved substitutions, and

[0129] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 14 due to two conserved substitutions.

[0130] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0131] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 17 due to two conserved substitutions.

[0132] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 18 due to two conserved substitutions.

[0133] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 19 due to two conserved substitutions.

[0134] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 20 due to two conserved substitutions.

[0135] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 21 due to two conserved substitutions, and

[0136] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 22 due to two conserved substitutions.

[0137] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0138] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 25 due to two conserved substitutions.

[0139] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises an amino acid sequence different from SEQ ID NO: 26 due to two conserved substitutions.

[0140] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 27 due to two conserved substitutions.

[0141] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 28 due to two conserved substitutions.

[0142] e. Light chain variable region CDR2, wherein the light chain variable region CDR2 comprises an amino acid sequence different from SEQ ID NO: 29 due to two conserved substitutions, and

[0143] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 30 due to two conserved substitutions.

[0144] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0145] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 33 due to two conserved substitutions.

[0146] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 34 due to two conserved substitutions.

[0147] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 35 due to two conserved substitutions.

[0148] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 36 due to two conserved substitutions.

[0149] e. Light chain variable region CDR2, wherein the light chain variable region CDR2 comprises an amino acid sequence different from SEQ ID NO: 37 due to two conserved substitutions, and

[0150] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 38 due to two conserved substitutions.

[0151] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0152] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 1 due to three conserved substitutions.

[0153] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 2 due to three conserved substitutions.

[0154] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 3 due to three conserved substitutions.

[0155] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 4 due to three conserved substitutions.

[0156] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 5 due to three conserved substitutions, and

[0157] f. Light chain variable region CDR3, which contains an amino acid sequence that differs from SEQ ID NO: 6 due to three conserved substitutions.

[0158] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0159] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 9 due to three conserved substitutions.

[0160] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 10 due to three conserved substitutions.

[0161] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 11 due to three conserved substitutions.

[0162] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 12 due to three conserved substitutions.

[0163] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 13 due to three conserved substitutions, and

[0164] f. Light chain variable region CDR3, which contains an amino acid sequence different from SEQ ID NO: 14 due to three conserved substitutions.

[0165] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0166] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 17 due to three conserved substitutions.

[0167] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 18 due to three conserved substitutions.

[0168] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 19 due to three conserved substitutions.

[0169] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 20 due to three conserved substitutions.

[0170] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 21 due to three conserved substitutions, and

[0171] f. Light chain variable region CDR3, which contains an amino acid sequence that differs from SEQ ID NO: 22 due to three conserved substitutions.

[0172] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0173] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 25 due to three conserved substitutions.

[0174] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 26 due to three conserved substitutions.

[0175] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 27 due to three conserved substitutions.

[0176] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 28 due to three conserved substitutions.

[0177] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 29 due to three conserved substitutions, and

[0178] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 30 due to three conserved substitutions.

[0179] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising:

[0180] a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 33 due to three conserved substitutions.

[0181] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 contains an amino acid sequence that differs from SEQ ID NO: 34 due to three conserved substitutions.

[0182] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 contains an amino acid sequence that differs from SEQ ID NO: 35 due to three conserved substitutions.

[0183] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 contains an amino acid sequence that differs from SEQ ID NO: 36 due to three conserved substitutions.

[0184] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising an amino acid sequence different from SEQ ID NO: 37 due to three conserved substitutions, and

[0185] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence that differs from SEQ ID NO: 38 due to three conserved substitutions.

[0186] In various other embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to human CD103, said antibody or antigen-binding fragment comprising:

[0187] a. A heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 7 and a light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 8; or

[0188] b. A heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 15 and a light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 16; or

[0189] c. A heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 23 and a light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 24; or

[0190] d. A heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 31 and a light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 32; or

[0191] e. The heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 39 and the light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 40;

[0192] Alternatively, in each case, the sequence has at least 95% (and more preferably 97% or 99%) sequence similarity or identity with a given SEQ ID NO, and any differences in such sequences are limited to amino acids that are not part of the CDR sequence within the SEQ ID NO.

[0193] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of an antibody comprising the amino acid sequence of SEQ ID NO: 7 and a light chain of an antibody comprising the amino acid sequence of SEQ ID NO: 8.

[0194] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of an antibody comprising the amino acid sequence of SEQ ID NO: 15 and a light chain of an antibody comprising the amino acid sequence of SEQ ID NO: 16.

[0195] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of an antibody comprising the amino acid sequence of SEQ ID NO: 23 and a light chain of an antibody comprising the amino acid sequence of SEQ ID NO: 24.

[0196] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of an antibody comprising the amino acid sequence of SEQ ID NO: 31 and a light chain of an antibody comprising the amino acid sequence of SEQ ID NO: 32.

[0197] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of an antibody comprising the amino acid sequence of SEQ ID NO: 39 and a light chain of an antibody comprising the amino acid sequence of SEQ ID NO: 40.

[0198] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 7, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 7; and a light chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 8, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 8.

[0199] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 15, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 15; and a light chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 16, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 16.

[0200] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 23, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 23; and a light chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 24, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 24.

[0201] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 31, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 31; and a light chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 32, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 32.

[0202] In one embodiment, the present invention provides an antibody that binds to human CD103, the antibody comprising: a heavy chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 39, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 39; and a light chain of the antibody comprising an amino acid sequence having at least 95% (and more preferably 97% or most preferably 99%) sequence identity with SEQ ID NO: 40, and any differences in such sequence being limited to amino acids that are not part of the CDR sequence in SEQ ID NO: 40.

[0203] In this context, "sequence similarity" is a combination of the degree of identity and the degree of conserved variation. The percentage of "sequence similarity" is the percentage of identical or conserved amino acids or nucleotides, i.e., "sequence similarity" = (percentage of sequence identity) + (percentage of conserved variation). Therefore, for the purposes of this invention, "conserved variation" and "identity" are considered as a kind of broader term "similarity". Thus, whenever the term "sequence similarity" is used, it encompasses both sequence "identity" and "conserved variation". According to some embodiments, regardless of conserved variation, the percentage of sequence similarity refers to the percentage of sequence identity. In some embodiments, the sequence variation allowed by the mentioned percentage of sequence identity is all or almost all conserved variation; in other words, when the sequence has 90% identity, the remaining 10% is all or almost all conserved variation. In this context, the term "almost all" means at least 75% of the allowed sequence variation is conserved variation, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. In some embodiments of the antibody heavy chain and / or light chain, the allowed sequence variation is within the framework region, not in the CDR.

[0204] In any of the above embodiments, the antibody or its antigen-binding fragment may be isolated, as the terminology is defined herein.

[0205] In any of the above embodiments, the antibody or its antigen-binding fragment is a recombinant antibody, as the term is defined herein.

[0206] In any of the above embodiments, the antibody or its antigen-binding fragment is a full-length antibody, as the terminology is defined herein.

[0207] The antibody or antigen-binding fragments of the present invention can be obtained from a variety of species. For example, the antibodies of the present invention may comprise immunoglobulin sequences of rabbit, mouse, rat, guinea pig, chicken, goat, sheep, donkey, human, llama, or camel sequences or combinations of such sequences (so-called chimeric antibodies). Most preferably, the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. Most preferably, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment.

[0208] The term antibody includes an antigen-binding portion that retains the ability to bind to an antigen, i.e., an "antigen-binding site" (e.g., fragment, subsequence, complementarity-determining region (CDR)), including (i) Fab fragments, which are composed of V L V H C L And C H (ii) A monovalent segment composed of a structural domain; (iii) A divalent segment containing two Fab segments connected by disulfide bonds in the hinge region; (iv) A segment composed of V H and C H (iv) The Fd fragment composed of the l-domain; L and V H The Fv fragment consists of a domain, the (v) dAb fragment (Ward et al., (1989) Nature 341:544-546), said fragment consisting of a VH domain; and (vi) a separated complementarity-determining region (CDR). The term “antibody” also includes single-chain antibodies. Preferred therapeutic antibodies are intact IgG antibodies. As used herein, the term “intact IgG” means a polypeptide belonging to an antibody class substantially encoded by the recognized immunoglobulin γ gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3. The known Ig domain in antibodies of the IgG class is VH. H Cγ1, Cγ2, Cγ3, V L And C L .

[0209] In any of the above embodiments, the antibody or its antigen-binding fragment is a human antibody or humanized antibody comprising two heavy chains and two light chains. In one embodiment, the antibody is IgG. In a preferred embodiment, the antibody is IgG1, IgG2, or IgG4, and preferably human IgG1, IgG2, or IgG4.

[0210] In one embodiment, the anti-CD103 antibody of the present invention comprises a full-length antibody structure having two light chains and two heavy chains as described above, wherein each light chain comprises a human κ light chain or a human λ light chain constant domain; and each heavy chain comprises a human IgG1 constant region.

[0211] In one embodiment, the anti-CD103 antibody of the present invention comprises a full-length antibody structure having two light chains and two heavy chains as described above, wherein each light chain comprises a human κ light chain or a human λ light chain constant domain; and each heavy chain comprises a human IgG2 constant region.

[0212] In one embodiment, the anti-CD103 antibody of the present invention comprises a full-length antibody structure having two light chains and two heavy chains as described above, wherein each light chain comprises a human κ light chain or a human λ light chain constant domain; and each heavy chain comprises a human IgG4 constant region.

[0213] In some embodiments, the anti-CD103 antibody of the present invention may be conjugated to at least one diagnostic marker for in vivo imaging studies. In some embodiments, the antigen-binding fragment of the anti-CD103 antibody of the present invention may be conjugated to at least one diagnostic marker for in vivo imaging studies. In some embodiments, the anti-CD103 antibody of the present invention may be conjugated to at least one therapeutic agent. In some embodiments, the antigen-binding fragment of the anti-CD103 antibody of the present invention may be conjugated to at least one therapeutic agent. In one embodiment, the therapeutic agent is a second antibody or a fragment thereof. In one embodiment, the therapeutic agent is a second antibody. In one embodiment, the therapeutic agent is an immunomodulator. In one embodiment, the therapeutic agent is a hormone. In one embodiment, the therapeutic agent is a cytotoxic agent. In one embodiment, the therapeutic agent is an enzyme. In one embodiment, the therapeutic agent is a radionuclide. In one embodiment, the therapeutic agent is a second antibody conjugated to at least one immunomodulator. In one embodiment, the therapeutic agent is a second antibody conjugated to at least one enzyme. In one embodiment, the therapeutic agent is a second antibody conjugated to at least one radiolabeled second antibody. In one embodiment, the therapeutic agent is a second antibody conjugated to at least one hormone. In one embodiment, the therapeutic agent is a second antibody conjugated to at least one antisense oligonucleotide. In one embodiment, the therapeutic agent is a second antibody conjugated to at least one cytotoxic agent. In one embodiment, the therapeutic agent is a second antibody conjugated to a combination thereof. In one embodiment, the therapeutic agent is a combination of a second antibody or a fragment thereof, an immunomodulator, a hormone, a cytotoxic agent, an enzyme, a radionuclide, or a second antibody conjugated to at least one immunomodulator, enzyme, radiolabel, hormone, antisense oligonucleotide, or cytotoxic agent. In another embodiment, the diagnostic marker is a diagnostic marker suitable for PET imaging. In another embodiment, the diagnostic marker is a diagnostic marker suitable for single-photon emission computed tomography (SPECT) imaging. In another embodiment, the diagnostic marker is a diagnostic marker suitable for MRI. In another embodiment, the diagnostic marker is a diagnostic marker suitable for optical imaging. In another embodiment, the diagnostic marker is a diagnostic marker suitable for (photo)acoustic imaging, such as... 11 C 13 N、 15 O、 99m Tc, 61 Cu、 62 Cu、 64 Cu、 67 Cu、 18 F, 19 F, 66 Ga、 67 Ga、 68 Ga、 72 Ga、 123 I, 124I, 111 In、 177 Lu、 44 Sc、 47 Sc、 86 Y、 88 Y、 90 Y、 45 Ti、 89 Zr, indocyanine green, IRDye 800CW, fluorescein (FITC), and magnetic (e.g., iron oxide) nanoparticles. This list is not intended to be limiting.

[0214] The present invention also provides isolated nucleic acids encoding either the anti-CD103 antibody or the antigen-binding fragment of the present invention.

[0215] The present invention also provides expression vectors comprising one or more nucleic acids of the present invention. The expression vector is a DNA molecule containing regulatory elements necessary for the transcription of a target nucleic acid in a host cell. Typically, the target nucleic acid is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancer elements. Such target nucleic acids are considered "operably linked" to regulatory elements when the regulatory elements control gene expression.

[0216] These isolated nucleic acids and the expression vectors containing them can be used to express the antibodies or antigen-binding fragments of the present invention in recombinant host cells. Therefore, the present invention also provides host cells containing the expression vectors of the present invention.

[0217] The present invention also provides a container or injection device comprising either the anti-CD103 antibody or the antigen-binding fragment of the present invention.

[0218] The present invention also provides a method for generating the anti-CD103 antibody or antigen-binding fragment of the present invention, the method comprising: culturing host cells containing said polynucleotides under conditions favorable to the expression of a heavy chain and / or light chain polynucleotide encoding the antibody (or its antigen-binding fragment) of the present invention; and optionally, recovering the antibody or antigen-binding fragment from the host cells and / or the culture medium. In one embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are located in a single vector. In another embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are located in different vectors.

[0219] In another aspect, the present invention relates to methods for imaging tissue or other biological samples. These methods include contacting the biological sample with an anti-CD103 antibody and detecting the presence or amount of binding of the antibody to CD103 present in the biological sample. Thus, the anti-CD103 antibody is used as an imaging agent.

[0220] In one related aspect, the present invention relates to methods for generating imaging agents comprising an anti-CD103 antibody and a diagnostic marker. These methods include forming a covalent association between the anti-CD103 antibody and the diagnostic marker. Alternatively, these methods include forming a non-covalent association between the anti-CD103 antibody and the diagnostic marker. In the case of radiolabeling, a bifunctional chelating agent comprising a first functional group for immobilizing a radioactive metal and a second functional group for covalently linking to the antibody can be used to chelate the cotope. Examples of such chelating agents include, but are not limited to, DOTA, NOA, TRITA, TETA, TACN, cyclen, cyclam, homocyclen, EDTA, DTPA, DOTP, and NOTMP. The functionalization of chelating agents provides a bonding chemistry that provides a terminal functional moiety, optionally free from the group consisting of: a protected or unprotected thioglycolic moiety, a protected or unprotected amine moiety, a protected or unprotected hydroxyl moiety, a primary amine reactive moiety, a thioglycolic reactive moiety, a photoreactive moiety, a carboxyl reactive moiety, an arginine reactive moiety, and a carbonyl reactive moiety. The functionalization of chelating agents provides a bonding chemistry that provides a terminal functional moiety (i.e., a protected thioglycolic moiety). The functionalization of chelating agents provides a bonding chemistry that provides a terminal functional moiety (i.e., an unprotected thioglycolic moiety). The functionalization of chelating agents provides a bonding chemistry that provides a terminal functional moiety (i.e., a protected amine moiety). The functionalization of chelating agents provides a bonding chemistry that provides a terminal functional moiety (i.e., an unprotected amine moiety). The functionalization of chelating agents provides a bonding chemistry that provides a terminal functional moiety (i.e., a protected hydroxyl moiety). Functionalized forms of chelating agents provide bonding chemistry for providing terminal functional moieties (i.e., unprotected hydroxyl moieties). Functionalized forms of chelating agents provide bonding chemistry for providing terminal functional moieties (i.e., amine reactive moieties). Functionalized forms of chelating agents provide bonding chemistry for providing terminal functional moieties (i.e., thiohydrogen reactive moieties). Functionalized forms of chelating agents provide bonding chemistry for providing terminal functional moieties (i.e., photoreactive moieties). Functionalized forms of chelating agents provide bonding chemistry for providing terminal functional moieties (i.e., carboxyl reactive moieties). Functionalized forms of chelating agents provide bonding chemistry for providing terminal functional moieties (i.e., arginine reactive moieties). Functionalized forms of chelating agents provide bonding chemistry for providing terminal functional moieties (i.e., carbonyl reactive moieties). Direct labeling methods are also covered, in which a reducing agent converts a disulfide bond into a free thiol, which binds to a radioactive label.

[0221] In various embodiments, the diagnostic marker may be selected from the group consisting of: enzymes, nucleic acids, fluorophores, biotin, avidin, streptavidin, digoxigenin, maltose, oligohistidine, 2,4-dinitrophenyl, phenylarsinate, metals, peptide tags, fluorescent or colored microspheres, fluorescent particles, and colored latex particles. This list is not intended to be limiting. In various embodiments, the diagnostic marker is an enzyme. In various embodiments, the diagnostic marker is a nucleic acid. In various embodiments, the diagnostic marker is a fluorophore. In various embodiments, the diagnostic marker is biotin. In various embodiments, the diagnostic marker is avidin. In various embodiments, the diagnostic marker is streptavidin. In various embodiments, the diagnostic marker is digoxigenin. In various embodiments, the diagnostic marker is maltose. In various embodiments, the diagnostic marker is oligohistidine. In various embodiments, the diagnostic marker is 2,4-dinitrophenyl. In various embodiments, the diagnostic marker is phenylarsinate. In various embodiments, the diagnostic marker is a metal. In various embodiments, the diagnostic marker is a peptide tag. In various embodiments, the diagnostic marker is a fluorescent microsphere. In various embodiments, the diagnostic marker is a colored microsphere. In various embodiments, the diagnostic marker is a fluorescent particle. In various embodiments, the diagnostic marker is a colored latex particle. Such markers can be conjugated to antibodies by means of a cross-linking agent containing maleimide, alkyl halide, aryl halide, α-haloacyl, activated aryl, pyridyl disulfide, carbonyl, carboxyl, thiol, thioester, disulfide, N-hydroxy-succinimide, or cyclic thiolactone, etc.

[0222] In some embodiments, the biological sample is in vivo tissue, and the method is an in vivo imaging method. In some embodiments, the biological sample is in vivo tissue, and the method is an in vivo imaging method such as PET imaging. In some embodiments, the biological sample is in vivo tissue, and the method is an in vivo imaging method, single-photon emission computed tomography (SPECT). In some embodiments, the biological sample is in vivo tissue, and the method is an in vivo imaging method, MRI. In these methods, the anti-CD103 antibody is a detectable marker according to the requirements of the imaging method used. Suitable diagnostic markers are described herein, including but not limited to... 11 C 13 N、 15 O、 99m Tc, 61 Cu、 62 Cu、 64 Cu、 67 Cu、 18 F, 19 F, 66 Ga、 67 Ga、 68 Ga、72 Ga、 123 I, 124 I, 111 In、 177 Lu、 44 Sc、 47 Sc、 86 Y、 88 Y、 90 Y、 45 Ti、 89 Zr, indocyanine green, IRDye800CW, fluorescein (FITC), and magnetic nanoparticles (e.g., iron oxide).

[0223] In various embodiments, the anti-CD103 antibody used as an imaging agent blocks CD103 from binding to its homologous receptor E-cadherin. In various embodiments, the anti-CD103 antibody used as an imaging agent does not block CD103 from binding to its homologous receptor E-cadherin. In various embodiments, the anti-CD103 antibody used as an imaging agent partially blocks CD103 from binding to its homologous receptor E-cadherin. Examples of each of these types of anti-CD103 antibodies are described below.

[0224] In various embodiments, the antibody of the present invention is used as an imaging agent in methods for imaging tissues or other biological samples.

[0225] In various embodiments, methods for imaging tissues or other biological samples use antigen-binding fragments of the antibodies of the present invention as imaging agents.

[0226] In various embodiments, methods for imaging tissues or other biological samples use antibodies or antigen-binding fragments thereof as imaging agents, said antibodies or antigen-binding fragments thereof comprising:

[0227] a. Heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence different from SEQ ID NO: 1 due to 1, 2 or 3 conserved substitutions,

[0228] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 2 or differs from the amino acid sequence of SEQ ID NO: 2 due to 1, 2 or 3 conserved substitutions.

[0229] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 3 or differs from the amino acid sequence of SEQ ID NO: 3 due to 1, 2 or 3 conserved substitutions.

[0230] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 4 or differs from the amino acid sequence of SEQ ID NO: 4 due to 1, 2 or 3 conserved substitutions.

[0231] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence different from SEQ ID NO: 5 due to 1, 2 or 3 conserved substitutions, and

[0232] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 6 or differs from the amino acid sequence of SEQ ID NO: 6 due to 1, 2 or 3 conserved substitutions;

[0233] or

[0234] g. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 9 or differs from the amino acid sequence of SEQ ID NO: 9 due to 1, 2 or 3 conserved substitutions.

[0235] h. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 10 or differs from the amino acid sequence of SEQ ID NO: 10 due to 1, 2 or 3 conserved substitutions.

[0236] i. Heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or differing from the amino acid sequence of SEQ ID NO: 11 due to 1, 2 or 3 conserved substitutions,

[0237] j. Light chain variable region CDR1, said light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or differing from the amino acid sequence of SEQ ID NO: 12 due to 1, 2 or 3 conserved substitutions,

[0238] k. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence different from SEQ ID NO: 13 due to 1, 2 or 3 conserved substitutions, and

[0239] 1. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 14 or differs from the amino acid sequence of SEQ ID NO: 14 due to 1, 2 or 3 conserved substitutions;

[0240] or

[0241] m. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 17 or differs from the amino acid sequence of SEQ ID NO: 17 due to 1, 2 or 3 conserved substitutions.

[0242] n. Heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 18 or differing from the amino acid sequence of SEQ ID NO: 18 due to 1, 2 or 3 conserved substitutions.

[0243] o. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 19 or differs from the amino acid sequence of SEQ ID NO: 19 due to 1, 2 or 3 conserved substitutions.

[0244] p. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or differs from the amino acid sequence of SEQ ID NO: 20 due to 1, 2 or 3 conserved substitutions.

[0245] q. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence different from SEQ ID NO: 21 due to 1, 2 or 3 conserved substitutions, and

[0246] r. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or differs from the amino acid sequence of SEQ ID NO: 22 due to 1, 2 or 3 conserved substitutions;

[0247] or

[0248] s. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 25 or differs from the amino acid sequence of SEQ ID NO: 25 due to 1, 2 or 3 conserved substitutions.

[0249] t. Heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 26 or differing from the amino acid sequence of SEQ ID NO: 26 due to 1, 2 or 3 conserved substitutions,

[0250] u. Heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 27 or differing from the amino acid sequence of SEQ ID NO: 27 due to 1, 2 or 3 conserved substitutions,

[0251] v. Light chain variable region CDR1, said light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 28 or differing from the amino acid sequence of SEQ ID NO: 28 due to 1, 2 or 3 conserved substitutions.

[0252] w. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence different from SEQ ID NO: 29 due to 1, 2 or 3 conserved substitutions, and

[0253] x. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 30 or differs from the amino acid sequence of SEQ ID NO: 30 due to 1, 2 or 3 conserved substitutions;

[0254] or

[0255] y. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 33 or differs from the amino acid sequence of SEQ ID NO: 33 due to 1, 2 or 3 conserved substitutions.

[0256] z. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 34 or differs from the amino acid sequence of SEQ ID NO: 34 due to 1, 2 or 3 conserved substitutions.

[0257] aa. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 35 or differs from the amino acid sequence of SEQ ID NO: 35 due to 1, 2 or 3 conserved substitutions.

[0258] bb. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 36 or differs from the amino acid sequence of SEQ ID NO: 36 due to 1, 2 or 3 conserved substitutions.

[0259] cc. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence different from SEQ ID NO: 37 due to 1, 2 or 3 conserved substitutions, and

[0260] dd. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 38 or differs from the amino acid sequence of SEQ ID NO: 38 due to 1, 2 or 3 conserved substitutions.

[0261] In various other embodiments, methods for imaging tissues or other biological samples use antibodies or antigen-binding fragments thereof as imaging agents, said antibodies or antigen-binding fragments thereof comprising:

[0262] a. A heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 7 and a light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 8; or

[0263] b. A heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 15 and a light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 16; or

[0264] c. A heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 23 and a light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 24; or

[0265] d. A heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 31 and a light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 32; or

[0266] e. The heavy chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 39 and the light chain of an antibody or antigen-binding fragment containing the amino acid sequence of SEQ ID NO: 40;

[0267] Alternatively, in each case, the sequence has at least 95% (and more preferably 97% or 99%) sequence similarity or identity with a given SEQ ID NO, and any differences in such sequences are limited to amino acids that are not part of the CDR sequence within the SEQ ID NO.

