HK1229353B - Anti-cxcr4 antibodies and antibody-drug conjugates - Google Patents

Description

Anti-CXCR4 antibodies and antibody-drug conjugates

The present invention relates to antibodies and related molecules that bind to chemokine receptor 4 (CXCR4). The present invention also relates to molecules comprising or consisting of full-length antibodies, antibody fragments, or variants thereof. The present invention further relates to amino acid and nucleic acid sequences encoding such antibodies. The present invention also relates to antibody conjugates (e.g., antibody-drug conjugates) comprising anti-CXCR4 antibodies, compositions comprising anti-CXCR4 antibodies, and methods of using anti-CXCR4 antibodies and their conjugates to treat conditions associated with CXCR4 expression (e.g., cancer). The present invention further comprises the use of the antibodies, antigen-binding fragments thereof, or antibody-drug conjugates, and corresponding methods for detecting and diagnosing pathological conditions associated with CXCR4 expression. In certain aspects, the condition is a carcinogenic condition associated with an increase in CXCR4 expression relative to normal or any other pathology associated with overexpression of CXCR4. In other aspects, the condition is inflammatory and immune conditions, allergic conditions, infections (such as HIV infection), autoimmune conditions (e.g., rheumatoid arthritis), fibrotic conditions (e.g., lung), and cardiovascular conditions. The present invention ultimately encompasses products and/or compositions or kits containing at least such antibodies or antibody-drug conjugates for use in the prognosis or diagnosis or treatment monitoring of such disorders.

Background Art

Chemokines are small secreted peptides that control the migration of leukocytes along the chemical gradient of the ligand (called the chemokine gradient), particularly during immune responses (Zlotnik et al., 2000, Immunity, 12: 121-127). They are divided into four categories based on the position of the Cys residue at the N-terminus. The CXC category consists of chemokines in which a pair of Cys residues are separated by a single residue. The most prominent members of this category are interleukin-8 (IL-8, CXCL8), stromal cell-derived factor-1 (SDF-1, CXCL12), gamma-interferon-inducible protein-10 (IP-10, CXCL10), platelet factor-4 (PF-4, CXCL4), neutrophil activation protein-2 (NAP-2, CXCL7), and melanoma growth stimulating activity (MGSA, CXCL1). The CC class of chemokines has two adjacent Cys at the N-terminus and includes macrophage inflammatory protein-1 (MIP-1α, CCL3; MIP-1α, CCL4), normal T cell expressed and secreted factor regulated by activation (RANTES, CCL5), and monocyte chemoattractant protein-1 (MCP-1, CCL2). _CX3C class chemokines contain two Cys separated by three residues at the N-terminus, represented by fractalkine/neuronal chemokine ( _CX3CL1 ). Class C chemokines contain a single Cys at the N-terminus, represented by lymphotactin/ATAC/SCM (CL1). Chemokine receptors are grouped according to their selectivity for binding to chemokines. For example, CXCR4 binds to SDF-1, while CXCR5 binds to chemokine 1 (BCA1), which attracts B cells. The interaction between CXCR4 and SDF-1 plays an important role in multiple stages of tumorigenesis, including tumor growth, invasion, angiogenesis and cancer metastasis.

CXCR4 is a seven-transmembrane G protein-coupled receptor (GPCR) (Herzog et al. DNA Cell Biol. 12:465 (1993); Rimland et al. Mol. Pharmacol. 40:869 (1991); WO03014153, WO02061087). Many medically important biological processes are mediated by signal transduction pathways involving G proteins (Lefkowitz, Nature, 351:353-354 (1991