[0268] In another aspect, the present invention relates to a method of using the anti-CD103 antibody of the present invention as a therapeutic agent. In a related aspect, the present invention relates to the use of the anti-CD103 antibody of the present invention in the manufacture of pharmaceutical agents. In another related aspect, the present invention relates to a method of inhibiting CD103 signaling using the anti-CD103 antibody of the present invention. In yet another related aspect, the present invention relates to a method of blocking CD103 binding to E-cadherin using the anti-CD103 antibody of the present invention.

[0269] In one embodiment, the method is for treating a CD103 signaling-mediated disorder in an individual in need, and the method includes administering an effective amount of the anti-CD103 antibody of the present invention to the individual, wherein the anti-CD103 antibody is optionally conjugated to a cytotoxic agent.

[0270] In one embodiment, the method is for treating a CD103 signaling-mediated disorder in an individual in need, and the method includes administering to the individual an effective amount of an antigen-binding fragment of the anti-CD103 antibody of the present invention, wherein the anti-CD103 antibody is optionally conjugated to a cytotoxic agent.

[0271] In one embodiment, the method is for preventing CD103 signaling-mediated disorders in individuals of need, and the method includes administering to the individual an effective amount of the anti-CD103 antibody of the present invention or an antigen-binding fragment thereof, wherein the anti-CD103 antibody is optionally conjugated to a cytotoxic agent.

[0272] In one embodiment, the method is for preventing CD103 signaling-mediated disorders in individuals of need, and the method includes administering to the individual an effective amount of an antigen-binding fragment of the anti-CD103 antibody of the present invention, wherein the anti-CD103 antibody is optionally conjugated to a cytotoxic agent.

[0273] In another embodiment, the method is used to inhibit CD103 signaling in cells, and the method includes contacting the cells with the anti-CD103 antibody of the present invention or its antigen-binding fragment.

[0274] In another embodiment, the method is used to inhibit CD103 binding to E-cadherin present on cells, and the method includes contacting the cells with the anti-CD103 antibody of the present invention or its antigen-binding fragment.

[0275] In another embodiment, the method is used to deplete cells expressing CD103 in an individual, and the method includes administering an effective amount of the anti-CD103 antibody of the present invention or an antigen-binding fragment thereof to the individual, wherein the anti-CD103 antibody is optionally conjugated to a cytotoxic agent.

[0276] In another embodiment, the method is a method for treating or preventing diseases selected from the group consisting of: hairy cell leukemia, HCLv, intestinal and extraintestinal lymphoma, enteropathy-associated T-cell lymphoma (EATL), T-lymphoblastic leukemia / lymphoma (T-ALL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell leukemia / lymphoma (ATLL), mycosis fungoides (MF), anaplastic large cell lymphoma ALCL, cutaneous T-cell lymphoma (CTCL), Sezary syndrome (SS), Alzheimer's disease, and Parkinson's disease. Diseases including multiple sclerosis, IgM polyneuropathy, myasthenia gravis, atopic dermatitis, allergic reactions, asthma, systemic inflammatory response syndrome (SIRS), sepsis, septic shock, atherosclerosis, celiac disease, dermatomyositis, scleroderma, interstitial cystitis, transplant rejection, graft-versus-host disease, Aicardi-Goutieres syndrome, Hutchison Guilford progeria syndrome, Singleton-Merten syndrome, proteasome-associated autoinflammatory syndrome, SAVI (STING-related vascular disease with onset in infancy), CANDLE (chronic atypical neutrophilic dermatitis with lipid metabolism disorders and hyperthermia) syndrome, frostbite-like lupus erythematosus, systemic lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, and Wegener's disease. The method includes administering to an individual an effective amount of the anti-CD103 antibody of the present invention or its antigen-binding fragment, wherein the anti-CD103 antibody is optionally conjugated to a cytotoxic agent. Other conditions include inflammatory bowel diseases (e.g., ulcerative colitis, Crohn's disease), idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, glomerulonephritis, autoimmune myocarditis, myasthenia gravis, vasculitis, type 1 diabetes, type 2 diabetes, Sjorgen's syndrome, X-linked reticulochromocytosis, polymyositis, spinal chondrodysplasia, and age-related macular degeneration.

[0277] In another embodiment, the method is for treating individuals in need of a disease selected from the group consisting of: hairy cell leukemia, HCLv, intestinal and extraintestinal lymphoma, enteropathy-associated T-cell lymphoma (EATL), T-lymphoblastic leukemia / lymphoma (T-ALL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell leukemia / lymphoma (ATLL), mycosis fungoides (MF), anaplastic large cell lymphoma ALCL, and cutaneous T-cell lymphoma (CTCL). Cezari syndrome (SS), Alzheimer's disease, Parkinson's disease, multiple sclerosis, IgM polyneuropathy, myasthenia gravis, atopic dermatitis, allergic reactions, asthma, systemic inflammatory response syndrome (SIRS), sepsis, septic shock, atherosclerosis, celiac disease, dermatomyositis, scleroderma, interstitial cystitis, transplant rejection, graft-versus-host disease, Ekaterie-Gutler syndrome, Haus-Gilles syndrome, Szymborskin syndrome, proteasome-associated autoinflammatory syndrome, SAVI (STING-related vascular disease with onset in infancy), CANDLE syndrome (chronic atypical neutrophilic dermatitis with lipid metabolism disorder and hyperthermia), frostbite-like lupus erythematosus, systemic lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, Wegener's disease, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, glomerulonephritis, autoimmune myocarditis, myasthenia gravis, vasculitis, type 1 diabetes, type 2 diabetes, Sjögren's syndrome, X-linked reticulochromocytosis, polymyositis, spinal chondrodysplasia, and age-related macular degeneration, and the method includes administering an effective amount of the anti-CD103 antibody of the present invention to an individual, wherein the anti-CD103 antibody is optionally conjugated to a cytotoxic agent.

[0278] In another embodiment, the method is for the prevention of diseases selected from the group consisting of: hairy cell leukemia, HCLv, intestinal and extraintestinal lymphoma, enteropathy-associated T-cell lymphoma (EATL), T-lymphoblastic leukemia / lymphoma (T-ALL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell leukemia / lymphoma (ATLL), mycosis fungoides (MF), anaplastic large cell lymphoma ALCL, and cutaneous T-cell lymphoma (CTCL). Cezari syndrome (SS), Alzheimer's disease, Parkinson's disease, multiple sclerosis, IgM polyneuropathy, myasthenia gravis, atopic dermatitis, allergic reactions, asthma, systemic inflammatory response syndrome (SIRS), sepsis, septic shock, atherosclerosis, celiac disease, dermatomyositis, scleroderma, interstitial cystitis, transplant rejection, graft-versus-host disease, Ekaterie-Gutler syndrome, Haus-Gilles syndrome, Szymborskin syndrome, proteasome-associated autoinflammatory syndrome, SAVI (STING-related vascular disease with onset in infancy), CANDLE syndrome (chronic atypical neutrophilic dermatitis with lipid metabolism disorder and hyperthermia), frostbite-like lupus erythematosus, systemic lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, Wegener's disease, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, glomerulonephritis, autoimmune myocarditis, myasthenia gravis, vasculitis, type 1 diabetes, type 2 diabetes, Sjögren's syndrome, X-linked reticulochromocytosis, polymyositis, spinal chondrodysplasia, and age-related macular degeneration, and the method includes administering an effective amount of the anti-CD103 antibody of the present invention to an individual, wherein the anti-CD103 antibody is optionally conjugated to a cytotoxic agent.

[0279] In various implementations, these methods utilize the antibodies of the present invention.

[0280] In various embodiments, these methods utilize the antigen-binding fragment of the antibody of the present invention.

[0281] In various embodiments, these methods use antibodies or antigen-binding fragments thereof, said antibodies or antigen-binding fragments comprising:

[0282] a. Heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence different from SEQ ID NO: 1 due to 1, 2 or 3 conserved substitutions,

[0283] b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 2 or differs from the amino acid sequence of SEQ ID NO: 2 due to 1, 2 or 3 conserved substitutions.

[0284] c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 3 or differs from the amino acid sequence of SEQ ID NO: 3 due to 1, 2 or 3 conserved substitutions.

[0285] d. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 4 or differs from the amino acid sequence of SEQ ID NO: 4 due to 1, 2 or 3 conserved substitutions.

[0286] e. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence different from SEQ ID NO: 5 due to 1, 2 or 3 conserved substitutions, and

[0287] f. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 6 or differs from the amino acid sequence of SEQ ID NO: 6 due to 1, 2 or 3 conserved substitutions;

[0288] or

[0289] g. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 9 or differs from the amino acid sequence of SEQ ID NO: 9 due to 1, 2 or 3 conserved substitutions.

[0290] h. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 10 or differs from the amino acid sequence of SEQ ID NO: 10 due to 1, 2 or 3 conserved substitutions.

[0291] i. Heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or differing from the amino acid sequence of SEQ ID NO: 11 due to 1, 2 or 3 conserved substitutions,

[0292] j. Light chain variable region CDR1, said light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or differing from the amino acid sequence of SEQ ID NO: 12 due to 1, 2 or 3 conserved substitutions,

[0293] k. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence different from SEQ ID NO: 13 due to 1, 2 or 3 conserved substitutions, and

[0294] 1. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 14 or differs from the amino acid sequence of SEQ ID NO: 14 due to 1, 2 or 3 conserved substitutions;

[0295] or

[0296] m. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 17 or differs from the amino acid sequence of SEQ ID NO: 17 due to 1, 2 or 3 conserved substitutions.

[0297] n. Heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 18 or differing from the amino acid sequence of SEQ ID NO: 18 due to 1, 2 or 3 conserved substitutions.

[0298] o. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 19 or differs from the amino acid sequence of SEQ ID NO: 19 due to 1, 2 or 3 conserved substitutions.

[0299] p. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or differs from the amino acid sequence of SEQ ID NO: 20 due to 1, 2 or 3 conserved substitutions.

[0300] q. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence different from SEQ ID NO: 21 due to 1, 2 or 3 conserved substitutions, and

[0301] r. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or differs from the amino acid sequence of SEQ ID NO: 22 due to 1, 2 or 3 conserved substitutions;

[0302] or

[0303] s. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 25 or differs from the amino acid sequence of SEQ ID NO: 25 due to 1, 2 or 3 conserved substitutions.

[0304] t. Heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 26 or differing from the amino acid sequence of SEQ ID NO: 26 due to 1, 2 or 3 conserved substitutions,

[0305] u. Heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 27 or differing from the amino acid sequence of SEQ ID NO: 27 due to 1, 2 or 3 conserved substitutions,

[0306] v. Light chain variable region CDR1, said light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 28 or differing from the amino acid sequence of SEQ ID NO: 28 due to 1, 2 or 3 conserved substitutions.

[0307] w. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence different from SEQ ID NO: 29 due to 1, 2 or 3 conserved substitutions, and

[0308] x. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 30 or differs from the amino acid sequence of SEQ ID NO: 30 due to 1, 2 or 3 conserved substitutions;

[0309] or

[0310] y. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 33 or differs from the amino acid sequence of SEQ ID NO: 33 due to 1, 2 or 3 conserved substitutions.

[0311] z. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 34 or differs from the amino acid sequence of SEQ ID NO: 34 due to 1, 2 or 3 conserved substitutions.

[0312] aa. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 35 or differs from the amino acid sequence of SEQ ID NO: 35 due to 1, 2 or 3 conserved substitutions.

[0313] bb. Light chain variable region CDR1, wherein the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 36 or differs from the amino acid sequence of SEQ ID NO: 36 due to 1, 2 or 3 conserved substitutions.

[0314] cc. Light chain variable region CDR2, said light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence different from SEQ ID NO: 37 due to 1, 2 or 3 conserved substitutions, and

[0315] dd. Light chain variable region CDR3, wherein the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 38 or differs from the amino acid sequence of SEQ ID NO: 38 due to 1, 2 or 3 conserved substitutions.

[0316] It should be understood that the present invention is capable of having embodiments other than those described, and can be practiced and performed in various ways. Furthermore, it should be understood that the wording and terminology used herein and in the abstract are for descriptive purposes and should not be considered limiting. Therefore, those skilled in the art will understand that the concepts on which this disclosure is based can readily be used as the basis for designing other structures, methods, and systems for carrying out several objectives of the invention. Accordingly, it is important that the claims be considered to include such equivalent structures, provided they do not depart from the spirit and scope of the invention. Attached Figure Description

[0317] Figure 1 : Cellular ELISA binding data of anti-hCD103 mAb with CHO.K1-hCD103 / hβ7, CHO.K1-rhCD103 / rhβ7 and CHO.K1-hα4 / hβ7.

[0318] Figure 2 Phylogenetic tree of different hCD103 VL and VH sequences.

[0319] Figure 3 CELISA binding data of hCD103.01A, hCD103.05A, hCD103.06A, Fab.hCD103.01.C1, Fab.hCD103.05.C1, and Fab.hCD103.06.C1 with CHO.K1.hCD103 / hβ7, CHO.K1, and recombinant human CD103 / β7.

[0320] Figure 4 AF647-hCD103.01A, AF647-hCD103.05A, AF647-hCD103.06A, AF647-Fab.hCD103.01.C1, AF647-Fab.hCD103.05.C1, and AF647-Fab.hCD103.06.C1 bound to CHO.K1.hCD103 / hβ7 cells, respectively.

[0321] Figure 5 Representative staining of CD8 and CD103 tumor digests under control antibody or anti-hCD103 mAb conditions.

[0322] Figure 6 The binding of anti-hCD103 mAb candidates to CD3+ cells (total T cell population), CD3+CD103+CD8+ cells (T cell subsets), and CD33+ cells (bone marrow-like population). N = 10 different tumor digests.

[0323] Figure 7 Staining of tumor digests containing CD8 and CD103 under control reagent or anti-hCD103 Fab conditions. The top two lines show staining under parental Fab (unlabeled) conditions and detection under second anti-Fab reagent conditions, while the bottom two lines show staining under AF647 conjugated Fab conditions and the results read directly.

[0324] Figure 8 CD103+CD8+ T cells were pre-incubated with our CD103 mAb or a commercial CD103 mAb (clone BerACT-8, BD bioscience) and subsequently incubated with its fluorescently labeled counterpart to investigate differences in affinity and competition between mAbs. The percentage of binding to the fluorescently labeled mAbs was determined using flow cytometry. Maximum binding was set at 100%.

[0325] Figure 9 The binding of CD103+ T cells to recombinant E-cadherin in the presence of anti-hCD103 mAb and control. Cells were pre-incubated with the antibody and then incubated with recombinant E-cadherin (left set of bars, before treatment), or cells were first incubated with recombinant E-cadherin and then the mAb was added (right set of bars, treatment).

[0326] Figure 10 :Df conjugation and binding of parental anti-hCD103 mAb (candidates hCD103.01A and hCD103.05A) or Fab fragment (candidates Fab.hCD103.01.C1 and Fab.hCD103.05.C1) to CHO.K1-hCD103 / hβ7.

[0327] Figure 11 :different 89 Zr level 89 Radiochemical purity of Zr-labeled anti-hCD103 mAb (candidates hCD103.01A and hCD103.05A).

[0328] Figure 12 : 89 Zr-labeled anti-hCD103 mAb (candidates hCD103.01A and hCD103.05A) binds to CHO.K1-hCD103 / hβ7 (red) and CHO.K1 (blue). Binding was measured as... 89 The amount of Zr-mAb binding activity.

[0329] Figure 13 AE: 89 Zr-hCD103.01A and 89 PET imaging protocol for Zr-hCD103.05A (13A), 2D visualization of PET imaging (coronas) (13B), in mice with CHO.K1-hCD103 / hβ7 or CHO.K1 WT 89 Zr-hCD103.01A and 89 The ratio of Zr-hCD103.05A targets to blood and the ratio of blood to target tissues 89 Zr-hCD103.01A and 89 Zr-hCD103.05A levels (13C and 13D), and comparative tissue allocation ratios (13E). Tumors (targets) here refer to CHO.K1-hCD103 / hβ7 (red and green) or CHO.K1 WT (gray), respectively. Mice with CHO.K1 WT were injected with… 89 Zr-hCD103.01A was used as a non-specific control group.

[0330] Figure 14 Six days after injection, in mice (n=3) carrying CHO.K1-hCD103 / hβ7 or CHO.K1 WT, 89 Zr-hCD103.01A and 89 Biodistribution results of Zr-hCD103.05A. Tumors here refer to CHO.K1-hCD103 / hβ7 (red or green) or CHO.K1 (gray), respectively. Mice with CHO.K1 WT were injected with... 89 Zr-hCD103.01A was used as a non-specific control group.

[0331] Figure 15 24 hours after injection, mice with CHO.K1-hCD103 / hβ7 (n=2) or CHO.K1 WT (n=3) 89 Zr-Fab.hCD103.01.C1 and 89Biodistribution results of Zr-Fab.hCD103.05.C1. Tumors here refer to CHO.K1-hCD103 / hβ7 (red or green) or CHO.K1 WT (gray), respectively. Mice with CHO.K1 WT were injected with... 89 Zr-Fab.hCD103.01.C1 was used as a non-specific control group. Detailed Implementation

[0332] Most (if not all) forms of cancer immunotherapy rely on inducing a T-cell-based immune response against antigens preferentially or selectively expressed in cancer cells and presented via major histocompatibility molecules (MHC) on the cell surface. This mode of action is perhaps largely exemplified by the keen response of patients with high tumor mutational burden (TMB) to treatment with monoclonal antibodies that block programmed death-1 (PD-1) or its ligand (PD-L1). In efforts to expand promising immunotherapies to more patients, more than 2,000 (combination) immunotherapy trials have been initiated for many types of cancer. Given the abundance of treatment options, there is an urgent need for biomarkers to guide drug development, treatment assessment, and evaluation of treatment efficacy.

[0333] A hallmark of successful immunotherapy is an increase in the activity and number of T cells (tumor-infiltrating lymphocytes; TILs) within tumor masses. Therefore, the TIL “load” in tumor lesions represents an attractive biomarker for supporting patient selection and monitoring in support of immunotherapy. Unfortunately, a broad spectrum of TILs exists, and not every T cell within a tumor participates in the anti-cancer immune response. In recent years, the integrin subunit CD103 has emerged as a marker of TILs, used to predict benefit against epithelial malignancies, including esophageal cancer, melanoma, lung cancer, breast cancer, bladder cancer, and all gynecological cancers. Importantly, CD103… + TIL contains CD39, which has previously been associated with anti-cancer effects in tumors. + PD-1 + and CD137 + TIL population. Mechanistic studies also confirmed that CD103 is induced after T cells are specifically activated against their homologous targets, and that CD103 is involved in successful anti-PD-1 therapy in patients with melanoma, esophageal squamous cell carcinoma, and non-small cell lung cancer. + Cells significantly expanded. Finally, CD103 was absent in other immune cell populations within the tumor and therefore provided excellent cell specificity. Overall, intratumoral CD103 detection provides an excellent biomarker for determining TIL load and response to immunotherapy.

[0334] Currently, the standard for assessing TIL load is immunohistochemistry (IHC) of tissue biopsies. However, several known barriers to biopsy-based techniques exist, such as poor lesion accessibility, patient burden, intralesional and interlesional tumor heterogeneity, and sampling errors. Furthermore, using current techniques, it is difficult / impossible to track T cell infiltration over time in tumor lesions. To overcome these barriers and obtain information on TIL load in all tumor lesions and toxically sensitive organs of a patient, non-invasive whole-body imaging techniques can be applied. Positron emission tomography (PET) is a molecular imaging technique that allows for repetitive, non-invasive clinical assessment of tumor signatures, such as the expression of hormone and growth factor receptors. PET is characterized by high spatial resolution, sensitivity, and the possibility of quantifying imaging signals. PET can detect CD103 in tumors. + Cell-specific monitoring requires the availability of appropriately sensitive radiopharmaceuticals. Therefore, we describe here the development of various anti-CD103-specific antibodies suitable for use with radiopharmaceuticals.

[0335] In addition to its diagnostic / prognostic significance, CD103 represents a therapeutic target for a variety of diseases. For example, CD103 is expressed in several lymphocyte subsets, including T cells, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. The interaction between CD103 and E-cadherin enables lymphocyte attachment to epithelial cells. While E-cadherin is constitutively expressed in epithelial cells, CD103 expression is induced in T cells following in vitro inflammatory stimulation. Blockade of CD103 is particularly relevant in conditions involving CD8+ and Th9 cell proliferation (such as inflammatory bowel disease) and in allogeneic transplant rejection. CD103 is also expressed by dendritic cells and on various T cell types, including malignant forms of these cells. In addition, tumor-associated CD103+ CD8 T cells can exhibit a tolerogenic phenotype, and CD103+ DCs show expression of immunomodulatory molecules and produce immunosuppressive factors such as IL-10, TGF-β, IL-35, and indoleamine 2,3-dioxygenase (IDO), thereby causing T cell weakness and apoptosis and induction of Tregs.

[0336] Therefore, molecules that bind to CD103 and interfere with its interaction between CD103 and E-cadherin are potential drug candidates for disease treatment. Similarly, molecules that bind to CD103 can, for example, be used as part of an antibody-drug conjugate to deplete CD103-expressing cells for therapeutic purposes.

[0337] definition

[0338] To facilitate a better understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0339] Unless the context clearly indicates otherwise, as used herein, including in the appended claims, the singular forms of words such as “a,” “an,” and “the” include their corresponding plural references.

[0340] "Administration" and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. Cell treatment encompasses contacting a reagent with cells, as well as contacting a reagent with a fluid, wherein the fluid contacts the cell. "Administration" and "treatment" also mean, for example, the in vitro and ex vivo treatment of cells with a reagent, diagnostic agent, conjugated compound, or with another cell.

[0341] "Treatment" means administering the agent, internally or externally, to a subject or patient who has one or more symptoms of a disease or is suspected of having a disease for which the therapeutic agent (such as a composition containing either an antibody or an antigen-binding fragment of the present invention) is therapeutically active. Typically, the agent is administered in an amount that effectively relieves one or more such symptoms by inducing regression of or inhibiting the progression of one or more disease symptoms in the treated subject or population to any clinically measurable degree. The amount of therapeutic agent that effectively relieves any particular disease symptom can vary depending on factors such as the disease condition, the patient's age and weight, and the drug's ability to elicit the desired response in the subject. Whether the disease symptoms have been relieved can be assessed by any clinical measurement typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptoms.

[0342] “Recombinant expression” of a protein refers to the transcription and translation of a foreign gene in a host organism to produce a protein referred to in this paper as a “recombinant protein”.

[0343] As used herein, the term "positron emission tomography (PET)" refers to a nuclear imaging technique used in the medical field to aid in the diagnosis of diseases. PET allows physicians to examine the entire patient immediately by generating images of many functions of the human body that are not available with other imaging techniques. In this respect, PET shows how the body works (physiologically or functionally) rather than simply how it looks. Applications of PET imaging include those in oncology, cardiology, and neurology. In PET, short-lived positron-emitting isotopes, referred to herein as radiopharmaceuticals, are injected into the patient. When these radioactive agents are administered to the patient, they are distributed throughout the body according to physiological pathways associated with their stable counterparts.

[0344] As used herein, the term "SPECT" refers to "Single-Photon Emission Computed Tomography," a nuclear medicine computed tomography imaging technique that uses gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera and is able to provide true 3D information. This information is typically presented as cross-sectional images through the patient, but can be freely formatted or manipulated as needed. The basic technique requires the delivery of gamma-emitting isotopes (called radionuclides) into the patient's body, usually by injection into the bloodstream.

[0345] For the purposes of this specification or claims, the term "detectable marker" means a marker molecule that is indirectly or directly linked to an antibody or its antigen-binding fragment according to this disclosure, wherein the marker molecule facilitates the detection of an antibody in which it is present. Therefore, "detectable marker" and "marker molecule" are used synonymously.

[0346] As used herein, the term "imaging agent" refers to a labeled portion that can be used to indicate the location of a marker and its associated entity in cells or tissues of an animal or human subject, or under in vitro conditions. While agents may include those that provide detectable signals (such as fluorescence, luminescence, radioactivity) or can be detected by methods such as MRI imaging, in the context of the probes and methods of use disclosed herein, the term "imaging agent" specifically refers to a marker detectable by imaging techniques such as PET or SPECT, such as, but not limited to, markers. 64 Cu、 67 Cu、 89 Zr、 124 l、 86 Y、 90 Y、 111 ln、 123 / 131 l、 177 Lu、 18 F, 99mTc, etc. In the most preferred embodiment of the immunoconjugated probe disclosed herein, the labeling agent is 89-zirconium (Zr), however, it is contemplated that any metal isotope (or any other PET-compatible labeling agent) that provides an image generated by PET and can be linked or conjugated to an antibody or antibody fragment targeting phosphatidylinositol proteoglycan-3 may be used.

[0347] The term "biological sample" refers to a tissue, body fluid, or other sample derived from an organism (e.g., a human patient) or from a component of an organism (e.g., cells). The sample can be any biological tissue or fluid. Samples can be "clinical samples," which are samples derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white blood cells), amniotic fluid, plasma, bone marrow, and tissue or fine-needle biopsy sections, urine, peritoneal fluid, and pleural fluid, or cells derived from these samples. Biological samples may also include tissue or tissue sections (such as frozen or paraffin-embedded sections) obtained for histological purposes. Biological samples may also be referred to as "patient samples."

[0348] In some embodiments, the biological sample may be a tumor within or removed from a biological organism. As used herein, the term "tumor" refers to the malignant or benign growth and proliferation of all proliferative cells, as well as all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe physiological conditions in mammals typically characterized by disordered cell growth. Specifically, the probes and compositions of this disclosure are mostly favorable for the detection of liver (hepatocellular carcinoma) cancer cells, and especially such cells carrying epitopes of phosphatidylinositol proteoglycan-3 membrane-binding protein.

[0349] Anti-CD103 antibody and its antigen-binding fragment

[0350] This invention provides antibodies that bind to human CD103 and uses of such antibodies. This invention also provides antigen-binding fragments that bind to human CD103 and uses of such fragments. In some embodiments, the anti-CD103 antibody is isolated.

[0351] Whether an antibody specifically binds to a peptide sequence (e.g., human CD103) can be determined using any analysis known in the art. Examples of analyses known in the art for determining binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET).

[0352] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. The term antibody includes an antigen-binding portion that retains the ability to bind to an antigen, i.e., an "antigen-binding site" (e.g., fragment, subsequence, complementarity-determining region (CDR)), including (i) Fab fragments, which are composed of V... L V HC L And C H (ii) A monovalent segment composed of a structural domain; (iii) A divalent segment containing two Fab segments connected by disulfide bonds in the hinge region; (iv) A segment composed of V H and C H (iv) The Fd fragment composed of the l-domain; L and V H The fragment consists of an Fv domain, a (v) dAb domain (Ward et al., (1989) Nature 341:544-546), said fragment consisting of a VH domain; and (vi) a separated complementarity-determining region (CDR). The term “antibody” also includes single-chain antibodies. Preferred therapeutic antibodies are intact IgG antibodies. As used herein, the term “intact IgG” means a polypeptide belonging to an antibody class substantially encoded by the recognized immunoglobulin γ gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3. The known Ig domain in antibodies of the IgG class is VH. H Cγ1, Cγ2, Cγ3, V L And C L .

[0353] This invention includes an anti-CD103 antigen-binding fragment and a method of using it.

[0354] As used in this article, in the case of IgG, a "full-length antibody" is a bivalent molecule consisting of two heavy chains and two light chains. Each heavy chain contains V... H The structural domain is followed by the constant structural domain (C H1 ), hinge area and two more constant (C) H2 and C H3 ) structural domain; and each light chain contains a V L A structural domain and a constant (C L ) domain. In the case of IgM, the full-length antibody is a decavalent or dodecvalent molecule containing 5 or 6 linked immunoglobulin monomers, each of which has two antigen-binding sites formed by a heavy chain and a light chain.

[0355] As used herein, unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound to the full-length antibody, such as a fragment retaining one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies; and multispecific antibodies formed from antibody fragments.

[0356] This invention includes an anti-CD103 Fab fragment and a method of using it. The "Fab fragment" comprises a light chain and a heavy chain of C. H 1. Variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fab fragment" can be a product of papain cleavage of the antibody.

[0357] This invention includes an anti-CD103 antibody, an antigen-binding fragment comprising an Fc region thereof, and a method of using the same. The "Fc" region contains a C-cell containing the antibody. H 3 and C H Two heavy-chain segments of the 2-domain structure. The two heavy-chain segments are linked by two or more disulfide bonds and by C... H The hydrophobic interactions of the three structural domains are combined.

[0358] This invention includes an anti-CD103 Fab' fragment and a method of using it. The "Fab' fragment" comprises a portion or segment of a light chain and a heavy chain, said heavy chain portion or segment containing V... H Domain and C H 1. A structural domain that also contains C H 1 and C H The region between the two structural domains allows interchain disulfide bonds to form between the two heavy chains of the two Fab' segments to form the F(ab')2 molecule.

[0359] This invention includes an anti-CD103 F(ab')2 fragment and a method of using it. The "F(ab')2 fragment" contains two light chains and two heavy chains, the two heavy chains containing C... H1 With C H2 Part of the constant region between the domains allows for the formation of interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments linked together by disulfide bonds between the two heavy chains. The "F(ab')2 fragment" can be a product of pepsin cleavage of the antibody.

[0360] This invention includes anti-CD103 Fv fragments and methods of using them. The "Fv region" contains variable regions from the heavy and light chains, but lacks a constant region.

[0361] This invention includes an anti-CD103 scFv fragment and its method of use. The term "single-chain Fv" or "scFv" antibody refers to a V containing an antibody. H and V L Antibody fragments containing domains, where these domains are present within a single polypeptide chain. Typically, Fv polypeptides also contain V... H With V LA polypeptide linker between the domains enables scFv to form the structure required for antigen binding. For a review of scFv, see Pluckthun (1994), *The Pharmacology of Monoclonal Antibodies*, Vol. 113, eds. Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315. See also International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

[0362] This invention includes anti-CD103 domain antibodies and methods of using them. "Domain antibody" is an immunofunctional immunoglobulin fragment containing only a variable region of the heavy chain or a variable region of the light chain. In some cases, two or more V... H The regions are covalently linked together to form a bivalent domain antibody. The two V's of the bivalent domain antibody are... H The region can target the same or different antigens.

[0363] This invention includes a bivalent anti-CD103 antibody and its method of use. A “bivalent antibody” contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific (see below).

[0364] This invention includes anti-CD103 biantibodies and methods of using them. As used herein, the term "biantibody" refers to a small antibody fragment having two antigen-binding sites on the same polypeptide chain (V). H -V L or V L -V H ) contains a variable structural domain (V) connected to the light chain. L The heavy chain variable structural domain (V) HBy using linkers that are too short to allow pairing between two domains on the same chain, a domain is forced to pair with a complementary domain on another chain, forming two antigen-binding sites. Biantibodies are described more fully, for example, in EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. Duobodies are described in Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150. A review of engineered antibody variants can be found broadly in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0365] Typically, when binding activity is expressed in moles, the antibody or antigen-binding fragment of the present invention modified in a certain way retains at least 10% of its said activity (when compared to the parent antibody). Preferably, the antibody or antigen-binding fragment of the present invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the CD103 binding affinity as a parent antibody. It is also contemplated that the antibody or antigen-binding fragment of the present invention may include conserved or non-conserved amino acid substitutions (referred to as “conserved variants” or “functionally conserved variants” of the antibody) that do not substantially alter its biological activity.

[0366] This invention includes isolated anti-CD103 antibodies and their antigen-binding fragments, as well as methods of using them. In this document, the term "isolated" is not intended to mean the complete absence of such biomolecules or the presence of water, buffer solutions, or salts, or to refer to components of pharmaceutical preparations containing antibodies or fragments. "Isolated" antibodies, antigen-binding fragments, nucleic acids, etc., are antibodies, antigen-binding fragments, nucleic acids, etc., that have been identified and isolated and / or recovered from one or more components in the natural environment. In a preferred embodiment, the antibodies, antigen-binding fragments, nucleic acids, etc., are purified to 75% by weight or more, more preferably to 90% by weight or more, more preferably to 95% by weight or more, and even more preferably to 98% by weight or more. Thus, the "isolated" biomolecules are at least partially free of other biomolecules from the cells or cell cultures in which they are produced. Such biomolecules include nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth media. The isolated antibodies or antigen-binding fragments may also be at least partially free of expression system components (such as biomolecules) from host cells or their growth media.

[0367] This invention includes anti-CD103 chimeric antibodies (e.g., human constant domain / mouse variable domain) and methods of using them. As used herein, a “chimeric antibody” is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species (US Patent No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). Typically, the variable domain is derived from an antibody from a laboratory animal (“parental antibody”), such as a rodent, and the constant domain sequence is derived from a human antibody, such that the resulting chimeric antibody is less likely to elicit a harmful immune response in human subjects compared to a parental (e.g., mouse) antibody.

[0368] This invention includes anti-CD103 humanized antibodies and their antigen-binding fragments (e.g., humanized rat or mouse antibodies) and methods of using them. As used herein, the term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse or rat) antibodies. Generally, humanized antibodies will contain substantially at least one and typically two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins, and all or substantially all framework (FR) regions are framework regions of human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the constant region (Fc) of human immunoglobulins. For more details on humanized antibodies, see, for example, Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21: 397-402 (2000).

[0369] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain defines the constant region primarily responsible for effector function. Typically, human light chains are classified as κ and λ light chains. Furthermore, human heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also includes a "D" region of approximately 10 more amino acids. See also... Fundamental Immunology Chapter 7 (edited by Paul, W., 2nd edition, Raven Press, NY (1989)).

[0370] The variable region of each light / heavy chain pair forms the antibody binding site. Therefore, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are usually the same.

[0371] Typically, the variable domains of both heavy and light chains contain three hypervariable regions, also known as complementarity-determining regions (CDRs), located within relatively conserved framework regions (FRs). CDRs are usually aligned with the framework regions to enable binding to specific epitopes. Generally, from the N-terminus to the C-terminus, the variable domains of both light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The allocation of amino acids to each domain is typically based on… Sequences of Proteins of Immunological Interest Kabat et al.; National Institutes of Health, Bethesda, MD; 5th edition; NIH Publication No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia et al., (1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 342:878-883.

[0372] As used herein, the term “hypervariant region” refers to the amino acid residues in an antibody or its antigen-binding fragment responsible for antigen binding. The hypervariant region comprises amino acid residues from the “complementarity-determining region” or “CDR” (i.e., CDRL1, CDRL2, and CDRL3 in the light chain variable domain and CDRH1, CDRH2, and CDRH3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Defining the CDR region of an antibody by sequence); also see Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (Defining the CDR region of an antibody by structure). As used herein, the term “framework” or “FR” residues refer to those variable domain residues other than the hypervariant residues defined herein as CDR residues.

[0373] "Isolated nucleic acid molecule" or "isolated polynucleotide" means a genomic, mRNA, cDNA, or synthetically derived DNA or RNA, or some combination thereof, that is not associated with all or part of a polynucleotide naturally present therein or with a polynucleotide not naturally associated with it. For the purposes of this disclosure, it should be understood that "nucleic acid molecule containing a specific nucleotide sequence" does not cover an entire chromosome. An isolated nucleic acid molecule "containing" the specified nucleic acid sequence may, in addition to the specified sequence, include coding sequences of up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operatively linked regulatory sequences that control the expression of the coding regions of the listed nucleic acid sequences, and / or may include vector sequences.

[0374] The phrase "control sequence" refers to a DNA sequence that is operatively linked to a coding sequence and is essential for expression in a specific host organism. Control sequences suitable for prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0375] When a nucleic acid or polynucleotide is placed in a functional relationship with another nucleic acid sequence, the nucleic acid or polynucleotide is "operably linked." For example, if it is described as a preprotein involved in the secretion of a polypeptide, then the DNA of the presequence or secretory leader is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of the sequence, then it is operably linked to the coding sequence; or if a ribosome binding site is positioned to facilitate translation, then it is operably linked to the coding sequence. Usually, but not always, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader, contiguous and located in the reading phase. However, enhancers do not have to be contiguous. Linkage is achieved by ligation at a convenient restriction site. If such a site is not available, then synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0376] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such nomenclature includes progeny. Therefore, the terms “transformation” and “transformed cell” include primary subject cells and cultures derived therefrom, regardless of the number of transductions. It should also be understood that, due to intentional or accidental mutations, not all progeny will have exactly the same DNA content. This includes mutant progeny with the same function or biological activity as selected in the initially transformed cells. Where different nomenclature is intended, it will be clear from the context.

[0377] As used herein, “germline sequence” refers to a sequence of unrearranged immunoglobulin DNA. Unrearranged immunoglobulin sequences from any suitable source can be used. For example, human germline sequences can be obtained from the JOINSOLVER germline database on the website of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. For example, mouse germline sequences can be obtained as described in Giudicelli et al. (2005) Nucleic Acids Res. 33: D256-D261.

[0378] Combining affinity

[0379] For example, but not limited to, the antibodies disclosed herein can be identified by surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET) at a density of 10 x 10⁻⁶. -9 M or lower K DIt binds to human CD103 bivalently. In one embodiment, the antibody disclosed herein can be approximately 5-10 x 10⁻⁶. -9 M of K D The antigen-binding fragment is divalently bound to human CD103. The Kd value can be determined by surface plasmon resonance (e.g., BIACORE). The Kd value can also be determined by similar techniques (e.g., KinExa or OCTET). For example, but not limited to, the antigen-binding fragment disclosed herein can be determined by surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET) at a Kd value of 10 x 10⁻⁶. -9 M or lower K D It is a bivalent binding fragment to human CD103. In one embodiment, the antigen-binding fragment disclosed herein can be approximately 5-10 x 10⁻⁶. -9 M of K D The Kd value is divalently bound to human CD103. The Kd value can be determined by surface plasmon resonance (e.g., BIACORE). The Kd value can also be determined by similar techniques (e.g., KinExa or OCTET). Affinity is calculated as K. D =k 解离 / k 缔合 (k 解离 K is the dissociation rate constant. 缔合 Let K be the association rate constant and K D (where r is the equilibrium constant). Affinity can be determined at equilibrium by measuring the binding fraction (r) of labeled ligands at various concentrations (c). The data are plotted using the Scatchard equation: r / c = K(nr), where r = the number of moles of ligand bound per mole of receptor at equilibrium; c = the concentration of free ligand at equilibrium; K = the equilibrium association constant; and n = the number of ligand binding sites per receptor molecule. By plotting r / c on the Y-axis relative to r on the X-axis, a Scatchard curve is generated. Measurement of antibody affinity using Scatchard analysis is well known in the art. See, for example, van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

[0380] Methods for preparing antibodies and their antigen-binding fragments

[0381] Therefore, the present invention includes a method for preparing the anti-CD103 antibody or its antigen-binding fragment of the present invention, the method comprising culturing hybridoma cells expressing the antibody or fragment under conditions favorable to the expression of such antibody or fragment, and optionally isolating the antibody or fragment from the hybridoma and / or growth medium (e.g., cell culture medium).

[0382] The anti-CD103 antibody disclosed herein can also be recombinantly generated (e.g., in an E. coli / T7 expression system, a mammalian cell expression system, or a lower eukaryotic expression system). In this embodiment, the antibody immunoglobulin molecule encoding the present invention (e.g., V...) can be... H or V L The nucleic acid insertion is based on a pET plasmid and expressed in the *E. coli* / T7 system. For example, the present invention includes a method for expressing an antibody or its antigen-binding fragment or its immunoglobulin chain in a host cell (e.g., a bacterial host cell, such as *E. coli*, such as BL21 or BL21DE3), the method comprising expressing a T7 RNA polymerase in the cell, the cell further comprising a polynucleotide operably linked to a T7 promoter encoding an immunoglobulin chain. For example, in one embodiment of the invention, the bacterial host cell (such as *E. coli*) comprises a polynucleotide operably linked to a lac promoter encoding a T7 RNA polymerase gene, and expression of the polymerase and chain is induced by incubating the host cell in the presence of IPTG (isopropyl-β-D-thiogalactopyranoside).

[0383] Several methods for generating recombinant antibodies are known in the art. One example of a method for preparing recombinant antibodies is disclosed in U.S. Patent No. 4,816,567.

[0384] Transformation can be performed using any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of one or more polynucleotides in liposomes, bio-projectile injection, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules can be introduced into mammalian cells using viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.

[0385] Therefore, the present invention includes a recombinant method for preparing the anti-CD103 antibody of the present invention or its antigen-binding fragment or its immunoglobulin chain, said recombinant method comprising introducing a polynucleotide encoding one or more immunoglobulin chains (e.g., heavy immunoglobulin chains and / or light immunoglobulin chains) encoding the antibody or fragment; culturing host cells (e.g., CHO or Pichia or Pichia pastoris) under conditions favorable to such expression; and optionally, isolating the antibody or fragment or chain from the host cells and / or the culture medium in which the host cells are grown.

[0386] Anti-CD103 antibodies can also be synthesized by any of the methods listed in U.S. Patent No. 6,331,415.

[0387] Eukaryotic and prokaryotic host cells (including mammalian cells) used as hosts to express the antibodies, fragments, or immunoglobulin chains disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These cell lines particularly include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include human cells, mouse cells, rat cells, dog cells, monkey cells, pig cells, goat cells, bovine cells, horse cells, and hamster cells. Specific preferred cell lines are selected by identifying which cell lines exhibit high expression levels. Other cell lines that can be used include insect cell lines (such as Sf9 cells), amphibian cells, bacterial cells, plant cells, and fungal cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia kolamae*, *Pichia membranaefaciens*, *Pichia minuta* (including *Ogataea minuta* and *Pichia lindneri*), *Pichia opuntiae*, *Pichia thermotolerans*, *Pichiasalictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichiasp.*, and *Saccharomyces*. *Cerevisiae*, *Saccharomyces* sp., *Hansenula polymorpha*, *Kluyveromyces* sp., *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium* sp., *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, and *Neurospora crassa*. Pichia, any of the following genera: Hansenula polymorpha, any of the following genera: Kluyveromyces, Candida albicans, any of the following genera: Trichoderma reesei, Microsporum aureum, any of the following genera: Fusarium, Yarrowia lipolytica, and Neurospora crassa.When a recombinant expression vector encoding a heavy chain or antigen-binding moiety or fragment thereof and / or a light chain or antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody is generated by culturing the host cell for a period sufficient to allow the antibody or fragment or chain to be expressed in the host cell or secreted into the culture medium in which the host cell grows.

[0388] Antibodies and their antigen-binding fragments, as well as immunoglobulin chains, can be recovered from the culture medium using standard protein purification methods. Furthermore, the expression of the antibodies and their antigen-binding fragments, as well as immunoglobulin chains (or other portions thereof), from the prepared cell lines can be enhanced using many known techniques. For example, the glutamine synthase gene expression system (GS system) is a commonly used method for enhancing expression under certain conditions. The GS system is discussed in whole or in part in conjunction with European Patent Nos. 0216846, 0256055, 0323997, and 0338841. Thus, in one embodiment of the invention, mammalian host cells (e.g., CHO) lack the glutamine synthase gene and are grown in the absence of glutamine in the culture medium; however, the polynucleotide encoding the immunoglobulin chain contains the glutamine synthase gene, which compensates for the lack of said gene in the host cells.

[0389] This invention includes a method for purifying the anti-CD103 antibody or its antigen-binding fragment of the present invention, the method comprising introducing a sample containing the antibody or fragment into a purification medium (e.g., cation exchange medium, anion exchange medium, hydrophobic exchange medium, affinity purification medium (e.g., protein-A, protein-G, protein-A / G, protein-L)) and collecting the purified antibody or fragment from a flow-through fraction of the sample that is not bound to the medium; or discarding the flow-through fraction and eluting the bound antibody or fragment from the medium and collecting the eluent. In one embodiment of the invention, the medium is located in a column in which the sample is applied. In one embodiment of the invention, the purification method is performed after recombinant expression of the antibody or fragment in host cells, for example, wherein the host cells are first lysed and optionally the lysate is purified to remove insoluble material, and then purified on a medium.

[0390] Generally, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern characteristic of glycoproteins produced in said cell line or transgenic animal. Therefore, the specific glycosylation pattern of an antibody will depend on the specific cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or containing the amino acid sequences provided herein constitute the present invention, regardless of the glycosylation pattern the antibody may have. Similarly, in certain embodiments, antibodies having a glycosylation pattern containing only non-trehalosylated N-glycans may be advantageous because these antibodies have been shown to typically exhibit greater efficacy than their trehalosylated counterparts both in vitro and in vivo (see, for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These antibodies having non-trehalosylated N-glycans are unlikely to be immunogenic because their carbohydrate structure is a normal component of the population present in human serum IgG.

[0391] This invention also includes the anti-CD103 antigen-binding fragment of the anti-CD103 antibody disclosed herein. The antibody fragment includes an F(ab)2 fragment, which can be generated by enzymatic cleavage of IgG with, for example, pepsin. The Fab fragment can be generated by, for example, reducing F(ab)2 with dithiothreitol or mercaptoethylamine.

[0392] Immunoglobulins can be assigned to different classes based on the amino acid sequence of the constant domain of the heavy chain. In some embodiments, different constant domains can be linked to humanized V derived from the CDR provided herein. L and V H The region contains at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. This invention comprises antibody-antigen binding fragments of any of these antibody classes or subclasses.

[0393] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, such as a human constant region, such as γ1, γ2, γ3, or γ4 human heavy chain constant regions or variants thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, such as a human light chain constant region, such as λ or κ human light chain regions or variants thereof. For example, but not limited to, the human heavy chain constant region may be γ4 and the human light chain constant region may be κ. In an alternative embodiment, the Fc region of the antibody is γ4 with the Ser228Pro mutation (Schuurman, J et al., Mol. Immunol. 38: 1-8, 2001).

[0394] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG1 isotype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG2 isotype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG4 isotype.

[0395] Antibody engineering

[0396] This also includes embodiments in which the anti-CD103 antibody and its antigen-binding fragment are engineered antibodies to include modifications to the framework residues within the variable domains of the antibody, thereby improving the properties of the antibody or fragment. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody or fragment. This is usually achieved by replacing non-CDR residues (i.e., framework residues) in the variable domains of the parental (e.g., rodent) antibody or fragment with similar residues from the immune profile of the species from which the antibody is to be used (e.g., human residues in the case of human therapeutics). Such antibodies or fragments are called “humanized” antibodies or fragments. In some cases, it is necessary to increase affinity or change the specificity of engineered (e.g., humanized) antibodies. One approach is to mutate one or more framework residues to a corresponding germline sequence. More specifically, antibodies or fragments that have undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody was derived. Such residues can be identified by comparing the framework sequence of the antibody or fragment with the germline sequence from which the antibody or fragment was derived. Another approach is to restore the original parent (e.g., rodent) residues at one or more sites in the engineered (e.g., humanized) antibody to, for example, restore binding affinity that may have been lost during the replacement of framework residues. (See, for example, U.S. Patent Nos. 5,693,762, 5,585,089, and 5,530,101).

[0397] In some implementations, anti-CD103 antibodies and their antigen-binding fragments are engineered (e.g., humanized) to include modifications in the framework and / or CDR to improve their properties. Such engineered changes can be based on molecular modeling. Molecular models of the variable regions of parental (non-human) antibody sequences can be constructed to understand the structural characterization of the antibody and to identify potential regions on the antibody that can interact with the antigen. Conventional CDRs are based on the alignment of immunoglobulin sequences and the identification of variable regions. (Kabat et al., (1991)) Sequences of Proteins of Immunological InterestKabat et al.; National Institutes of Health, Bethesda, MD; 5th edition; NIH Publication No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and co-workers carefully examined the rings in the crystal structure of the antibody and the proposed conformation of the hypervariable ring. Chothia et al., (1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 342:878-883. Variations exist between regions classified as “CDR” and “hypervariable ring”. Subsequent studies (Raghunathan et al., (2012) J. Mol Recog. 25, 3, 103-113) analyzed several antibody-antigen crystal complexes and observed that the antigen-binding region in the antibody does not necessarily strictly conform to the “CDR” residues or the “hypervariant” loop. Molecular models of the variable regions of non-human antibodies can be used to guide the selection of regions that can potentially bind to antigens. In practice, model-based potential antigen-binding regions differ from conventional “CDR” or “hypervariant” loops. Molecular modeling can be performed using commercial scientific software (such as Discovery Studio (BIOVIA, Dassault Systems)). Human scaffolds can be selected based on the best match between the scaffold and the non-human sequence in the CDR. For FR4 (scaffold 4) in VH, the VJ region of the human germline is compared with the corresponding non-human region. In the case of FR4 (scaffold 4) in VL, the J-κ and J-λ regions of the human germline sequence are compared with the corresponding non-human regions. After identifying a suitable human scaffold, the CDR is transplanted into the selected human scaffold. In some cases, certain residues at the VL-VH interface may be retained as in the non-human (parental) sequence. Molecular models can also be used to identify residues that could potentially alter the CDR conformation and thus bind to the antigen. In some cases, these residues are retained as in the non-human (parental) sequence. Molecular models can also be used to identify solvent-exposed amino acids that could cause unwanted effects such as glycosylation, deamidation, and oxidation. Developability filtering can be introduced shortly before the design phase to eliminate / minimize these potential problems.

[0398] Another type of framework modification involves mutating one or more residues within a framework region or even one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is further described in detail in U.S. Patent No. 7,125,689.

[0399] In certain embodiments, it will be necessary to change certain amino acids containing exposed side chains to other amino acid residues to give the final antibody greater chemical stability, thereby avoiding deamidation or isomerization. Deamidation of asparagine can occur at the NG, DG, NG, NS, NA, NT, QG, or QS sequences and result in the formation of isoaspartic residues, which will bend into the polypeptide chain and reduce its stability (the isoaspartic effect). Isomerization can occur at the DG, DS, DA, or DT sequences. In some embodiments, the antibodies of this disclosure do not contain deamidation or asparagine isomerization sites.

[0400] For example, asparagine (Asn) residues can be changed to Gln or Ala to reduce the likelihood of isoaspartic acid formation at any Asn-Gly sequence, particularly within the CDR. A similar problem can occur at Asp-Gly sequences. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartic acid formation can weaken or completely eliminate antibody binding to its target antigen. See Presta (2005) J. Allergy Clin. Immunol. 116:731, p. 734. In one embodiment, asparagine is changed to glutamine (Gln). It may also be necessary to modify the amino acids near the asparagine (Asn) or glutamine (Gln) residues to reduce the likelihood of deamidation, which occurs at a higher rate when small amino acids are present near asparagine or glutamine. See Bischoff and Kolbe (1994) J. Chromatog. 662:261. Furthermore, any methionine residues in the CDR (typically solvent-exposed Met) can be replaced with Lys, Leu, Ala, or Phe, or other amino acids, to reduce the likelihood of methionine sulfur being oxidized. This oxidation can decrease antigen-binding affinity and also contribute to molecular heterogeneity in the final antibody formulation. Ibid. Additionally, to prevent or minimize the cleavage of potentially fragile Asn-Pro peptide bonds, it may be necessary to replace any Asn-Pro combinations found in the CDR with Gln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions are then screened to ensure that the substitutions do not reduce the antibody's affinity or specificity for CD103 or other desired biological activities to unacceptable levels.

[0401] Table 1. Exemplary stabilized CDR variants

[0402]

[0403] Another type of framework modification involves mutating one or more residues within the framework region to prevent aggregation. The risk of antibody aggregation can be assessed using spatial aggregation tendency, see Chennamsetty, N et al. (2010) J. Phys. Chem. 114, 6614-6624. This method requires calculating the solvent accessible area (SAA) for each atom. The molecular aggregation score is then calculated as the sum of the scores of all atoms. For a molecule of a given radius and size, this is an approximate indication of its overall aggregation tendency. Residues with high aggregation scores are replaced with residues with lower scores (e.g., more hydrophilic amino acids).

[0404] Antibody engineering in the Fc region

[0405] The antibodies disclosed herein (e.g., humanized antibodies) and their antigen-binding fragments can also be engineered to include modifications within the Fc region, typically altering one or more properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or effector function (e.g., antigen-dependent cytotoxicity). Furthermore, the antibodies disclosed herein and their antigen-binding fragments can be chemically modified (e.g., one or more chemical moieties can be linked to the antibody) or modified to alter their glycosylation, thereby altering one or more properties of the antibody or fragment. Each of these embodiments is further described in detail below. The residues in the Fc region are numbered according to the EU index of Kabat.

[0406] The antibodies and their antigen-binding fragments disclosed herein also include antibodies and fragments with modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Patent Nos. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702. Such modifications can be used to enhance or suppress various immune system responses, potentially having beneficial effects in diagnosis and treatment. Modifications to the Fc region include amino acid alterations (substitution, deletion, and insertion), glycosylation or deglycosylation, and the addition of multiple Fc regions. Modifications to the Fc can also alter the half-life of antibodies in therapeutic antibodies, thereby enabling less frequent use and thus increasing convenience and reducing material usage. See Presta (2005) J. Allergy Clin. Immunol. 116:731, pp. 734-35.

[0407] In one embodiment, the antibody or antigen-binding fragment of the present invention is an IgG4 isotype antibody or fragment containing a serine-to-proline mutation at position 228 in the hinge region of the heavy chain constant region (S228P; EU index; SEQ ID NO: 66). This mutation has been reported to eliminate the heterogeneity of inter-heavy chain disulfide bonds in the hinge region (Angal et al. (1993). Mol. Immunol. 30:105-108; position 241 is based on the Kabat numbering system).

[0408] In one embodiment of the invention, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region increases or decreases. This method is further described in U.S. Patent No. 5,677,425. Changing the number of cysteine ​​residues in the hinge region of CH1 can, for example, facilitate the assembly of light and heavy chains or increase or decrease the stability of antibodies.

[0409] In another embodiment, the Fc hinge region of the antibody or antigen-binding fragment of the present invention is mutated to shorten the biological half-life of the antibody or fragment. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment, such that the antibody or fragment has impaired binding to Staphylococcus aureus protein A (SpA) relative to the native Fc-hinge domain SpA binding. This method is further described in detail in U.S. Patent No. 6,165,745.

[0410] In another embodiment, the antibody or antigen-binding fragment of the present invention is modified to increase its biological half-life. Various methods are possible. For example, one or more of the following mutations may be introduced as described in U.S. Patent No. 6,277,375: T252L, T254S, T256F. Alternatively, to increase the biological half-life, the antibody may be modified in the CH1 or CL region to contain a rescue receptor-binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022.

[0411] In other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change one or more effector functions of the antibody or antigen-binding fragment. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with different amino acid residues, such that the antibody has an altered affinity for the effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail in U.S. Patent Nos. 5,624,821 and 5,648,260.

[0412] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 may be replaced with different amino acid residues, such that the antibody exhibits altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is further described in detail in U.S. Patent No. 6,194,551.

[0413] In another instance, altering one or more amino acid residues at positions 231 and 239 changes the antibody's ability to fix complement. This method is further described in PCT Publication WO 94 / 29351.

[0414] The protein of the present invention (preferably an antibody, and most preferably an IgG antibody or a fragment thereof) may have altered (e.g., relative to an unmodified antibody) FcγR binding properties (examples of binding properties include, but are not limited to, binding specificity, equilibrium dissociation constant (K)). D ), dissociation and association rates (k, respectively) 解离 and k 缔合 ( ), binding affinity and / or cohesive affinity), and some changes are more or less desired. The equilibrium dissociation constant (K) is known in the art. D ) is defined as k 解离 / k 缔合 And K a For K D The reciprocal of.

[0415] The affinity and binding properties of the Fc region to its ligands can be determined by a variety of in vitro analytical methods (biochemical or immunological analyses) known in the art for determining Fc-FcγR interactions (i.e., specific binding of the Fc region to FcγR). These in vitro analytical methods include, but are not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., BIACORE®, OCTET®, or KinExa® assays), as well as other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labeling on one or more of the components being tested and / or employ a variety of detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labeling.

[0416] In some embodiments, the protein of the present invention binds to one or more human FcγRs. In some embodiments, the protein of the present invention binds to one or more human FcγRs selected from the group consisting of: FcγRI, FcγRIIB, FcγRIIC, FcγRIIIA-F158, and FcγRIIIA-V158, with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having a wild-type human IgG1 heavy chain constant domain (SEQ ID NO: 119) FcγRI. In some embodiments, the protein of the present invention binds to human FcγRIs with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having a wild-type human IgG1 heavy chain constant domain (SEQ ID NO: 119) FcγRI. In some embodiments, the protein of the present invention binds to human FcγRIIB with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG1 heavy chain constant domain (SEQ ID NO: 119). In some embodiments, the protein of the present invention binds to human FcγRIIC with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG1 heavy chain constant domain (SEQ ID NO: 119). In some embodiments, the protein of the present invention binds to human FcγRIIIA-F158 with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG1 heavy chain constant domain (SEQ ID NO: 119). In some embodiments, the protein of the present invention binds to human FcγRIIIA-V158 with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG1 heavy chain constant domain (SEQ ID NO: 119). In some embodiments, the protein of the present invention binds to one or more human FcγRs selected from the group consisting of: FcγRI, FcγRIIB, FcγRIIC, FcγRIIIA-F158, and FcγRIIIA-V158. In some embodiments, the protein of the present invention binds to human FcγRI with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG4 heavy chain constant domain (SEQ ID NO: 66).In some embodiments, the protein of the present invention binds to human FcγRIIB with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG4 heavy chain constant domain (SEQ ID NO: 66). In some embodiments, the protein of the present invention binds to human FcγRIIC with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG4 heavy chain constant domain (SEQ ID NO: 66). In some embodiments, the protein of the present invention binds to human FcγRIIIA-F158 with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG4 heavy chain constant domain (SEQ ID NO: 66). In some embodiments, the protein of the present invention binds to human FcγRIIIA-V158 with an affinity at least 10 times, preferably at least 30 times, and more preferably at least 100 times smaller than an equivalent protein having the Fc region of the wild-type human IgG4 heavy chain constant domain (SEQ ID NO: 66).

[0417] In various embodiments, the protein of the present invention comprises an immunoglobulin Fc region, said immunoglobulin Fc region comprising an immunoglobulin C2 region and an immunoglobulin C3 region, as well as an immunoglobulin hinge region. For example, the immunoglobulin Fc region may be an IgG Fc region, an IgE Fc region, or an IgA Fc region. In some preferred embodiments, the protein comprises two immunoglobulin Fc regions, each immunoglobulin Fc region comprising an immunoglobulin C2 region and an immunoglobulin C3 region, as well as an immunoglobulin hinge region, wherein the hinge region of one immunoglobulin Fc region binds to the hinge region of the other immunoglobulin Fc region to form a dimerized Fc structure. Most preferably, such a protein is a human IgG protein or a humanized IgG protein.

[0418] In some embodiments, the protein of the present invention comprises a mutant IgG4 Fc region, and preferably the protein is an IgG comprising two mutant IgG4 Fc regions to form a dimer Fc structure. For example, the mutant IgG4 Fc region may comprise one of the mutations or combinations of mutations listed in Table 2. The numbering system for constant regions mentioned in this table is the EU index numbering system as described in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In the table, the first letter and number indicate the unmodified amino acid and its position, and the second letter indicates the substituted amino acid at said position. For entries comprising combinations of more than one mutation, each mutation in the combination is separated by a " / ". Deletions are indicated by "Δ".

[0419] Table 2:

[0420]

[0421] In some embodiments, the protein of the present invention comprises a mutant IgG1 Fc region, and preferably the protein is an IgG comprising two mutant IgG1 Fc regions to form a dimer Fc structure. For example, the mutant IgG1 Fc region may comprise one of the mutations listed in Table 3. The constant region numbering system mentioned in this table is the EU index numbering system as described in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In the table, the first letter and number indicate the unmodified amino acid and its position, and the second letter indicates the substituted amino acid at said position.

[0422] Table 3:

[0423]

[0424]

[0425]

[0426] In some implementations, the mutant IgG1 Fc region may contain one of the mutation combinations listed in Table 4. The constant region numbering system mentioned in this table is the EU index numbering system as described in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In the table, the first letter and number indicate the unmodified amino acid and its position, and the second letter indicates the substituted amino acid at said position. For each of more than one combination of mutations, each mutation in the combination is separated by a " / " and deletions are indicated by "Δ".

[0427] Table 4:

[0428]

[0429]

[0430] In some embodiments, the protein of the present invention comprises a wild-type or mutant IgG2 Fc region, and preferably the protein is an IgG comprising two wild-type or mutant IgG2 Fc regions to form a dimer Fc structure. The mutant IgG2 Fc region may comprise one of the mutations or combinations of mutations listed in Table 5. The numbering system for constant regions mentioned in this table is the EU index numbering system as described in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). In the table, the first letter and number indicate the unmodified amino acid and its position, and the second letter indicates the substituted amino acid at said position. For entries comprising combinations of more than one mutation, each mutation in the combination is separated by a " / ".

[0431] Table 5:

[0432]

[0433] Preparation of antibodies with modified glycosylation

[0434] In another embodiment, the antibody or antigen-binding fragment of the present invention comprises a specific glycosylation pattern. For example, non-trehalosylated or deglycosylated antibodies or fragments can be prepared (i.e., antibodies lacking trehalose or glycosylation, respectively). The glycosylation pattern of the antibody or fragment can be altered to, for example, increase the affinity or co-existence of the antibody or fragment for the CD103 antigen. Such modifications can be achieved, for example, by altering one or more glycosylation sites within the sequence of the antibody or fragment. For example, one or more amino acid substitutions can be performed such that the removal of one or more glycosylation sites in the variable region framework eliminates the glycosylation at said site. Such deglycosylation can increase the affinity or co-existence of the antibody or fragment for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0435] The antibody-antigen binding fragments disclosed in this article may also include those produced in lower eukaryotic host cells, particularly fungal host cells, such as yeast and filamentous fungi, that have been genetically engineered to produce glycoproteins with mammalian-like or human-like glycosylation patterns (see, for example, Choi et al., (2003) Proc.Natl. Acad. Sci. 100: 5022-5027; Hamilton et al., (2003) Science 301: 1244-1246; Hamilton et al., (2006) Science 313: 1441-1443; Nett et al., Yeast 28(3):237-52 (2011); Hamilton et al., Curr Opin Biotechnol. 18(5): 387-92 (2007)). One particular advantage of these genetically modified host cells over currently used mammalian cell lines is their ability to control the glycosylation patterns of glycoproteins produced within the cells, enabling the production of glycoprotein compositions in which a specific N-glycan structure is dominant (see, for example, U.S. Patent Nos. 7,029,872 and 7,449,308). Host cells using these genetic modifications have produced antibodies that are predominantly composed of specific N-glycan structures (see, for example, Li et al., (2006) Nat. Biotechnol. 24: 210-215).

[0436] In certain embodiments, the antibodies and their antigen-binding fragments disclosed herein also include those antibodies and their antigen-binding fragments produced in lower eukaryotic host cells and comprising trehalosylated and non-trehalosylated hybrid and complex N-glycans, said N-glycans comprising bipartite and multitenantaneous substances, including but not limited to N-glycans such as GlcNAc. (1-4) Man3GlcNAc2;Gal (1-4) GlcNAc(1-4) Man3GlcNAc2;NANA (1-4) Gal (1-4) GlcNAc (1-4) Man3GlcNAc2.

[0437] In certain embodiments, the antibodies and their antigen-binding fragments provided herein may comprise an antibody or fragment having at least one heterozygous N-glycan selected from the group consisting of: GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In a particular aspect, the heterozygous N-glycan is the dominant N-glycan in the composition.

[0438] In certain embodiments, the antibodies and their antigen-binding fragments provided herein comprise antibodies and fragments having at least one complex N-glycan selected from the group consisting of: GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In a particular aspect, the complex N-glycan is the dominant N-glycan substance in the composition. In other aspects, the complex N-glycan is a specific N-glycan substance comprising about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycan in the composition. In one embodiment, the antibody and its antigen-binding fragment provided herein comprise a complex N-glycan, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycan comprises the structure NANA2Gal2GlcNAc2Man3GlcNAc2, wherein such structure is non-trehalosylated. Such structures can be generated, for example, in engineered Pichia pastoris host cells.

[0439] In a specific embodiment, the N-glycan is trehalose-based. Generally, trehalose is linked to the GlcNAc α1,3-terminus of the N-glycan, to the GlcNAc α1,6-terminus of the N-glycan, to the Gal α1,2-terminus of the N-glycan, to the GlcNac α1,3-terminus of the N-glycan, or to the GlcNAc α1,4-terminus of the N-glycan.

[0440] Therefore, in certain aspects of the above glycoprotein compositions, the glycoform is in the form of α1,3-linked or α1,6-linked trehalose to produce glycoforms selected from the group consisting of: Man5GlcNAc2(Fuc), GlcNAcMan5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc). Gal2GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc); in α1,3- or α1,4-linked trehalose forms to produce glycoforms selected from the group consisting of: GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, and GlcNAc2(Fuc)Man3GlcNAc2. 1-2 Man3GlcNAc2, GalGlcNAc2(Fuc 1-2 )Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc 1-2 Man3GlcNAc2 and NANA2Gal2GlcNAc2(Fuc 1-2 Man3GlcNAc2; or in the form of α1,2-linked trehalose to produce glycoforms selected from the group consisting of: Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc) 1-2 )GlcNAc2Man3GlcNAc2, NANAGal2(Fuc 1-2 )GlcNAc2Man3GlcNAc2 and NANA2Gal2(Fuc 1-2 )GlcNAc2Man3GlcNAc2.

[0441] In other respects, antibodies (e.g., humanized antibodies) or their antigen-binding fragments contain high-mannose N-glycans, including but not limited to Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or N-glycans composed of the Man3GlcNAc2 N-glycan structure.

[0442] Among the other aspects mentioned above, complex N-glycans also include trehalosylated and non-trehalosylated bipartite and multi-tentacle substances.

[0443] As used herein, the terms “N-glycan” and “glycoform” are used interchangeably and refer to N-linked oligosaccharides, such as N-linked oligosaccharides linked to asparagine residues of a polypeptide via an asparagine-N-acetylglucosamine bond. N-linked glycoproteins contain N-acetylglucosamine residues with an amide nitrogen linked to an asparagine residue in the protein. The dominant sugars present on glycoproteins are glucose, galactose, mannose, trehalose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (e.g., N-acetyl-neuraminic acid (NANA)). Glycogroup processing occurs simultaneously with translation in the lumen of the ER and, for N-linked glycoproteins, continues post-translational in the Golgi apparatus.

[0444] N-glycans share a common pentasaccharide core of Man3GlcNAc2 (“Man” stands for mannose; “Glc” for glucose; and “NAc” for N-acetyl; GlcNAc for N-acetylglucosamine). Typically, the N-glycan structure presents a non-reducing end on the left and a reducing end on the right. The reducing end of an N-glycan is a terminal appendage of an Asn residue containing a glycosylation site on the protein. N-glycans vary in the number of branched chains (tentacles) containing peripheral sugars (e.g., GlcNAc, galactose, trehalose, and sialic acid) added to the Man3GlcNAc2 (“Man3”) core structure, also known as a “trimannose core,” “pentasaccharide core,” or “oligomannose core.” N-glycans are classified according to their branched chain composition (e.g., high-mannose, complex, or hybrid). “High-mannose” N-glycans have five or more mannose residues. "Complex" N-glycans typically have at least one GlcNAc linked to a 1,3-mannose arm and at least one GlcNAc linked to a 1,6-mannose arm of a "trimannose" core. Complex N-glycans may also have galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues, optionally modified with sialic acid or derivatives (e.g., "NANA" or "NeuAc", where "Neu" refers to neuraminic acid and "Ac" refers to acetyl). Complex N-glycans may also have intrachain substitutions comprising a "dichotomous" GlcNAc and a core trehalose ("Fuc"). Complex N-glycans may also have multiple tentacles on the "trimannose core," often referred to as "multi-tentacle glycans." "Hybrid" N-glycans have at least one GlcNAc at the end of the 1,3-mannose arm of the trimannose core and zero or more mannoses on the 1,6-mannose arm of the trimannose core. Various N-glycans are also referred to as "glycans".

[0445] For complex N-glycans, the terms “G-2”, “G-1”, “G0”, “G1”, “G2”, “A1” and “A2” have the following meanings. "G-2" refers to an N-glycan structure that can be characterized as Man3GlcNAc2; the term "G-1" refers to an N-glycan structure that can be characterized as GlcNAcMan3GlcNAc2; the term "G0" refers to an N-glycan structure that can be characterized as GlcNAc2Man3GlcNAc2; the term "G1" refers to an N-glycan structure that can be characterized as GalGlcNAc2Man3GlcNAc2; the term "G2" refers to an N-glycan structure that can be characterized as Gal2GlcNAc2Man3GlcNAc2; the term "A1" refers to an N-glycan structure that can be characterized as NANAGal2GlcNAc2Man3GlcNAc2; and the term "A2" refers to an N-glycan structure that can be characterized as NANA2Gal2GlcNAc2Man3GlcNAc2. Unless otherwise specified, the terms “G-2”, “G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” refer to N-glycans lacking a trehalose residue linked to a GlcNAc residue at the reducing end of the N-glycan. When the term includes “F”, “F” indicates that the N-glycan contains a trehalose residue at the GlcNAc residue at the reducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2F all indicate that the N-glycan also includes a trehalose residue linked to a GlcNAc residue at the reducing end of the N-glycan. Lower eukaryotes (such as yeast and filamentous fungi) typically do not produce N-glycans that produce trehalose.

[0446] For the purpose of multi-tentacle N-glycans, the term "multi-tentacle N-glycan" refers to an N-glycan that also contains: GlcNAc residues on mannose residues at the non-reducing ends of the 1, 6, or 1, 3 arms of the N-glycan, or GlcNAc residues on each of the non-reducing ends of the 1, 6, and 1, 3 arms of the N-glycan. Therefore, multi-tentacle N-glycans can be characterized by the formula GlcNAc(2-4)Man3GlcNAc2, Gal(1-4)GlcNAc(2-4)Man3GlcNAc2, or NANA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2. The term "1-4" refers to 1, 2, 3, or 4 residues.

[0447] For the purpose of bipartite N-glycans, the term "bipartite N-glycan" refers to an N-glycan in which GlcNAc residues are attached to mannose residues at the reducing end of the N-glycan. A bipartite N-glycan can be characterized by the formula GlcNAc3Man3GlcNAc2, where each mannose residue is attached to a GlcNAc residue at its non-reducing end. In contrast, when a multi-tentacle N-glycan is characterized as GlcNAc3Man3GlcNAc2, the formula indicates that two GlcNAc residues are attached to mannose residues at the non-reducing end of one of the two arms of the N-glycan, and one GlcNAc residue is attached to a mannose residue at the non-reducing end of the other arm of the N-glycan.

[0448] In some embodiments, the protein of the present invention comprises a deglycosylated Fc region. For example, the IgG1 Fc region can be deglycosylated by deletion or substitution of residue N297.

[0449] Antibody physical properties

[0450] The antibodies and their antigen-binding fragments disclosed in this paper may also contain one or more glycosylation sites in the variable regions of light or heavy chain immunoglobulins. Such glycosylation sites can increase the immunogenicity of the antibody or fragment or alter the antibody's pK by changing antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al. (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al. (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing NXS / T sequences.

[0451] Each antibody or antigen-binding fragment will have a unique isoelectric point (pI), which is typically within a pH range of 6 to 9.5. The pI of IgG1 antibodies is typically in the pH range of 7–9.5, and the pI of IgG4 antibodies is typically in the pH range of 6–8.

[0452] Each antibody or antigen-binding fragment will have a characteristic melting temperature, and a higher melting temperature indicates greater overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol). 3 :361-71). Generally speaking, TM1 The initial unfolding temperature can be greater than 60°C, 65°C, or 70°C. The melting temperature of antibodies or fragments can be determined using differential scanning calorimetry (Chen et al. (2003) Pharm Res). 20 :1952-60; Ghirlando et al (1999) Immunol Lett 68 :47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40 :343-9) to measure.

[0453] In another implementation, antibodies and their antigen-binding fragments that do not degrade rapidly are selected. Degradation of the antibody or fragment can be assessed using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) AnalChem). 67 :3626-32) to measure.

[0454] In another implementation, antibodies and their antigen-binding fragments with minimal aggregation effects are selected, as aggregation can lead to the triggering of unwanted immune responses and / or altered or adverse pharmacokinetic properties. Typically, aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of antibodies and fragments is acceptable. Aggregation can be measured using several techniques, including size exclusion column chromatography (SEC), high-performance liquid chromatography (HPLC), and light scattering.

[0455] Antibody conjugates

[0456] The anti-CD103 antibody disclosed herein may also be conjugated to a chemical portion. The anti-CD103 antigen-binding fragment disclosed herein may also be conjugated to a chemical portion. The chemical portion may be, in particular, a polymer. The chemical portion may be, in particular, a radioactive nucleotide. The chemical portion may be, in particular, a cytotoxic factor. In a particular embodiment, the chemical portion is a polymer that increases the half-life of the antibody or fragment in the subject. Suitable polymers include, but are not limited to, hydrophilic polymers. Such hydrophilic polymers may include, but are not limited to, polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa). Such hydrophilic polymers may include, but are not limited to, dextran. Such hydrophilic polymers may include, but are not limited to, monomethoxy polyethylene glycol (mPEG). Lee et al., (1999) (Bioconj. Chem. 10:973-981) disclosed PEG-conjugated single-chain antibodies. Wen et al. (2001) (Bioconj. Chem. 12:545-553) disclosed a method for conjugating antibodies with PEG linked to a radioactive metal chelating agent (diethylenetriaminepentaacetic acid (DTPA)).

[0457] The antibodies and their antigen-binding fragments disclosed herein can also be conjugated with markers such as the following: 99 Tc, 99m Tc, 86 Y、 88 Y、 90 Y、 111 In、 32 P, 14 C 123 I, 124 I, 125 I, 3 H, 131 I, 11 C 15 O、 13 N、 18 F, 19 F, 35 S, 51 Cr 57 To 226 Ra、 60 Co、 59 Fe、 57 Se、 152 Eu、 61 Cu、 62 Cu、 64 Cu、 67 Cu、 66 Ga、 67 Ga、 68 Ga、 72 Ga、45 Ti、 89 Zr、 217 Ci、 211 At、 212 Pb, 177 Lu、 44 Sc、 47 Sc、 109 Pd, 234 Th and 40 K, 157 Gd, 55 Mn, 52 Tr and 56 Fe.

[0458] The antibodies disclosed herein may also be PEGylated to, for example, increase their biological (e.g., serum) half-life. The antigen-binding fragments disclosed herein may also be PEGylated. To PEGylate an antibody or fragment, typically the antibody or fragment is reacted with a reactive form of polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) under conditions where one or more PEG groups become linked to the antibody or antibody fragment. In certain embodiments, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to cover any of the PEG forms already used to derive other proteins, such as mono(C1-C10)alkoxy-PEG or aryloxy-PEG or PEG-maleimide. In some embodiments, the antibody or fragment to be PEGylated is a deglycosylated antibody or fragment. Methods for PEGylating proteins are known in the art and applicable to the antibodies of this invention. See, for example, EP 0 154 316 and EP 0 401 384.

[0459] The antibodies disclosed herein can also be conjugated with fluorescent labels. The antibodies disclosed herein can also be conjugated with chemiluminescent labels. The antigen-binding fragments disclosed herein can also be conjugated with fluorescent labels. This includes fluorophores (such as rare earth chelates, fluorescein, and their derivatives), rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, fluorescein, and amines. 152 Eu, dansyl, umbelliferone, fluorescein, luminal label, isoluminal label, aromatic acridine ester label, imidazole label, acridine salt label, oxalate ester label, jellyfish luminescent protein label, 2,3-dihydrophthalazinedione, biotin / avidin, spin label, and stable free radicals.

[0460] The antibodies of the present invention can also be conjugated to cytotoxic agents. The antigen-binding fragments of the antibodies of the present invention can also be conjugated to cytotoxic agents. The antibodies and their antigen-binding fragments of the present invention can also be conjugated to cytotoxic agents, such as auristatin F, paclitaxel, docetaxel, vincristine, CC-1065, SN-38, topotecan, morpholino doxorubicin, lysoxin, cyanomorpholino doxorubicin, dolastatin-10, echinomycin, comprehendingin, chalicheamicin, maytansine, DM-1, fusiformin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, absinthecin A chain, saccharin A chain, α-acinthin, and aleurites. Fordii) proteins and compounds (e.g., fatty acids), caryophyllin protein, Phytolacca americana protein PAPI, PAPII and PAP-S, momordica charantia inhibitors, jatropha toxin, croton toxin, saponaria officinalis inhibitors, lindolin, aspergillin, phenolmycin and enomycin. This list is not intended to be limiting. Antibodies of the present invention can be conjugated to aurestatin F. Antibodies of the present invention can be conjugated to parcetaxel. Antibodies of the present invention can be conjugated to docetaxel. Antibodies of the present invention can be conjugated to vincristine. Antibodies of the present invention can be conjugated to CC-1065. Antibodies of the present invention can be conjugated to SN-38. Antibodies of the present invention can be conjugated to topotecan. Antibodies of the present invention can be conjugated to morpholino doxorubicin. Antibodies of the present invention can be conjugated to lysin. Antibodies of the present invention can be conjugated to cyanomorpholino doxorubicin. The antibodies of the present invention can be conjugated to dolastatin-10. The antibodies of the present invention can be conjugated to echinococcin. The antibodies of the present invention can be conjugated to compressin. The antibodies of the present invention can be conjugated to chalcogenin. The antibodies of the present invention can be conjugated to maytansin. The antibodies of the present invention can be conjugated to DM-1. The antibodies of the present invention can be conjugated to fusiformin. The antibodies of the present invention can be conjugated to diphtheria toxin. The antibodies of the present invention can be conjugated to the A chain of Pseudomonas aeruginosa exotoxin. The antibodies of the present invention can be conjugated to the A chain of pectin. The antibodies of the present invention can be conjugated to the A chain of absinthecin. The antibodies of the present invention can be conjugated to the A chain of senna root toxin. The antibodies of the present invention can be conjugated to α-acramucin. The antibodies of the present invention can be conjugated to tung oil protein and compounds (e.g., fatty acids). The antibodies of the present invention can be conjugated to caryophyllin protein.The antibodies of the present invention can be conjugated to Papigenin (PAPI). The antibodies of the present invention can be conjugated to Papigenin (PAPII). The antibodies of the present invention can be conjugated to Papigenin (PAP-S). The antibodies of the present invention can be conjugated to bitter melon inhibitors. The antibodies of the present invention can be conjugated to jatropha toxin. The antibodies of the present invention can be conjugated to croton toxin. The antibodies of the present invention can be conjugated to soapwort inhibitors. The antibodies of the present invention can be conjugated to lindanemycin. The antibodies of the present invention can be conjugated to aspergillin. The antibodies of the present invention can be conjugated to phenytoin. The antibodies of the present invention can be conjugated to enoxacin. The antigen-binding fragment of the antibodies of the present invention can be conjugated to auratestatin F. The antigen-binding fragment of the antibodies of the present invention can be conjugated to parcetaxel. The antigen-binding fragment of the antibodies of the present invention can be conjugated to docetaxel. The antigen-binding fragment of the antibodies of the present invention can be conjugated to vincristine. The antigen-binding fragment of the antibodies of the present invention can be conjugated to CC-1065. The antigen-binding fragment of the antibodies of the present invention can be conjugated to SN-38. The antigen-binding fragment of the antibody of the present invention can be conjugated to topotecan. The antigen-binding fragment of the antibody of the present invention can be conjugated to morpholino doxorubicin. The antibody of the present invention can be conjugated to lysin. The antigen-binding fragment of the antibody of the present invention can be conjugated to cyanomorpholino doxorubicin. The antigen-binding fragment of the antibody of the present invention can be conjugated to dolastatin-10. The antigen-binding fragment of the antibody of the present invention can be conjugated to echinococcal. The antigen-binding fragment of the antibody of the present invention can be conjugated to compressin. The antigen-binding fragment of the antibody of the present invention can be conjugated to chalcoxim. The antigen-binding fragment of the antibody of the present invention can be conjugated to maytansin. The antigen-binding fragment of the antibody of the present invention can be conjugated to DM-1. The antigen-binding fragment of the antibody of the present invention can be conjugated to fusiformin. The antigen-binding fragment of the antibody of the present invention can be conjugated to diphtheria toxin. The antigen-binding fragment of the antibody of the present invention can be conjugated to the Pseudomonas aeruginosa exotoxin A chain. The antibody of the present invention can be conjugated to the pectin A chain. The antigen-binding fragment of the antibody of the present invention can be conjugated to the absinthecin A chain. The antigen-binding fragment of the antibody of the present invention can be conjugated to the A chain of *Saccharinus spp.* The antigen-binding fragment of the antibody of the present invention can be conjugated to α-octococcin. The antigen-binding fragment of the antibody of the present invention can be conjugated to tung oil protein and compounds (e.g., fatty acids). The antigen-binding fragment of the antibody of the present invention can be conjugated to caryophyllin protein. The antigen-binding fragment of the antibody of the present invention can be conjugated to *Phytolacca americana* protein (PAPI). The antigen-binding fragment of the antibody of the present invention can be conjugated to *Phytolacca americana* protein (PAPII). The antigen-binding fragment of the antibody of the present invention can be conjugated to *Phytolacca americana* protein (PAP-S). The antigen-binding fragment of the antibody of the present invention can be conjugated to bitter melon inhibitor. The antigen-binding fragment of the antibody of the present invention can be conjugated to *Jatropha curcas* toxin. The antigen-binding fragment of the antibody of the present invention can be conjugated to croton toxin. The antigen-binding fragment of the antibody of the present invention can be conjugated to soapwort inhibitor. The antigen-binding fragment of the antibody of the present invention can be conjugated to lindanemycin. The antigen-binding fragment of the antibody of the present invention can be conjugated to aspergillin.The antigen-binding fragment of the antibody of the present invention can be conjugated to phenolmycin. The antigen-binding fragment of the antibody of the present invention can be conjugated to enoxamycin.

[0461] The antibodies of this invention can also be conjugated to anticancer agents. These include, for example, erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatin(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), parcitabine (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), and trastuzumab. (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]non-2,7,9-trien-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethylamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®). Other commercially or clinically available anticancer agents include oxaliplatin (ELOXATIN®, Sanofi) and bortezomib (VELCADE®, Millennium Pharm).Sutent (SUNITINIB®, SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, ArrayBioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK 222584 (Novartis), Fulvestrant (FASLODEX®, AstraZeneca), Leucovorin / Folic Acid acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (cremophor-free), and albumin-engineered nanoparticle formulations of parcitaxel (American Pharmaceutical Partners). Schaumberg, Ill.The following are listed: vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa, and cyclophosphamide (CYTOXAN®, NEOSAR®); vinorelbine (NAVELBINE®); capecitabine (XELODA®, Roche); tamoxifen (including NOLVADEX®; tamoxifen citrate, FARESTON® (toremifine citrate)); MEGASE®. (Megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIIDEX® (anastrozole; AstraZeneca). This list is not intended to be restrictive.

[0462] The antigen-binding fragment of the antibody of the present invention can also be conjugated to anticancer agents. These anticancer agents include, for example, erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatin(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), and parcitabine (TAXOL®, Bristol-Myers Squibb Oncology, Princeton). NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]non-2,7,9-trien-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethylamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®). Other commercially or clinically available anticancer agents include oxaliplatin (ELOXATIN®, Sanofi) and bortezomib (VELCADE®, Millennium Pharm).Sunitinib® (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, ArrayBioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin / folinic acid The following are listed: rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo SmithKline), lonafanib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tepififibib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (without clamifloxacin), and albumin-engineered nanoparticle formulations of parcitabine (American Pharmaceutical Partners, Schaumberg, Ill).Vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU 5271; Sugen), tesiromoximide (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canphosphatamide (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); vinorelbine (NAVELBINE®); capecitabine (XELODA®, Roche), tamoxifen (including NOLVADEX®; tamoxifen citrate, FARESTON® (toremifene citrate), MEGASE® (medroxyprogesterone acetate), AROMASIN® (exemestane; Pfizer), formestan, fadroxil, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (Anastrozole; AstraZeneca). This list is not intended to be restrictive.

[0463] The antibody and antigen binding fragments described in this article can be labeled using paramagnetic chelates, microparticles, and superparamagnetic particles; and incorporated into ultrasonic bubbles, microparticles, microspheres, emulsions, etc.

[0464] Metal chelating agents are molecules having one or more polar groups that act as ligands for and complex with paramagnetic metals. Suitable chelating agents are those known in the art and include acids having methylenephosphonic acid groups, methylene carbonate hydroxylamine groups, carboxyethylidene groups, or carboxymethylene groups. Examples of chelating agents include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA), 1-substituted 1,4,7-tricarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Other chelating ligands are ethylidene bis-(2-hydroxy-phenylglycine) (EHPG) and its derivatives, including 5-C1-EHPG, 5Br-EHPG, 5-Me-EHPG, 5t-Bu-EHPG, and 5sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and its derivatives, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and diphenylmethyl-DTPA; bis-2-(hydroxyphenylmethyl)-ethylene-diaminediacetic acid (HBED) and its derivatives; and macrocyclic compounds containing at least 3 carbon atoms, more preferably at least 6 carbon atoms, and at least two heteroatoms (O and / or N), said macrocyclic compounds may consist of one ring or two or three rings linked together at heterocyclic elements, such as benzo-DOTA, dibenzo-DOTA, and benzo-NOTA. (wherein Nota is 1,4,7-triazacyclononane N,N′,N″-triacetic acid), benzo-TETA, benzo-DOTMA (wherein DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid)), and benzo-TETMA (wherein TETMA is 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyltetraacetic acid)); derivatives of 1,3-propylene-diaminetetraacetic acid (PDTA) and triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatechin ester (LICAM); and 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM).Examples of representative chelating agents and chelating groups covered by the present invention are described in WO 98 / 18496, WO 86 / 06605, WO 91 / 03200, WO 95 / 28179, WO 96 / 23526, WO 97 / 36619, PCT / US98 / 01473, PCT / US98 / 20182, and U.S. Patent Nos. 4,899,755, 5,474,756, 5,846,519, and 6,143,274, all of which are incorporated herein by reference.

[0465] Any method known in the art for conjugating the antibodies and their antigen-binding fragments of the present invention to various parts can be used, including those described in Hunter et al., (1962) Nature 144:945; David et al., (1974) Biochemistry 13:1014; Pain et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. The methods used for conjugating antibodies and fragments are conventional and well-known in the art.

[0466] Chemical crosslinking agents can be classified based on the following:

[0467] 1. Functional groups and chemical specificity;

[0468] 2. The length and composition of cross-linking bridges;

[0469] 3. Are the cross-linking groups similar (homobifunctional) or different (heterobifunctional)?

[0470] 4. Does the reaction involve a chemical reaction or a photochemical reaction?

[0471] 5. Whether the reagent is cleavable; and

[0472] 6. The reagent may be radiolabeled or labeled with another label.

[0473] Reactive groups on antibodies and labels that can be targeted using cross-linking agents include primary amines, carbonyl groups, carbohydrates, and carboxylic acids. Furthermore, many reactive groups can be non-selectively coupled using cross-linking agents such as photoreactive phenyl azide compounds. For information on suitable reagents, see Pierce 2003-2004 Applications Handbook and Catalog # 1600926, which is incorporated herein by reference.

[0474] Many factors must be considered to determine the optimal cross-linking agent to target molar ratio. Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is usually required to increase the immunogenicity of the antigen. However, when conjugating antibodies or enzymes, a low to medium degree of conjugation may be optimal to ensure that the protein's biological activity is preserved. The number of reactive groups on the protein surface is also important to consider. If many target groups are present, a lower cross-linking agent to protein ratio can be used. For a limited number of potential targets, a higher cross-linking agent to protein ratio may be necessary. For small molecular weight proteins, this translates to more cross-linking agent per gram.

[0475] Protein conformational changes associated with specific interactions can also be analyzed by performing cross-linking studies before and after the interaction. Comparisons are made by using cross-linking agents with different arm lengths and analyzing the success of conjugation. When protein conformation changes, it may be necessary to use cross-linking agents with different reactive groups and / or spacer arms to make the hindered amino acids available for cross-linking.

[0476] Crosslinking agents with spacer arms or bridges of varying lengths connecting reactive ends are available. The most obvious property of a bridge is its ability to handle spatial considerations of the parts to be joined. Because spatial effects indicate the distance between potential reactive sites for crosslinking, bridges of different lengths can be considered for different interactions. Shorter spacer arms are often used in intramolecular crosslinking studies, while intermolecular crosslinking prefers crosslinking agents with longer spacer arms.

[0477] In some cases, it may be advantageous for the crosslinker to include a polymeric portion (e.g., polyethylene glycol (“PEG”) homopolymer, polypropylene glycol homopolymer, other alkyl-polyethylene oxide, dipolyethylene oxide, and copolymers or blocking copolymers of poly(olefin oxides)). See, for example, U.S. Patents 5,643,575, 5,672,662, 5,705,153, 5,730,990, 5,902,588, and 5,932,462; and Topchieva et al., Bioconjug. Chem. 6: 380-8, 1995). For instance, U.S. Patent 5,672,662 discloses a bifunctional crosslinker comprising a PEG polymeric portion and a single ester bond. In water, such molecules are believed to provide a half-life of approximately 10 to 25 minutes.

[0478] Designing crosslinking agents involves selecting the functional moiety to be used. The selection of the functional moiety depends entirely on the available target sites on the substance to be crosslinked. Some substances (such as proteins) may have many sites that can be used for targeting (e.g., lysine ε-amino, cysteine ​​sulfhydryl, glutamic acid carboxyl, etc.), and specific functional moieties can be selected empirically to best preserve the biological properties of interest (e.g., antibody binding affinity, enzyme catalytic activity, etc.).

[0479] Coupling via amine groups

[0480] Typically, imine esters and N-hydroxysuccinimide (“NHS”) esters are used as amine-specific functional moieties. NHS esters produce stable products upon reaction with primary or secondary amines. Coupling is highly efficient at physiological pH, and NHS-ester crosslinkers are more stable in solution than their imine ester counterparts. Bifunctional NHS-ester conjugations are commonly used in one- or two-step reactions to crosslink amine-containing proteins. Primary amines are the principle targets of NHS-esters. Accessible α-amine groups at the N-terminus of proteins react with NHS-esters to form amides. However, because α-amines on proteins are not always available, reactions with the side chains of amino acids become important. Although all five amino acids have nitrogen in their side chains, only the ε-amino group of lysine reacts significantly with NHS-esters. When an NHS-ester crosslinker reacts with a primary amine, a covalent amide bond is formed, releasing N-hydroxysuccinimide.

[0481] via thiol coupling

[0482] Maleimides, alkyl and aryl halides, α-haloacyl groups, and pyridyl disulfides are typically used as the thioglycolic functional moiety. The maleimide group exhibits specificity for the thiol group when the pH of the reaction mixture is maintained between pH 6.5 and 7.5. At pH 7, maleimides react with thioglycolic groups 1000 times faster than with amines. Maleimides do not react with tyrosine, histidine, or methionine. When free thioglycolic groups are not present in sufficient quantities, they can often be generated by reducing available disulfide bonds.

[0483] Through carboxyl coupling

[0484] Carbodiimides couple a carboxyl group to a primary amine or hydrazide, resulting in the formation of an amide or hydrazone bond. Unlike other conjugation reactions, carbodiimides do not form cross-linking bridges with the molecule being coupled; instead, peptide bonds are formed between the available carboxyl group and the available amine group. They can target the carboxyl terminus of proteins as well as the glutamic acid and aspartic acid side chains. Polymerization can occur in the presence of excess cross-linking agents because proteins contain both carboxyl and amine groups. Since no cross-linking bridges are formed, and the amide bond is identical to the peptide bond, reversing the cross-linking is impossible without damaging the protein.

[0485] Non-selective labeling

[0486] Photoaffins are chemically inert compounds, but become reactive when exposed to ultraviolet or visible light. Aryl azides are photoaffins that photolyze at wavelengths between 250 and 460 nm to form reactive aryl azenes. The aryl azenes react non-selectively to form covalent bonds. Reducing agents must be used with caution because they can reduce the azide group.

[0487] carbonyl-specific crosslinking agent

[0488] At pH 5–7, carbonyl groups (aldehydes and ketones) react with amines and acylhydrazides. The reaction with acylhydrazides is faster than with amines, making this suitable for site-specific crosslinking. Carbonyl groups are not readily present in proteins; however, mild oxidation of the sugar moiety using sodium metasulfonate will convert adjacent hydroxyl groups into aldehydes or ketones.

[0489] Laboratory and diagnostic uses

[0490] The anti-CD103 antibodies disclosed herein can be used as affinity purifiers. The anti-CD103 antigen-binding fragments disclosed herein can also be used as affinity purifiers. In this method, the anti-CD103 antibody and its antigen-binding fragment are immobilized on a solid phase (such as cross-linked dextran gel, glass or agarose resin, or filter paper) using methods well known in the art. The immobilized antibody or fragment is contacted with a sample containing the CD103 protein (or a fragment thereof) to be purified, and the support is subsequently washed with a suitable solvent that removes substantially all material from the sample except for the CD103 protein, which binds to the immobilized antibody or fragment. Finally, the support is washed with a solvent that elutes the bound CD103 (e.g., protein A). Such immobilized antibodies and fragments form part of this invention.

[0491] An antigen for generating a second antibody is also provided, which can be used, for example, for Western blotting and other immunoassays discussed herein.

[0492] Anti-CD103 antibodies (such as humanized antibodies) and their antigen-binding fragments can also be used for diagnostic analysis of the CD103 protein, such as detecting its expression in specific cells, tissues, or serum (e.g., myeloid cells, such as monocytes, macrophages, neutrophils, basophils, eosinophils, and dendritic cells). Such diagnostic methods can be used for the diagnosis of a variety of diseases.

[0493] This invention includes an ELISA (enzyme-linked immunosorbent assay) incorporating the use of the anti-CD103 antibody or its antigen-binding fragment disclosed herein.

[0494] For example, such methods include the following steps:

[0495] (a) Coating a substrate (e.g., the surface of a microtiter plate well, such as a plastic plate) with an anti-CD103 antibody or its antigen-binding fragment;

[0496] (b) Apply the sample to the substrate to test for the presence of CD103;

[0497] (c) Wash the plate to remove unbound material from the sample;

[0498] (d) Apply a detectable labeled antibody (e.g., enzyme-linked antibody) that is also specific to the CD103 antigen;

[0499] (e) Wash the substrate to remove unbound labeled antibodies;

[0500] (f) If the labeled antibody is enzyme-linked, then apply a chemical that is converted into a fluorescent signal by the enzyme; and

[0501] (g) Detect the presence of labeled antibodies.

[0502] The detection of a marker associated with the substrate indicates the presence of the CD103 protein.

[0503] In another embodiment, the labeled antibody or its antigen-binding fragment is labeled with a peroxidase, which reacts with ABTS (e.g., 2,2'-azono-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or 3,3',5,5'-tetramethylbenzidine to produce a color change, which is detectable. Alternatively, the labeled antibody or fragment is labeled with a detectable radioisotope (e.g., 3 H) label, the detectable radioactive isotope can be detected by a scintillation counter in the presence of a scintillator.

[0504] The anti-CD103 antibody or its antigen-binding fragment of the present invention can be used in Western blotting or immunoblotting procedures. Such procedures form part of the present invention and include, for example:

[0505] (1) Optionally, a protein from a sample in which CD103 is to be tested (e.g., separated by PAGE or SDS-PAGE electrophoresis of proteins in the sample) is transferred onto a membrane or other solid substrate using a method known in the art (e.g., semi-dry blotting or jar blotting); and the membrane or other solid substrate in which the presence of the bound CD103 or a fragment thereof is to be tested is brought into contact with the anti-CD103 antibody or an antigen-binding fragment thereof of the present invention.

[0506] (2) Wash the membrane once or multiple times to remove unbound anti-CD103 antibodies or fragments and other unbound substances; and

[0507] (3) Detect the bound anti-CD103 antibody or fragment.

[0508] Such membranes may be in the form of nitrocellulose or vinyl (e.g., polyvinylidene fluoride (PVDF)) membranes, to which the proteins for which the presence of CD103 is to be tested in non-denaturing PAGE (polyacrylamide gel electrophoresis) or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels have been transferred (e.g., after electrophoretic separation in the gel). The membrane may optionally be blocked, for example with skim milk powder, before being contacted with an anti-CD103 antibody or fragment to allow binding to non-specific protein binding sites on the membrane.

[0509] The detection of the bound antibody or fragment indicates the presence of CD103 protein on the membrane or substrate and in the sample. The bound antibody or fragment can be detected by binding the antibody or fragment to a detectable labeled second antibody (anti-immunoglobulin antibody) and then detecting the presence of the second antibody.

[0510] Immunohistochemical analysis can also be performed using the anti-CD103 antibody and its antigen-binding fragment disclosed herein. Such methods form part of this invention and include, for example,

[0511] (1) Contacting the cells (e.g., samples containing myeloid cells, such as monocytes, macrophages, neutrophils, basophils, eosinophils, and dendritic cells) to be tested for the presence of CD103 protein with the anti-CD103 antibody or its antigen-binding fragment of the present invention; and

[0512] (2) Detect antibodies or fragments on or in cells.

[0513] If the antibody or fragment itself is a detectable label, then it can be detected directly. Alternatively, the antibody or fragment can be bound by a second antibody to the detectable label.

[0514] In vivo tumor imaging can also be performed using certain anti-CD103 antibodies and their antigen-binding fragments disclosed herein. Such methods may include injecting a radiolabeled anti-CD103 antibody or its antigen-binding fragment into a patient to be tested for the presence of a tumor associated with CD103 expression (e.g., CD103 expression on the surface of tumor cells), followed by nuclear imaging of the patient's body to detect, for example, the presence of the antibody or fragment labeled at a locus containing a high concentration of the antibody or fragment bound to the tumor. Detection of said locus indicates the presence of CD103. + Tumors and tumor cells.

[0515] Imaging techniques include SPECT (single-photon emission computed tomography) or PET (positron emission tomography). Labeling includes, for example, iodine-123 (…). 123 I) and Technetium-99m ( 99m Tc), for example, combined with SPECT imaging or 11 C 13 N、 15 O or 18 F, for example, in combination with PET imaging or indium-111 (see, for example, Gordon et al., (2005) International Rev. Neurobiol. 67:385-440).

[0516] Pharmaceutical compositions and their application and therapeutic uses

[0517] The antibodies of the present invention inhibit CD103 signaling, and therefore, in one aspect of the invention, certain antibodies disclosed herein are candidates for the treatment or prevention of certain diseases and ailments. The present invention provides a method for treating diseases and ailments in which the course of the disease or ailment can be affected by CD103 signaling. The method comprises administering a therapeutically effective amount of the antibody of the present invention to a subject requiring such treatment.

[0518] Integrin family heterodimers play multiple and redundant roles in T cell activation, homing, and effector delivery. The CD103 integrin heterodimer was initially identified by its expression on T cells in the vertebrate intestinal mucosa, where it is expressed at high levels by >95% of intestinal intraepithelial lymphocytes (iIEL) and approximately 40% of lamina propria lymphocytes. CD103 recognizes the epithelial cell-specific ligand E-cadherin. In normal mice and humans, CD8+ T cells residing in the intestinal epithelium express high levels of CD103, and CD103 is widely expressed in intraepithelial lymphocytes, tumor-infiltrating lymphocytes, and certain dendritic cells. Previous studies have demonstrated that CD103 plays a crucial role in cell lysis induced by tumor-specific infiltrating lymphocytes by triggering cytolytic granule polarization and exocytosis through its interaction with its tumor cell ligand E-cadherin. Furthermore, CD103-E-cadherin binding promotes T cell attachment to tumor cells and induces co-stimulation in activated cytotoxic T cells. These findings suggest that CD103 could be a target for enhancing tumor immunity.

[0519] In various embodiments, the antibody of the present invention blocks the binding of E-cadherin; depletes CD103+ cells; depletes CD103+CD8+ effector cells; and / or depletes tissue-resident memory T cells (T cells). RM ).

[0520] The present invention also provides a method for treating a CD103 signaling-mediated disorder, the method comprising administering an effective amount of one or more antibodies of the present invention to a patient in need. In some embodiments, the CD103 signaling-mediated disorder is an autoimmune, inflammatory or neurodegenerative disorder or cancer (see Rayburn, ER et al., Mol Cell Pharmacol. 2009; 1(1): 29-43 and Urbanska, AM et al., Cell Biochem Biophys. 2015 Jul; 72(3): 757-69).

[0521] CD103 in allogeneic transplant rejection

[0522] FACS analysis of resected kidney specimens revealed high levels of CD103 expression in a large subset of CD8 effectors infiltrating allogeneic grafts that had experienced rejection. Interestingly, in cases of chronic allogeneic transplant nephropathy, the CD103+CD8+ effector was most abundant in allogeneic kidney grafts that had experienced rejection. Importantly, the CD103+CD8+ effector was absent in the peripheral lymphoid compartment (i.e., peripheral blood lymphocytes) and was therefore undetectable by conventional immunosurveillance methods. However, CD103 mRNA was expressed in cells isolated from the urine of allogeneic kidney transplant recipients with clinical rejection, consistent with the tubular intratubular localization of the CD103+CD8+ effector during rejection. The aforementioned clinical observations are consistent with the major role of CD103 in promoting the destruction of transplanted epithelial compartments by the CD8 effector population and support the hypothesis that CD103 expression is necessary for CD8-mediated destruction of transplanted epithelial elements. The antibodies of the present invention, as described herein, can be used to treat allogeneic transplant rejection. The antibodies of the present invention, as described herein, can be used to prevent allogeneic transplant rejection. The antigen-binding fragments of the present invention, as described herein, can be used to treat allogeneic transplant rejection. The antigen-binding fragments of the present invention, as described herein, can be used to prevent allogeneic transplant rejection.

[0523] Tissue-resident memory T cells

[0524] It has long been known that chronic skin inflammation (especially psoriasis and FDE) frequently recurs at previously affected sites. Therefore, immune memory has been proposed to be associated with sudden-onset reactive and chronic inflammatory conditions. Regarding T... RM Triggering characteristics of cells (long-term survival and low migration in peripheral tissues) have been proposed for skin T cells. RM Cells can actively participate in the recurrence of inflammatory skin conditions.

[0525] Initial flare-ups of psoriatic lesions often recur at previously resolved sites, and locally resident memory T cells have been proposed to play a role in their development and sudden onset. CD8+ in psoriatic lesions + T cells are highly activated and express large amounts of CD69 and CD103. In contrast, these proteins are constitutively expressed by very few T cells in peripheral blood; furthermore, it is evident that T cells... EM Cells interact with the angiotensin E-selectin and are transported to the skin during infection or flare-ups. More importantly, current research indicates that TCRαβ is present at sites of psoriasis remission, and even in outwardly normal skin. + Resident T cells accumulate, and they are able to produce IL-17 and IFN-γ to trigger a psoriasis-like response. These findings support the important role of lesion-resident T cells in the development of psoriasis. The antibodies of the present invention described herein can be used to treat psoriasis. The antibodies of the present invention described herein can be used to prevent psoriasis. The antigen-binding fragments of the present invention described herein can be used to treat psoriasis. The antigen-binding fragments of the present invention described herein can be used to prevent psoriasis.

[0526] Inflammatory bowel disease and CD103

[0527] A growing consensus among pathological processes driving intestinal inflammation and autoimmunity is the loss of immunological homeostasis secondary to qualitative or quantitative deficiencies in the regulatory T cell (Treg) aggregate. Tregs can be broadly classified into two groups based on their developmental origin: thymic Tregs (tTregs) or peripherally induced Tregs (pTregs). T cells can be converted into Foxp3-expressing CD4+CD25+ Tregs via T cell receptor (TCR) co-stimulation in the presence of transforming growth factor β (TGF-β).14 In the presence of TGF-β, IL-2, and retinoic acid (RA), pTreg conversion in gut-associated lymphoid tissue (GALT) is enhanced when primordial CD4+ T cells encounter antigens.1516 CD103+ DCs are influenced by the gut microenvironment to produce or activate TGF-β and provide RA to facilitate this process.1718 In the absence of CD103 expression, DCs fail to induce Treg development and produce pro-inflammatory cytokines. In UC patients, colonic CD4+ has been reported to be significantly reduced. + CD103 expression on T cells is associated with increased production of pro-inflammatory Th1, Th17, and Th1 / Th17 cytokines, and CD103 expression is also associated with increased production of these cytokines in UC patients. + DCs have the ability to drive Th1 / Th2 / Th17 cell responses. Therefore, the efficacy of targeting integrins can be achieved by eliminating CD103, which causes colitis. +Dendritic cells and blocking lymphocyte recruitment are used to explain this. The antibodies of the present invention, as described herein, can be used to treat inflammatory bowel disease. The antibodies of the present invention, as described herein, can be used to prevent inflammatory bowel disease. The antigen-binding fragments of the present invention, as described herein, can be used to treat inflammatory bowel disease. The antigen-binding fragments of the present invention, as described herein, can be used to prevent inflammatory bowel disease.

[0528] CD103+ lymphoproliferative disorders

[0529] In the diagnosis of B-cell lymphoproliferative disorders (BC-LPD), including B-cell chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and hairy cell leukemia (HCL), flow cytometry immunophenotyping is important. In the diagnostic evaluation of low-grade B-cell lymphoproliferative processes, a positive CD103 positivity indicator is confirmed for the diagnosis of hairy cell leukemia (HCL) or its variant HCLv.

[0530] In addition to these B-cell disorders, CD103 positivity is also a characteristic feature of a subset of T-cell sarcomas. In a study of 184 cases representing the majority of entities within the current World Health Organization T-cell sarcoma category, 46% of gastrointestinal lymphomas, 40% of adult T-cell leukemia / lymphomas, and 6.9% of other sarcomas showed CD103 positivity. Similarly, blastic plasmacytoid dendritic cell sarcoma (BPDCN) expresses CD103.

[0531] The antibodies of the present invention described herein can be used to treat lymphoproliferative disorders. The antigen-binding fragments of the present invention described herein can be used to treat lymphoproliferative disorders. Lymphoproliferative disorders expressing CD103 that can be treated with the antibodies of the present invention include, but are not limited to, hairy cell leukemia, HCLv, intra- and extra-intestinal lymphomas, enteropathy-associated T-cell lymphoma (EATL), T-lymphoblastic leukemia / lymphoma (T-ALL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell leukemia / lymphoma (ATLL), mycosis fungoides (MF), anaplastic large cell lymphoma ALCL, cutaneous T-cell lymphoma (CTCL), and blastic plasmacytoid dendritic cell neoplasm.

[0532] CD103 in tumorigenesis

[0533] Tumor-associated CD103+ CD8 T cells have been reported to exhibit a tolerogenic phenotype in the presence of increased CTLA-4 and IL-10 expression and decreased expression of IFN-γ, TNF-α, and granzyme. Furthermore, CD103 has been described as a marker of CD4+-regulated cells and is present on tolerogenic DCs. Anti-CD103 antibodies that reduce CD103+ CD8 T cells in mice, directly targeting CD103, have been reported to provide therapeutic effects in B16 melanoma and MC38 CRC models. Antibodies of the present invention as described herein can be used to treat tumorigenesis. Antibodies of the present invention as described herein can be used to prevent tumorigenesis. Antigen-binding fragments of the present invention as described herein can be used to treat tumorigenesis. Antigen-binding fragments of the present invention as described herein can be used to prevent tumorigenesis. Antibodies of the present invention as described herein can be used to slow the progression of tumorigenesis.

[0534] Therapeutic application of the CD103 antibody of the present invention

[0535] This invention provides the use of one or more antibodies of the present invention for inhibiting CD103 signaling in cells.

[0536] This invention provides the use of one or more antibodies of the present invention for inhibiting CD103 binding to E-cadherin and cells expressing E-cadherin.

[0537] The present invention also provides the use of one or more antibodies of the present invention for the treatment of CD103-mediated diseases.

[0538] This invention provides the use of one or more antibodies of the invention for depleting cells expressing CD103.

[0539] The present invention also provides the use of one or more antibodies of the present invention in the manufacture of a pharmaceutical agent for one of the foregoing uses.

[0540] The antibodies of the present invention, as described herein, can be used to treat a variety of diseases, wherein modulation of CD103 signaling can provide therapeutic benefits. In some aspects, the compounds of the present invention inhibit CD103 signaling and can be used to treat diseases selected from the group consisting of: atopic dermatitis, allergic reactions, asthma, systemic inflammatory response syndrome (SIRS), sepsis, septic shock, atherosclerosis, celiac disease, dermatomyositis, scleroderma, interstitial cystitis, transplant rejection, graft-versus-host disease, Ekaterie-Gutteres syndrome, Hodgson-Gilles syndrome, Simpson-Meyer syndrome, proteasome-associated autoinflammatory syndrome, SAVI (Sting-related vascular disease with infancy), and CANDLE. (Chronic atypical neutrophilic dermatosis with lipid metabolism disorders and hyperthermia) syndrome, frostbite-like lupus erythematosus, systemic lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, Wegener's disease, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, glomerulonephritis, autoimmune myocarditis, myasthenia gravis, vasculitis, type 1 diabetes mellitus, type 2 diabetes mellitus, Sjögren's syndrome, X-linked reticulochromocytosis, polymyositis, spinal chondrodysplasia, age-related macular degeneration, Alzheimer's disease, and Parkinson's disease. In some embodiments, the compounds of the present invention can be used to treat Ecardi-Gutteres syndrome, X-linked reticulochromia, dermatomyositis, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, or type I or type II diabetes. The antibodies of the present invention as described herein are preferably used to treat inflammatory bowel disease. The antibodies of the present invention as described herein are preferably used to treat psoriasis.

[0541] This invention provides a method for treating an autoimmune disease in a subject, the method comprising administering a therapeutically effective amount of one or more antibodies of the invention to a subject in need. In some embodiments, the autoimmune disease may be type I interferon disorders (e.g., Ekady-Gutteres syndrome, Sjögren's syndrome, Szymborsson-Medell's syndrome, proteasome-associated autoinflammatory syndrome, SAVI (Sting-associated vascular disease with infancy), Candle syndrome, frostbite-like lupus erythematosus, systemic lupus erythematosus, vertebral chondrodysplasia), rheumatoid arthritis, juvenile rheumatoid arthritis, idiopathic thrombocytopenic purpura, autoimmune myocarditis, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, psoriasis, type 1 diabetes, or type 2 diabetes.

[0542] This invention provides a method for treating an inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of one or more antibodies of the invention to the subject in need. For example, inflammatory diseases may be selected from the group consisting of: atopic dermatitis, allergic reactions, asthma, systemic inflammatory response syndrome (SIRS), sepsis, septic shock, atherosclerosis, celiac disease, dermatomyositis, scleroderma, interstitial cystitis, transplant rejection, graft-versus-host disease, Ekaterie-Gutteres syndrome, Hodgson-Gilles syndrome, Szymborskin syndrome, proteasome-associated autoinflammatory syndrome, SAVI (Sting-related vascular disease with infancy), and CANDLE. (Chronic atypical neutrophilic dermatosis with lipid metabolism disorders and hyperthermia) syndrome, frostbite-like lupus erythematosus, systemic lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, Wegener's disease, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, glomerulonephritis, autoimmune myocarditis, myasthenia gravis, vasculitis, type 1 diabetes mellitus, type 2 diabetes mellitus, Sjögren's syndrome, X-linked reticulochromocytosis, polymyositis, spinal chondrodysplasia, age-related macular degeneration, Alzheimer's disease, and Parkinson's disease. In some embodiments, the compounds of the present invention can be used to treat Ekady-Guterres syndrome, X-linked reticulochromia, dermatomyositis, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, or type I or type II diabetes.

[0543] The present invention also provides a method for treating a subject with a neurodegenerative disease, the method comprising administering a therapeutically effective amount of one or more antibodies of the present invention to the subject in need. For example, neurodegenerative diseases may include Alzheimer's disease, Parkinson's disease, multiple sclerosis, IgM polyneuropathy, or myasthenia gravis.

[0544] The present invention also provides a method for treating malignant tumors expressing CD103. For example, malignant tumors can be T-cell and B-cell lymphomas, and particularly hairy cell leukemia, HCLv, intra- and extra-intestinal lymphomas, enteropathy-associated T-cell lymphoma (EATL), T-lymphoblastic leukemia / lymphoma (T-ALL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell leukemia / lymphoma (ATLL), mycosis fungoides (MF), anaplastic large cell lymphoma ALCL, cutaneous T-cell lymphoma (CTCL), and Cezari syndrome (SS).

[0545] To prepare the anti-CD103 antibody and antigen-binding fragment of the present invention into a pharmaceutical or sterile composition, the antibody or its antigen-binding fragment is mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0546] Formulations of therapeutic and diagnostic agents can be prepared by mixing with carriers, excipients, or stabilizers in forms acceptable for example, such as lyophilized powders, slurries, aqueous solutions, or suspensions (see, for example, Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, NY).

[0547] The toxicity and therapeutic efficacy of the antibodies of the present invention, administered alone or in combination with another therapeutic agent, can be determined in cell cultures or laboratory animals by, for example, methods used to determine LD50. 50 (50% of the lethal dose for the population) and ED 50 The standard drug procedure determines the dose that is effective in 50% of the population. The dose-to-dose ratio between toxicity and therapeutic effect is the therapeutic index (LD50). 50 / ED 50 Data obtained from these cell culture analyses and animal studies can be used to formulate a range of dosages for human use. The preferred dosages for such compounds include those with minimal toxicity. 50 The cyclic concentration range is specified. Dosage may vary within this range depending on the dosage form and route of administration.

[0548] In another embodiment, another therapeutic agent is administered to the subject in combination with the anti-CD103 antibody of the present invention or its antigen-binding fragment, according to Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

[0549] The mode of administration can be varied. Routes of administration include oral, rectal, mucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrasheath, directly intracardiac, intravenous, intraperitoneal, intranasal, intraocular, inhalation, inhalation, surface, transdermal, transdermal, or intra-arterial.

[0550] In certain embodiments, the anti-CD103 antibody or its antigen-binding fragment of the present invention may be administered via invasive routes, such as injection. In other embodiments of the invention, the anti-CD103 antibody or its antigen-binding fragment or pharmaceutical composition thereof may be administered intravenously, subcutaneously, intramuscularly, intra-arterially, intratumorally, or by inhalation or aerosol delivery. Administration via non-invasive routes (e.g., orally; for example, in the form of pills, capsules, or tablets) is also within the scope of the invention.

[0551] This invention provides a container (e.g., a plastic or glass vial, such as one with a cap or chromatographic column, a hollow needle, or a syringe barrel) containing any of the antibodies or antigen-binding fragments of the present invention or their pharmaceutical compositions. The invention also provides an injection device containing any of the antibodies or antigen-binding fragments of the present invention or their pharmaceutical compositions. The injection device is a means of introducing a substance into a patient via a parenteral route (e.g., intramuscular, subcutaneous, or intravenous). For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector), which includes, for example, a barrel or container for receiving a fluid (e.g., an antibody or fragment or its pharmaceutical composition) to be injected, a needle for piercing the skin and / or blood vessel to inject the fluid, and a piston for discharging the fluid from the barrel and through the needle eye. In one embodiment of the invention, the injection device containing the antibody or its antigen-binding fragment or its pharmaceutical composition is an intravenous (IV) injection device. Such devices comprise antibodies or fragments or pharmaceutical compositions thereof in a cannula or cannula / needle, which may be connected to a tube that may be connected to a bag or reservoir for containing fluid (e.g., saline; or a lactated Ringer's solution containing NaCl, sodium lactate, KCl, CaCl2, and optionally glucose) introduced into the patient via the cannula or cannula / needle. In one embodiment of the invention, the antibody or fragment or pharmaceutical composition thereof may be introduced into the device after the cannula and needle have been inserted into a vein of the subject and the cannula has been removed from the inserted cannula. For example, the intravenous device may be inserted into a peripheral vein (e.g., in the hand or arm); the superior or inferior vena cava or the right atrium of the heart (e.g., the central vein); or inserted subpinnately, into the internal jugular vein or femoral vein, and advanced, for example, toward the heart until it reaches the superior vena cava or the right atrium (e.g., the central vein). In one embodiment of the invention, the injection device is an autoinjector; a jet injector or an external infusion pump. An injector uses a high-pressure, narrow jet of liquid that penetrates the epidermis to deliver antibodies or fragments, or drug compositions thereof, into the patient's body. An external infusion pump is a medical device that delivers antibodies or fragments, or drug compositions thereof, into the patient's body in a controlled manner. External infusion pumps can be electrically or mechanically driven. Different pumps operate in different ways; for example, a syringe pump contains fluid in a syringe reservoir and a movable piston controls fluid delivery; an elastic pump contains fluid in a stretchable balloon reservoir and pressure from the elastic walls of the balloon drives fluid delivery; in a peristaltic pump, a set of rollers presses downwards along a flexible tube of a certain length, propelling fluid forward; in a multichannel pump, fluid can be delivered from multiple reservoirs at multiple rates.

[0552] The pharmaceutical compositions disclosed herein can also be administered using needle-free subcutaneous injection devices, such as those disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Such needle-free devices containing pharmaceutical compositions are also part of this invention. The pharmaceutical compositions disclosed herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include those disclosed in the following references: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable-flow-rate implantable infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196, which discloses a permeable drug delivery system having multiple compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art and those incorporating the pharmaceutical compositions of the present invention are within the scope of this invention.

[0553] Alternatively, the anti-CD103 antibody or antigen-binding fragment of the present invention can be administered locally rather than systemically, for example, by directly injecting the antibody or fragment into the tumor. Furthermore, the antibody or fragment can be administered in a targeted drug delivery system (e.g., in liposomes coated with tissue-specific antibodies targeting, for example, tumors). The liposomes will be targeted and selectively absorbed by the affected tissue. Such methods and liposomes are part of the present invention.

[0554] Administration regimens depend on several factors, including the serum or tissue conversion rate of the therapeutic antibody or antigen-binding fragment, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody or fragment to achieve improvement in the condition of the target disease while minimizing undesirable side effects. Therefore, the amount of biological agent delivered depends in part on the specific therapeutic antibody and the severity of the disease being treated. Guidelines for selecting appropriate doses of therapeutic antibodies or fragments are available (see, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

[0555] The determination of the appropriate dosage is made by the clinician, for example, using parameters or factors known or suspected to affect treatment in the field. Typically, the dosage is started at a slightly less than optimal dose and then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include, for example, symptoms of inflammation or levels of inflammatory cytokines produced. Generally, it is desirable to derive the biologics used from the same species as the animal being targeted for treatment, thereby minimizing any immune response to the agent. In the case of human subjects, humanized and fully human antibodies, for example, may be required.

[0556] The antibodies or antigen-binding fragments disclosed herein may be delivered by continuous infusion or by doses administered, for example, daily, 1-7 times per week, weekly, bi-weekly, monthly, bi-monthly, quarterly, semi-annually, or annually. Doses may be delivered, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is typically at least 0.05 μg per kilogram of body weight, and more typically at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / mL, 10 mg / kg, 25 mg / kg, 50 mg / kg or more (see, for example, Yang et al. (2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67: 451-456; Portielji et al. (20003) Cancer Immunol. Immunother. 52: 151-144). Doses may also be provided to achieve predetermined target concentrations of the anti-CD103 antibody in the serum of the subject, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / mL or more. In other embodiments, the anti-CD103 antibody of the present invention may be administered to each subject at doses of 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg weekly, bi-weekly, “every four weeks,” monthly, bi-monthly or quarterly, for example subcutaneously or intravenously.

[0557] As used herein, the term "effective dose" refers to the amount by which the anti-CD103 or its antigen-binding fragment of the present invention effectively induces a measurable improvement in one or more symptoms of a disease (e.g., cancer or cancer progression) when administered alone or in combination with another therapeutic agent to cells, tissues, or subjects. An effective dose also refers to an amount of antibody or fragment sufficient to cause at least partial improvement in symptoms (e.g., tumor shrinkage or elimination, lack of tumor growth, increased survival time). When applied to an individual active ingredient administered alone, the effective dose refers to the individual ingredient. When applied in combination, the effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, whether administered in combination, consecutively, or simultaneously. An effective dose of the therapeutic agent will result in an improvement of diagnostic metrics or parameters of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. An effective dose may also improve subjective measures in cases where subjective measures are used to assess disease severity.

[0558] medicine box

[0559] A kit is also provided, comprising one or more components, including but not limited to anti-CD103 antibodies or antigen-binding fragments as discussed herein, and the one or more components combined with one or more other components, including but not limited to pharmaceutically acceptable carriers and / or therapeutic agents as discussed herein. The antibodies or fragments and / or therapeutic agents may be formulated as pure compositions or combined with pharmaceutically acceptable carriers in pharmaceutical compositions.

[0560] In one embodiment, the kit includes the anti-CD103 antibody of the present invention or its antigen-binding fragment or its pharmaceutical composition thereof in a container (e.g., in a sterile glass or plastic vial) and / or the therapeutic agent and its pharmaceutical composition in another container (e.g., in a sterile glass or plastic vial).

[0561] In another embodiment, the kit contains a combination of the invention in a single common container, the combination of the invention comprising an anti-CD103 antibody or antigen-binding fragment thereof of the invention, optionally formulated together with one or more therapeutic agents to form a pharmaceutical composition, and a pharmaceutically acceptable carrier.

[0562] If the pillbox includes a pharmaceutical composition for parenteral administration to a subject, then the pillbox may include a device for making such administration. For example, the pillbox may include one or more hypodermic needles or other injection devices as discussed above.

[0563] The medicine box may include a packaging insert containing information about the pharmaceutical composition and dosage form contained within. Typically, this information helps patients and physicians use the packaged pharmaceutical composition and dosage form effectively and safely. For example, the insert may provide information about combinations of the present invention such as: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and use, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, how to supply, appropriate storage conditions, references, manufacturer / marketer information, and patent information.

[0564] Test kit

[0565] Diagnostic or detection reagents and kits containing one or more such reagents are also provided for various detection analyses, including, for example, immunoassays such as ELISA (sandwich or competitive modes)). Kit components may be pre-attached to a solid carrier or applied to the surface of the solid carrier when the kit is used. In some embodiments of the invention, the signal-generating component may be pre-associated with an antibody or fragment of the invention or may need to be combined with one or more components (e.g., buffer, antibody-enzyme conjugate, enzyme substrate, etc.) before use. The kit may also include other reagents, such as blocking agents, washing agents, enzyme substrates, etc., for reducing nonspecific binding to the solid surface. The solid surface may be in the form of tubes, beads, microtiter plates, microspheres, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In certain aspects, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or reduces a chemiluminescent or chromogenic substrate is a component of the signal-generating component. Such enzymes are well known in the art. The kit may contain any of the capture agents and detection reagents described herein. Optionally, the kit may also include instructions for performing the methods of the invention.

[0566] The test kits disclosed herein can also be prepared to contain at least one of the antibodies, peptides, antigen-binding fragments, or polynucleotides disclosed herein, along with instructions for using the composition as a test reagent. Containers for such kits typically include at least one vial, test tube, flask, bottle, syringe, or other suitable container into which one or more of the test compositions can be placed and preferably appropriately aliquoted. The kits disclosed herein will also typically include components for tightly sealing one or more vials for commercial sale, such as injection or blow-molded plastic containers in which one or more desired vials are held. Where radiolabeling, chromogenic, fluorescent, or other types of detectable labels or test components are included in the kit, the labeling agent may be provided in the same container as the test or therapeutic composition itself, or alternatively placed in a second, different container component into which the second composition can be placed and appropriately aliquoted. Alternatively, the test reagent and labeling may be prepared in a single container component, and in most cases, the kit will also typically include components for tightly sealing one or more vials for commercial sale and / or for convenient packaging and delivery.

[0567] General methods

[0568] Sambrook, Fritsch, and Maniatis (2nd ed. 1982 & 1989, 3rd ed. 2001) *Molecular Cloning, A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) *Molecular Cloning*, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) *Recombinant DNA*, Vol. 217, Academic Press, San Diego, CA) describes standard methods in molecular biology. Standard methods are also presented in Ausbel et al. (2001) *Current Protocols in Molecular Biology*, Vol. 1–4, John Wiley and Sons, Inc., New York, NY. These references describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0569] Methods for protein purification have been described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, preparation of fusion proteins, and protein glycosylation have been described (see, for example, Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391). The preparation, purification, and fragmentation of polyclonal and monoclonal antibodies have been described (Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, ibid.). Standard techniques for characterizing ligand / receptor interactions are readily available (see, for example, Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0570] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, for example, Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter et al. (2000) J. Immunol. 165:6205; He et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; US Patent No. 6,329,511).

[0571] An alternative to humanization is to use human antibody libraries displayed on phages or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

[0572] Single-chain antibodies and biantibodies have been described (see, for example, Malecki et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al. (2001) J. Biol. Chem. 276:7346-7350; Desmyter et al. (2001) J. Biol. Chem. 276:26285-26290; Hudson and Kortt (1999) J. Immunol. Methods 231:177-189; and U.S. Patent No. 4,946,778). Bifunctional antibodies have been provided (see, for example, Mack et al. (1995) Proc. Natl. Acad. Sci. USA 92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel et al. (2001) Protein Engineering 14:815-823; Segal et al. (2001) J. Immunol. Methods 248:1-6; Brennan et al. (1985) Science 229:81-83; Raso et al. (1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207; Traunecker et al. (1991) EMBO). J.10:3655-3659; and U.S. Patent Nos. 5,932,448, 5,532,210 and 6,129,914.

[0573] Bispecific antibodies were also provided (see, for example, Azzoni et al. (1998) J. Immunol. 161:3493; Kita et al. (1999) J. Immunol. 162:6901; Merchant et al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J. Biol. Chem. 275:38633; Zheng et al. (2001) J. Biol. Chem. 276:12999; Propst et al. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol. 17:875).

[0574] Antigen purification is not necessary for antibody production. Animals can be immunized with cells containing the antigen of interest. Spleen cells can then be isolated from the immunized animal and fused with myeloma cell lines to produce hybridomas (see, for example, Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., ibid.; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

[0575] Antibodies can be conjugated to, for example, small drug molecules, enzymes, liposomes, and polyethylene glycol (PEG). Antibodies can be used for therapeutic, diagnostic, kit, or other purposes, and include antibodies conjugated to, for example, dyes, radioisotopes, enzymes, or metals (e.g., colloidal gold) (see, for example, Le Doussal et al. (1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

[0576] Methods used for flow cytometry (including fluorescence activated cell sorting (FACS)) are available (see, for example, Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and probes), peptides, and antibodies, used as, for example, diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0577] The standard approach to histology of the immune system has been described (see, for example, Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0578] Software packages and databases for identifying, for example, antigen fragments, leader sequences, protein folds, functional domains, glycosylation sites, and sequence alignment are available (see, for example, GenBank, Vector NTI® package (Informax, Inc., Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne et al. (2000) Bioinformatics 16: 741-742; Menne et al. (2000) Bioinformatics Applications Note 16: 741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0579] Example

[0580] The following examples are used to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.

[0581] Example 1: Reagent

[0582] Table 6 provides details of the reagents and antibodies used in the following examples:

[0583]

[0584]

[0585] Example 2: Raw materials and cell lines

[0586] Chinese hamster ovary (CHO)-K1 cells and human adenocarcinoma cell line MCF7 were obtained from the American Type Culture Collection (ATCC). Cells were isolated until screening for microbial contamination and Mycoplasma spp. was confirmed negative. CHO-K1 cells were grown in DMEM / F12 (Gibco), 1% PenStrep (Gibco), and 5% NCBS (Biowest) and incubated at 37°C in a humidified atmosphere with 5% CO2. MCF7 cells were grown in EMEM (ATCC), 1% PenStrep (Gibco), and 10% FBS (Gibco) and incubated at 37°C in a humidified atmosphere with 5% CO2. CHO-K1.hCD103 / hβ7 cell lines were generated by transfecting CHO-K1 cells with pCI-neo and pcDNA3.1(+)-hygro vectors encoding full-length open reading frames of human integrin αE (UniProt P38570) and human integrin β7 (UniProt P26010), respectively. Stable clones were obtained by limiting dilution in CHO medium supplemented with genimycin (50 μg / mL, Gibco) and hygromycin B (50 μg / mL, Invitrogen). CHO-K1.hα4 / hβ7 cells were generated by transiently transfecting CHO-K1 cells with pCI-neo and pcDNA3.1(+)-hygro vectors encoding full-length open reading frames of human integrin α4 (UniProt P13612) and human integrin β7, respectively. CHO-K1 cells expressing CHO-K1.rhCD103 / rhβ7 were generated by transiently transfecting CHO-K1 cells with pCI-neo and pcDNA3.1(+)-hygro vectors encoding full-length open reading frames of rhesus integrin αE (UniProt H9Z8N2) and rhesus integrin β7 (NCBI XP_015007317.1), respectively.

[0587] The human non-small cell lung cancer cell line A549 was obtained from the American Type Culture Collection (ATCC). Cells were isolated until screening for microbial contamination and Mycoplasma spp. and confirmed negative. Cells were grown in DMEM / F-12, GlutaMAX™ supplement + 5% FCS + 25 mM HEPES (for CHO-K1), and RPMI + 10% FCS (for A549), and incubated at 37°C in a humidified atmosphere with 5% CO2. CDH1 (E-cadherin) knockout was performed on the A549 cell line via non-liposome transfection (Fugene) using a guide RNA, a fully functional CAS9 cassette, and a plasmid containing GFP (pSpCas9(BB)-2A-GFP (PX458) (Ran et al., Nature Protocols 8:2281-2308 (2013)) (Addgene plasmid number 48138; n2t.net / addgene:48138; RRID:Addgene_48138)). GFP-positive single-cell clones were isolated using a Moflo Astrios sorter (Beckman Coulter). Disruption was confirmed by Sanger sequencing in cases of insertion / deletion tracking.

[0588] CD103-positive T cells were generated as follows. Human peripheral blood mononuclear cells (PBMCs) (Sanquin) were isolated by Ficoll-Paque density gradient centrifugation of the erythrocyte sedimentation rate (ESR) amber layer from healthy volunteers (Ficoll-Paque PLUS, GE Healthcare Life Sciences, Marlborough, MA, USA) after obtaining informed consent. Next, CD8-positive T cells were negatively selected using the MagniSort™ Human CD8 T Cell Enrichment Kit according to a standard protocol (Thermo Fisher Scientific). Cells were then stimulated with 10 μg / mL PHA, 6000 U / mL IL-2, and 10 ng / mL recombinant TGFβ, and cultured in RPMI supplemented with 10% FCS and penicillin / streptomycin (100 U / mL). Cells were cultured for at least 10 days to obtain >80% CD103-positive CD8 cells.

[0589] Fresh tumor material was obtained from an ovarian cancer patient who underwent tumor debulking surgery. Approximately 1 mm was cut using a scalpel. 3Tumor fragments were extracted and enzymatically digested at 37°C (supplemented with 1 mg / ml collagenase type IV (Lifetechnologies), 31 U / ml rhDNAase (Pulmozyme, Genentech, California, USA), and 10% FCS in RPMI) for 30 minutes or overnight at room temperature. The digestion medium containing remaining tumor fragments was then filtered through a 70 μm cell sieve (Corning, Amsterdam, The Netherlands). For flow cytometry analysis, cells were pelleted, washed, and cryopreserved until further use.

[0590] Spleens from immune-active Balb / c and C57 / BL6 mice and thymus from Balb / c mice were harvested, and the tissues were then minced on a 70 μm sieve with a piston. Red blood cells were removed using erythrocyte lysis buffer (Biolegend). The cells were then pelleted, washed, and cryopreserved until further use.

[0591] Example 3: Production of Monoclonal Antibodies

[0592] To generate human CD103 antibodies, mice were immunized with cDNA plasmid constructs encoding the full-length open reading frame of human CD103 (integrin α-E) and human integrin β-7. pCI-neo and pcDNA3.1(+) were custom-synthesized and obtained from GeneArt / ThermoFisher (Regensburg, Germany). Mice were immunized via gene gun immunization at Envigo (Horst, The Netherlands) using a Helios gene gun (BioRad, Hercules, CA, USA) and DNA-coated gold bullets (BioRad) following the manufacturer's instructions. In short, 1 μm gold particles were coated with commercial expression vectors of pCI-neo-hCD103 and pcDNA3.1(+)-hβ7 cDNA and mouse Flt3L and mouse GM-CSF (both derived from Aldevron) at a 1:1:1:1 ratio. A total of 50 μg of plasmid DNA was used to coat 25 mg of gold particles. Specifically, 7-8 week old female BALB / c mice (Harlan) were immunized in the ear with a gene gun, with each ear receiving 3 administration cycles.

[0593] Antibody titers were assessed using a stable CHO-K1.hCD103 / hβ7 cell line via cell ELISA (“CELISA”). Cells were loaded at 8 x 10⁻⁶ cells per cell line. 4Cells / well were seeded into 96-well flat-bottom tissue culture plates and cultured at 37°C, 5% CO2, and 95% humidity until cell confluence. Cells were incubated with diluted mouse serum for 1 hour at 37°C, 5% CO2, and 95% humidity for each sample. Next, cells were washed with phosphate-buffered saline (PBS) / 0.05% Tween-20 (PBS-T) and incubated with goat-anti-mouse IgG-HRP conjugate (Southern Biotech) for 1 hour at 37°C, 5% CO2, and 95% humidity. Subsequently, cells were washed three times with PBS-T and anti-hCD103 / hβ7 immunoreactivity was visually observed in the presence of a TMB-stabilized chromogen (Invitrogen). The reaction was stopped with 0.5 M H2SO4, and absorbance was read at 450 and 610 nm. As detected after two DNA immunizations, the anti-hCD103 / hβ7 titer was higher than 1:2500 in each individual mouse serum sample. All mice were then immunized a third time and sacrificed 4 days later. Spleen and lymphoid cell populations depleted of red blood cells were prepared according to the published protocol.

[0594] To select B cells that produce anti-hCD103 antibodies, a selection strategy was designed and developed that preferentially binds to B cells expressing antibodies specifically binding to hCD103 that exhibit cross-reactivity with monkey CD103. Since the cynomolgus monkey CD103 sequence is unknown, rhesus monkey CD103 was used for cross-reactivity studies. Spleen cells and lymphocytes from hCD103 / hβ7 immunized mice were incubated with hCD103-negative MCF-7 cells seeded in T25 culture flasks and irradiated at 30 Gy. After 1 hour, unbound cells were gently removed by moving the flask back and forth. The culture medium containing unbound cells was then transferred to a new T25 flask containing irradiated CHO-K1.hα4 / hβ7 cells (transiently transfected). This procedure was repeated once more on ice for negative selection of hβ7-reactive B cells. Next, the culture medium containing unbound B cells was incubated with CHO-K1.hCD103 / hβ7 cells irradiated with 30 Gy. After incubation on ice for 1.5 hours, unbound cells were removed using multiple washing steps with culture medium. Subsequently, bound lymphocytes were harvested using trypsin-EDTA (Sigma) in T25 flasks containing CHO-K1.hCD103 / hβ7 cells. In 96-well flat-bottom tissue culture plates, selected B cells were mixed with 10% (v / v) T cell supernatant and 50,000 irradiated (25 Gy) EL-4 B5 feeder cells in a final volume of 200 μl of culture medium. On day 4, the cell culture medium was replaced. On day 8, the supernatant was screened for hCD103 / hβ7 reactivity by cell ELISA as described below. CHO-K1.hCD103 / hβ7, CHO.K1.rhCD103 / rhβ7 (transient transfection), and CHO-K1.hα4 / hβ7 (transient transfection) were seeded in 96-well flat-bottom tissue culture plates in DMEM-F12 (Gibco) supplemented with 10% fetal bovine serum (Hyclone) and 80 U Pen / Strep (Gibco) and cultured at 37°C, 5% CO2, and 95% humidity until confluence. Subsequently, the culture medium was removed, and the cells were incubated with supernatant from B cell culture at 37°C, 5% CO2, and 95% humidity for 1 hour. Next, the cells were washed with PBS-T and incubated with goat-anti-mouse IgG-HRP conjugate (SouthernBiotech) at 37°C, 5% CO2, and 95% humidity for 1 hour. Subsequently, the cells were washed three times with PBS-T and their immunoreactivity against anti-hCD103 / hβ7 and anti-hα4 / hβ7 was visually observed in the presence of TMB stable chromogen (Invitrogen).The reaction was stopped with 0.5 M H2SO4 and the absorbance was read at 450 and 610 nm.

[0595] Immortification of B cell clones from hCD103 / hβ7 reactive supernatants that were non-responsive or had very low responsiveness to hα4 / hβ7 was achieved via microelectrofusion with minimal bias, following a published procedure (Steenbakkers et al. (1992) Mol. Biol. Rep. 19: 125). In simple terms, B cells were electrofused with 10... 6 Sp2 / 0-Ag14 mouse myeloma cells (ATCCCRL-1581) were mixed in electrofusion equimolar buffer (Eppendorf). Electrofusion was performed in 50 μL fusion chambers using an alternating electric field of 15 s, 1 MHz, 23 Vrms AC, followed by a square high-field DC pulse of 10 as, 180 volts DC, and again an alternating electric field of 15 s, 1 MHz, 23 Vrms AC. The contents of the chambers were transferred to hybridoma selection medium and plated in 96-well plates under limiting dilution. On day 8 post-electrofusion, the hybridoma supernatant was screened for hCD103 / hβ7, rhCD103 / rhβ7, and hα4 / hβ7 binding activity by cell ELISA as described above. Hybridomas secreting CD103-specific antibodies in the supernatant were frozen at -180°C (-1 batch) and subcloned by limiting dilution to protect their integrity and stability. Stable hybridomas were frozen at -180°C (-LD1 batch) until the cell layers merged.

[0596] Selected stable hybridomas were cultured in serum-free medium for 7 days; the supernatant was harvested and the antibody was purified using MabSelect Sure Protein A resin according to the manufacturer's instructions (GE Healthcare). Antibody concentration was quantified spectrophotometrically. Antibody monomericity was assessed by SEC-HPLC. Hybridomas were isotyped using the supernatant of the hybridoma culture. In short, isotyping was performed using a mouse monoclonal antibody isotype kit (Biorad) based on dipsticks immobilized with goat-anti-mouse antibody bands to each of the common mouse isotype and light chain. All recovered antibodies were identified as mouse IgG1. Antibody sequences were elucidated by sequencing the variable region of the mouse IgG1 hybridoma material at LakePharma (CA, USA) using the following methods: total RNA was extracted from hybridoma cells, allowing cDNA synthesis. Rapid amplification of cDNA ends (RACE) was performed, allowing cloning of positive fragments in the TOPO (Thermo Fisher Scientific) vector. The TOPO clones were sequenced and the sequences were annotated using VBASE2.

[0597] The mAb discovery series yielded six distinct mAb candidates that exhibited specific binding to the human CD103 (integrin α-E) domain of the integrin heterodimer hCD103 / hβ7. The six selected candidates were generated from hybridomas and purified. Figure 1 Cellular binding data of purified anti-hCD103 mAbs with CHO.K1-hCD103 / hβ7, CHO.K1-rhCD103 / rhβ7, and CHO.K1-hα4 / hβ7 are presented. Expression of integrin heterodimers in transfected CHO-K1 cell lines was confirmed using commercial mAbs targeting integrin CD103, integrin α-4, and integrin β-7. Selected hybridomas were sequenced, and phylogenetic trees were constructed using Discovery Studio. Figure 2 This indicates that all VH and VL sequences are unique and have varying degrees of similarity. As shown, the antibody exhibits binding to hCD103 but not to hβ7. The binding of various candidates to rhesus monkey CD103 / rhβ7 is different.

[0598] Example 4: Generation of anti-hCD103 Fab fragments

[0599] Anti-hCD103 Fab candidates were generated using ImmunoPrecise (Oss, the Netherlands). Synthetic vectors encoding the VH and VL domains of candidates hCD103.01A, hCD103.05A, and hCD103.06A were synthesized and subsequently cloned into ImmunoPrecise's human IgG1-Fab-K and human κ light chain vectors, respectively, followed by transfection into HEK293 cells. Fab fragments from the harvested supernatant were purified using CaptureSelect IgG-CH1 affinity matrix to remove endotoxins. Fab concentration was quantified spectrophotometrically, and Fab purity was assessed by SDS-PAGE and HP-SEC. Endotoxin levels were determined by LAL analysis.

[0600] Example 5: Fluorescent labeling of mAb and Fab

[0601] Fab and mAb were conjugated with a 6-molar excess of Alexa Fluor 647-NHS (Thermo Scientific). Briefly, the mAb / Fab was reburied to 0.2 M sodium bicarbonate at pH 8.3 using a Zeba 7K MWCO column. A 6-molar excess of Alexa Fluor 647-NHS (from a 10 mg / mL stock solution in DMSO) was added. The reaction was allowed to proceed in the dark at room temperature for 1 h. Unreacted Alexa Fluor-NHS was removed using a Zeba 7K MWCO column. Antibody and AF647 concentrations were measured spectrophotometrically at 280 nm and 650 nm, respectively. The amount of residual unreacted AF647 was determined by HP-SEC using a dual-detector system (280 nm and 650 nm). Labeling rates of + / - 4 dyes per mAb and + / - 2-3 dyes per Fab were observed.

[0602] Example 6: Immunoreactivity of CHO cells expressing human CD103 / β7

[0603] As previously described, label-free mAbs and Fab were analyzed by CELISA targeting cell binding to CHO.K1-hCD103 / hβ7. Next, flow cytometry was used to perform cell binding assays to determine the binding morphology of AF647-labeled mAbs / Fab on CHO.K1-hCD103 / hβ7 and CHO.K1. 1x10 5 The detached cells were incubated with mAb / Fab at 4°C for 30 min. After washing, the cells were resuspended in 1% BSA / DPBS / 1xDAPI and analyzed on FACS-CantoII (BD Biosciences).

[0604] Figure 3 The binding data of various unlabeled mAbs / Fabs in cellular ELISA are presented. hCD103.01 mAb / Fab and hCD103.05 mAb bind efficiently to both CHO.K1.hCD103 / β7 and recombinant hCD103 / β7, hCD103.05 Fab binds slightly less to both CHO.K1.hCD103 / β7 and recombinant hCD103 / β7, and hCD103.06 mAb / Fab binds weakly / very little to recombinant hCD103 / β7 (Acro Biosystems), while hCD103.06 mAb binds strongly to CHO.K1.hCD103 / hβ7.

[0605] Figure 4 Data on the binding of various AF647-labeled mAbs / Fab reagents during flow cytometry are presented. No dose-dependent binding of mAbs and Fab reagents was observed on untransfected CHO.K1 cells (data not shown).

[0606] Example 7: Immunoreactivity to recombinant human CD103 / β7

[0607] Immunoreactivity to human CD103 / β7 was assessed by ELISA using 96-well MaxiSorp plates coated with recombinant hCD103 / hβ7 Fc-protein (Acro-Biosystems). The protein-coated 96-well plates were blocked for 1.5 hours at 37°C in protein-free blocking buffer (Pierce). The plates were washed and incubated with mAb / Fab in PBS containing 0.1% Tween-20 for 1 hour at room temperature. Next, the plates were washed with PBS-T and incubated with goat-anti-mouse IgG-HRP conjugate (Southern Biotech) (for mAb) and goat-anti-human Fab-HRP conjugate (Jackson Immuno Research) (for Fab fragment) in PBS containing 0.1% Tween-20 for 1 hour at room temperature. Subsequently, the wells were washed three times with PBS-T, and anti-hCD103 immunoreactivity was visually observed in the presence of a TMB-stabilized chromogen (Invitrogen). The reaction was stopped with 0.5 MH2SO4 and the absorbance was read at 450 and 610 nm.

[0608] Example 8: Flow Cytometry Analysis

[0609] In the binding assay of anti-CD103 mAb and Fab fragment in tumor digests, samples were aliquoted and stained with live / dead markers and commercial antibodies against human CD3, CD8α, CD33, and CD103, or commercial antibodies against CD3, CD8α, and CD33, along with our anti-CD103 mAb or Fab plus a second assay. In the binding assay of anti-CD103 to mouse CD103-positive T cells, spleen and thymus single-cell suspensions were aliquoted and stained with live / dead markers and commercial antibodies against mouse CD8 and CD103. Other aliquots were stained with a relevant isotype control or used as a fluorescence minus one control (“FMO” control: cells stained with all fluorophores minus one; relevant isotype controls contain directly labeled nonspecific isotype antibodies or a combination of nonspecific isotype antibodies and a second assay). The percentage of binding of the fluorescently labeled mAbs was determined using flow cytometry. The maximum binding was set to 100%. Measurements were performed on a BDFACSVerse (BD Biosciences). Data analysis was performed using FlowJo v10 (Tree Star) and surface receptor levels were expressed as mean fluorescence intensity (MFI).

[0610] Using ex vivo human tumor digests, the baseline commercial anti-CD103 mAb (BD Bioscience) routinely used in flow cytometry was evaluated against CD103 mAb, with gating to CD3+CD8+ T cells. Anti-CD103 mAb readily identified CD103+CD8+ T cell subsets at the same frequencies observed with commercial anti-CD103 mAb. Figure 5 No binding to CD4+ T cells or CD33+ (myeloid) cells was detected in these digests. For clone 01A, the mAb showed the highest binding to the CD103+ CD8+ T cell subset in ten independent patients, while clone 03A showed the lowest binding. Figure 6 Additionally, for Fab.hCD103.01.C1, recombinant Fab fragments from antibody clones 01A, 05A, and 06A showed the strongest binding to CD103+CD8+ T cell subsets, while Fab.hCD103.06.C1 showed the lowest binding in human tumor digests. Figure 7 This is consistent with the mAb binding data.

[0611] To assess differences in affinity and competition between mAbs, CD103+CD8+ T cells were pre-incubated for 1 hour at 4°C with either our anti-CD103 mAb or a commercial anti-CD103 mAb in FACS medium, and subsequently incubated for 1 hour at 4°C with their fluorescently labeled counterparts. The percentage of binding to the fluorescently labeled mAbs was determined using flow cytometry. Maximum binding was set at 100%.

[0612] Flow cytometry was used to determine whether the CD103 mAb clones cross-reacted with mouse CD103+ T cells. Mouse spleen and thymus were examined for CD103 expression using commercial anti-mouse CD103 mAb. Approximately half of the CD8 cells were CD103 positive. However, the anti-human CD103 mAb clones did not show specific binding to mouse CD103 (data not shown). Competition analysis indicated that, in our experimental group, binding to most mAbs, except for clones 03A and 06A, inhibited binding to the commercial CD103 mAb and vice versa, indicating binding to the same region on CD103 (…). Figure 8 However, differences in binding characteristics were observed. In the competition analysis, clones 01A and 02A blocked binding to most other mAb clones, while clones 03A, 05A, 06A, and 07A did not, indicating different binding epitopes. Furthermore, after saturation with the same clones, fluorescently labeled 05A, 06A, and 07A showed binding, indicating lower binding affinity.

[0613] The previously described protocol was used to determine the internalization and dissociation of anti-CD103 mAb and Fab fragments, as well as the membrane transformation of CD103. In short, CHO.K1-hCD103 / hβ7 cells or CD103-positive T cells were stained on ice with anti-CD103 mAb and Fab fragments (20 μg / mL final concentration). After staining; 1) cells were washed with ice-cold FACS buffer and incubated at 4°C for 1 hour with a secondary antibody diluted 1:50 in FACS medium to measure surface expression. 2) Cells were washed with ice-cold FACS buffer, incubated at 37°C for 4 hours in medium, and then incubated at 4°C for 1 hour with the secondary antibody to measure non-internalized CD103-antibody complexes, as the secondary antibody binds only to surface-bound CD103 mAb or Fab fragments. 3) Cells were washed with ice-cold FACS buffer, incubated in medium at 37°C for 4 hours, and then sequentially incubated with CD103 mAb or Fab fragments and a second antibody to measure non-internalized, re-emerged, and possibly de novo synthesized receptors. Paired samples were measured under each treatment condition, and corrections were made for background fluorescence and non-specific binding to the second antibody. Measurements were performed on a BD FACSVerse or BD Accuri C6 (BD Biosciences). Data analysis was performed using FlowJo v10 (Tree Star), and surface receptor expression was expressed as mean fluorescence intensity (MFI). Incubation at 37°C for four hours allowed varying amounts of residual mAb and Fab fragments to bind to the cell surface. Decreased mAb and Fab fragments could indicate internalization. However, the same experiments using directly labeled mAb and Fab fragments confirmed that the reduction in residual mAb or Fab fragments on the cell surface was attributed to dissociation. Notably, CD103 surface expression levels changed only slightly with incubation with mAb or Fab fragments (data not shown).

[0614] Example 9: CD103+ T cell attachment analysis

[0615] The following is an analysis of CD103+ T cell attachment. One day prior to the experiment, the cells were placed at 4°C in an environment containing 1 mM Ca. 2+ and Mg 2+ The 96-well plates were plated overnight with 100 μL of recombinant E-cadherin at a concentration of 2 μg / mL in Dulbecco's PBS (DPBS). Next, the wells were blocked with DPBS containing 1% bovine serum albumin (BSA) for at least 1 hour. (As previously...) The description describes labeling CD103+ T cells with CFSE (Thermo Fisher Scientific) and resuspending them in RPMI + 10% FCS + 1 mM Mn. 2+ In this process, CFSE-labeled cells were pre-incubated on ice with 10 μg / mL antibody or Fab fragment for 30 min, followed by incubation at 37°C in E-cadherin-coated wells (50,000 cells / well) for 30 min, or cells were directly transferred to E-cadherin-coated wells at 37°C for 30 min, followed by treatment with 10 μg / mL antibody or Fab fragment at 37°C for 30 min.

[0616] In the attachment analysis using A549 wild-type and E-cadherin knockout cells, tumor cells (30,000 cells / well) were seeded in 96-well plates one day prior to the experiment. Next, CFSE-labeled CD103+ T cells were pre-incubated on ice with 10 μg / mL antibody for 30 min, followed by incubation at 37°C in the wells seeded with tumor cells for 60 min.

[0617] After incubation, unbound cells were removed by inverting the plate and washing with DPBS. Finally, cells were fixed with DPBS containing 3.7% formalin. Images were captured using a conventional fluorescence microscope (Invitrogen™ EVOS™ FL imaging system). The number of bound T cells was quantified using ImageJ software (version 1.50).

[0618] like Figure 9 As shown, CD103 mAb clones 01A, 02A, 03A, and 07A exhibited the strongest inhibition of T cell binding, while 06A partially inhibited binding to E-cadherin. Clone 05A was the only clone that did not impede CD103-mediated T cell attachment. Similar effects were observed in Fab.hCD103.01.C1, Fab.hCD103.05.C1, and Fab.hCD103.06.C1 (data not shown). CRISPR knockout of CDH1 (E-cadherin) in A549 tumor cells reduced CD103+ T cell attachment; however, no significant effect of our CD103 mAb was observed in E-cadherin wild-type and E-cadherin knockout cells.

[0619] Example 10: Cell-based ELISA

[0620] One day prior to the procedure, CD103 / β7 transfected CHO cells (30,000 cells / well) were seeded in 96-well plates. Subsequently, serial dilutions of CD103 mAb, Fab fragment, and isotype control were added to each well of the 96-well plate and incubated at 37°C for 1 h. The wells were washed with PBS and incubated at 37°C for 1 h with either rabbit anti-mouse / IgG-HRP (1:4000, Dako) or Fab-specific goat anti-human / IgG-HRP (1:4000; Sigma Aldrich). Next, the wells were washed with PBS and TMB substrate (KPL) was added. The staining reaction was stopped by adding 1M HCl solution, and absorbance was measured using a microplate reader (Thermo Scientific).

[0621] Example 11: 89 Zr-hCD103.01A 89 Zr-hCD103.05A 89 Zr-Fab.hCD103.01.C1 and 89 Zr- hCD103.05.C1 tracer development and quality control

[0622] hCD103.01A, hCD103.05A, Fab.hCD103.01.C1, and Fab.hCD103.05.C1 were incubated with 3 or 4 times molar excess of TFP-N-Suc-deferoxamine-Fe (Df, ABX GmbH, Hamburg, Germany) and clinical grade [product name missing]. 89 Zr (Perkin Elmer, Groningen, The Netherlands) will follow up. 89 Zr labeling. Different amounts of antibody or Fab fragment per milligram were used, ranging from 250 to 1000 MBq / mg. 89 Zr was used to determine the maximum achievable specific activity. Radiochemical purity (RCP) was assessed using a trichloroacetic acid (TCA) precipitation test. Among the three tested... 89 Zr levels (250, 500, and 750 MBq) 89 Zr) below, 89 The radiochemical purity of Zr-Fab.hCD103.01.C1 and Fab.hCD103.05.C1 was >96%. Df-mAb and -Fab conjugates were analyzed for aggregation and fragmentation using size exclusion ultra-high performance liquid chromatography (SE-UPLC). The Waters SE-UPLC system was equipped with a dual-wavelength absorbance detector, an online radioactivity detector, and a TSK-GEL G3000SWXL column (JSB, Eindhoven, The Netherlands).

[0623] The CD103 binding affinity of the two Df-conjugated CD103 mAb and Fab fragments is similar to that of their unmodified counterparts. Figure 10Furthermore, without further purification, both the Df-conjugated mAb and Fab fragments achieved 500 MBq at a radiochemical purity of >95%. 89 Specific activity of Zr / mg ( Figure 11 Therefore, when used for biodistribution studies, these tracers are suitable for PET imaging at doses as low as 10 μg (PET imaging) or even smaller. In vitro, hCD103.01A and hCD103.05A showed specific binding to CD103-transfected CHO-K1 model cell lines (CHO.K1-hCD103 / hβ7), but not to CHO-K1 wild-type cells (CHO.WT). Figure 12 ).

[0624] Example 12: Animal Research

[0625] Figure 13 Showcase A 89 Zr-hCD103.01A and 89 An exemplary PET imaging protocol for Zr-hCD103.05A. Male nude mice (BALB / cOlaHsd-Foxn1nu, Envigo, The Netherlands) were subcutaneously (sc) inoculated with CHO.K1 or CHO.CD103 (300 μL 1:1 PBS and 5 x 10 μL high growth factor matrix gel (BD Biosciences, Breda, The Netherlands). 6 (Number). Allow xenografts to grow to at least 200 mm. 3 In microPET imaging using mAb, 8.7 ± 0.48 μg of the drug was administered intravenously (iv) via the penile vein to mice (n=3 per group) with xenografts. 89 Zr-CD103.01A or 8.76 ± 0.42 μg 89 Zr-CD103.05A. MicroPET scans were performed using a Focus 220 PET scanner (CTISiemens) at 1, 3, and 6 days post-injection (pi), followed by in vitro biodistribution analysis after the final scan. In the biodistribution assay using the Fab fragment, approximately 10 μg was intravenously (iv) injected into mice with xenografts (n=2 or 3 per group) via the penile vein. 89 Zr-Fab.hCD103.01.C1 or 89 Zr-Fab.hCD103.05.C1 was then subjected to in vitro biodistribution analysis 24 hours later.

[0626] Rescanning was performed and in vivo quantification was performed using AMIDE (version 1.0.4, Stanford University, Stanford, CA, USA). MicroPET data are presented as average normalized intake values ​​(SUV average). Regions of interest (ROIs) were plotted against the tumor based on ex vivo weight, assuming a tissue density of 1 g / ml. In hematopoietic pooling measurements, a fixed-size area was plotted at the center of the heart, and for the liver and spleen, fixed-size elliptical ROIs were plotted in representative portions of the organs. After the final scan, mice were euthanized and organs of interest were collected for biodistribution studies. Organs and injected tracer standards were counted and weighed in a well-calibrated LKB-1282-Compu-γ system (LKB WALLAC). Ex vivo tissue activity was expressed as a percentage of the injected dose per gram of tissue (%ID / g) after decay correction.

[0627] CD103 membrane expression in CHO.K1-hCD103 / hβ7 is similar to CD103 expression in TILs (data not shown). PET scans of mice with CHO.K1-hCD103 / hβ7 tumors showed that... 89 Zr-hCD103.01A and 89 Zr-hCD103.05A tumor uptake increases over time. Figure 13 B), the highest tumor uptake and lowest background organ uptake were observed on day 6 post-injection. 89 Median mean standardized intake of Zr-hCD103.01A (SUV) 平均 Tumor: 2.7, median SUV 平均 Blood: 0.9, and 89 Zr-hCD103.05A mid-range SUV 平均 Tumor: 3.0, median SUV 平均 Blood: 0.9; Figure 13 C Figure 13 D and Figure 13E CHO.K1 WT xenografts 89 Zr-hCD103.01A did not show accumulation (mean tumor SUV: 1.5, mean blood SUV: 1.4) and was used as a non-specific control. Similarly, in vitro biodistribution analysis on day 6 showed high CD103 specificity. 89 Zr-hCD103.01A and 89Zr-hCD103.05A tumor uptake (%ID / g percentage of injected dose per gram of tissue compared to CHO.K1 WT was 7.8, and %ID / g of CHO.K1-hCD103 / hβ7 were 17.0 and 32.8, respectively) and there were no major reservoir organs ( Figure 14 For the Fab fragment, in vitro biodistribution analysis 24 hours post-injection showed CD103 specificity. 89 Zr-Fab.hCD103.01.C1 and 89 Tumor uptake of Zr-Fab.hCD103.05.C1 (%ID / g was 0.64 for CHO.K1 WT, and 2.44 and 1.27 for CHO.K1-hCD103 / hβ7, respectively) Figure 15 ).

[0628] Example 13: Use of CD103-targeting radiopharmaceuticals for monitoring treatment

[0629] Before initiating (immunotherapy), perform a 'CD103 PET' scan, alone or in combination with a low-dose CT scan. Quantify the intake of the CD103 radiopharmaceutical. CD103 PET-CT scans can be repeated and quantified at intervals during (immunotherapy). Use changes in baseline intake, treatment-induced intake, or treatment-induced intake relative to baseline to guide clinical decisions. These decisions may include, but are not limited to, continuation of therapy, discontinuation of therapy, or dose adjustment.

[0630] Example 14: Sequence

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638] Example 15: References

[0639]

[0640]

[0641]

[0642]

[0643]

[0644]

[0645] All references cited herein are incorporated herein by reference to the extent that each individual publication, database entry (e.g., gene bank sequence or GeneID entry), patent application, or patent is specifically and individually indicated to be incorporated herein by reference. The applicant intends, pursuant to 37 CFR §1.57 (b)(1), that such incorporated statement refers to each individual publication, database entry (e.g., gene bank sequence or GeneID entry), patent application, or patent, each of which is clearly identified in accordance with 37 CFR §1.57 (b)(2), even if such reference is not directly adjacent to a specific statement incorporated herein by reference. The inclusion of specific statements incorporated herein by reference (if any) does not in any way diminish the general statement incorporated herein by reference. References herein are not intended to acknowledge that the references are relevant prior art, nor do they constitute any admission of the content or dates of such publications or documents. In the event of a conflict between the definitions of claimed terms provided in the references and those provided herein, the definitions provided herein shall be used to interpret the claimed invention.

[0646] While the invention has been described and illustrated sufficiently in detail for those skilled in the art to carry out and use it, various alternatives, modifications, and improvements will be apparent without departing from the spirit and scope of the invention. The embodiments provided herein represent preferred embodiments and are exemplary, and are not intended to limit the scope of the invention. Modifications and other uses will be apparent to those skilled in the art. These modifications are covered within the spirit and scope of the invention and are defined by the claims.

[0647] It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0648] All patent applications, patents, publications, and other references mentioned in this specification indicate the level of skill of one skilled in the art to which this invention pertains and are each incorporated herein by reference. References cited herein are not to be acknowledged as prior art to the claimed invention.

[0649] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, this specification (including definitions) shall prevail.

[0650] Unless otherwise indicated herein or clearly contradicted by the context, the articles “a,” “an,” and “described” used in the specification and claims should be understood to cover both the singular and plural. Unless otherwise indicated, the terms “comprising,” “having,” “having,” “including,” and “containing” in “having a chemical formula” should be treated as open-ended terms (i.e., meaning “including but not limited to”). Furthermore, whenever “comprising” or another open-ended term is used in an embodiment, it should be understood that the same embodiment may be more narrowly claimed using the intermediate term “consistently comprising” or the closed term “consisting of.”

[0651] The terms “about,” “approximately,” or “approximately” when used in conjunction with numerical values ​​mean that they encompass a set or range of values. For example, “about X” includes a range of values ​​of X ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1%, where X is a single numerical value. In one embodiment, the term “about” refers to a range of values ​​that are 10% more or less than a specified value. In another embodiment, the term “about” refers to a range of values ​​that are 5% more or less than a specified value. In yet another embodiment, the term “about” refers to a range of values ​​that are 1% more or less than a specified value.

[0652] Unless otherwise stated herein, the ranges of stated values ​​are intended merely as a way of individually referring to each individual value belonging to the range, and each individual value is incorporated into this specification as if it were individually enumerated herein. Unless otherwise specified, the ranges used herein include both limits of the range. For example, the terms "between X and Y" and "the range from X to Y includes X and Y and integers therebetween." On the other hand, when a series of individual values ​​are mentioned in this disclosure, this disclosure also considers any range that includes either of the two individual values ​​as its two endpoints. For example, the expression "a dose of about 100 mg, 200 mg, or 400 mg" may also mean "a dose varying from 100 to 200 mg," "a dose varying from 200 to 400 mg," or "a dose varying from 100 to 400 mg."

[0653] The invention described illustratively herein may be practiced in the absence of any element or elements, or any limitation or limitations, not specifically disclosed herein. Thus, by way of example, in each case herein, any of the terms “comprising,” “substantially consisting of,” and “consisting of” may be replaced by any of the other two terms. The terms and expressions used are intended to be descriptive rather than limiting, and their use is not intended to exclude any equivalents or portions thereof of the features shown and described, but it should be recognized that various modifications are possible within the claimed scope of the invention. Therefore, it should be understood that while the invention has been specifically disclosed by way of preferred embodiments and optional features, modifications and alterations can be made to the concepts disclosed herein by those skilled in the art, and such modifications and alterations are considered to be within the scope of the invention as defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to human CD103, wherein the antibody or antigen-binding fragment comprises: a. Heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 consists of the amino acid sequence of SEQ ID NO:

1. b. Heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 consists of the amino acid sequence of SEQ ID NO:

2. c. Heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 is composed of the amino acid sequence of SEQ ID NO:

3. d. Light chain variable region CDR1, wherein the light chain variable region CDR1 is composed of the amino acid sequence of SEQ ID NO:

4. e. Light chain variable region CDR2, wherein the light chain variable region CDR2 is composed of the amino acid sequence of SEQ ID NO: 5, and f. Light chain variable region CDR3, which consists of the amino acid sequence of SEQ ID NO:

6.

2. An antibody or antigen-binding fragment thereof that binds to human CD103, wherein the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment is SEQ ID NO: 7, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment is SEQ ID NO:

8.

3. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody is an intact IgG.

4. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody is scFv, Fab, or F(ab')2.

5. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody comprises a wild-type or mutant IgG2 Fc region.

6. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody comprises a mutant IgG1 Fc region.

7. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody comprises a mutant IgG4 Fc region.

8. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment is humanized.

9. One or more nucleic acids, wherein the one or more nucleic acids encode an antibody according to any one of claims 1-8.

10. An expression system comprising one or more nucleic acids encoding an antibody according to any one of claims 1-8 and a regulatory sequence operatively linked thereto, the expression system being configured to express the antibody in a host cell.

11. A host cell comprising the expression system according to claim 10, wherein the host cell is a bacterial cell, a human cell, a mammalian cell, or a Pichia pastoris cell.

12. The host cell as described in claim 11, wherein the host cell is HEK293 cell or Chinese hamster ovary cell.

13. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1-8 and a pharmaceutically acceptable carrier.

14. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1-8 and a diluent.

15. A method for generating an antibody or antigen-binding fragment according to any one of claims 1-8, the method comprising: Host cells containing the polynucleotides are cultured under conditions that favor the expression of the heavy and / or light chains of the polynucleotides encoding either the antibody or the antigen-binding fragment.

16. The method of claim 15, wherein the method comprises recovering the antibody or antigen-binding fragment from the host cell and / or culture medium.

17. The method of claim 15 or 16, wherein the antibody or antigen-binding fragment does not block CD103 from binding to E-cadherin.

18. The method of claim 15 or 16, wherein the antibody or antigen-binding fragment at least partially blocks CD103 binding to E-cadherin.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8 in the preparation of a detection reagent for detecting the presence of CD103 in a biological sample. The detection includes contacting the sample with the antibody or its antigen-binding fragment; and The presence or amount of the antibody or antigen-binding fragment bound to CD103 present in the biological sample in the form of a complex is detected, wherein the detection of the complex indicates the presence or amount of CD103 present in the biological sample.

20. The use according to claim 19, wherein the antibody or antigen-binding fragment comprises a diagnostic marker, wherein the diagnostic marker is selected from the group consisting of: 11 C 13 N、 15 O、 99m Tc, 61 Cu、 62 Cu、 64 Cu、 67 Cu、 18 F, 19 F, 66 Ga、 67 Ga、 68 Ga、 72 Ga、 123 I, 124 I, 111 In、 177 Lu、 44 Sc、 47 Sc、 86 Y、 88 Y、 90 Y、 45 Ti、 89 Zr, indocyanine green, IRDye 800CW, fluorescein, and magnetic nanoparticles.

21. The use according to claim 20, wherein the fluorescein is FITC.

22. The use according to claim 20, wherein the magnetic nanoparticles are magnetic iron oxide nanoparticles.

23. The use according to any one of claims 20-22, wherein the detection step comprises performing PET imaging, single-photon emission computed tomography (SPECT) imaging, MRI, optical imaging, or acoustic imaging.

24. The use according to any one of claims 20-22, wherein the detection step includes performing photoacoustic imaging.

25. The use according to any one of claims 19-22, wherein the antibody or antigen-binding fragment does not block CD103 from binding to E-cadherin.

26. The use according to any one of claims 19-22, wherein the antibody or antigen-binding fragment at least partially blocks CD103 binding to E-cadherin.

27. The use according to any one of claims 19-22, wherein the detection step comprises an in vivo imaging method for detecting the complex.

28. The use according to claim 27, wherein the detection step comprises an in vivo imaging method for detecting the complex in a tumor.

29. A developer comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-8 is detectably labeled.

30. The imaging agent of claim 29, wherein the antibody or antigen-binding fragment does not block CD103 from binding to E-cadherin.

31. The imaging agent of claim 29, wherein the antibody or antigen-binding fragment at least partially blocks CD103 binding to E-cadherin.

32. The imaging agent of claim 29, wherein the antibody or antigen-binding fragment is the antibody or antigen-binding fragment of any one of claims 1 or 2.

33. The imaging agent according to any one of claims 29-31, wherein the antibody or antigen-binding fragment is detectably labeled by a marker selected from the group consisting of: 11 C 13 N、 15 O、 99m Tc, 61 Cu、 62 Cu、 64 Cu、 67 Cu、 18 F, 19 F, 66 Ga、 67 Ga、 68 Ga、 72 Ga、 123 I, 124 I, 111 In、 177 Lu、 44 Sc、 47 Sc、 86 Y、 88 Y、 90 Y、 45 Ti、 89 Zr, indocyanine green, IRDye800CW, fluorescein, and magnetic nanoparticles.

34. The developer of claim 33, wherein the fluorescein is FITC.

35. The developer according to claim 33, wherein the magnetic nanoparticles are magnetic iron oxide nanoparticles.

Images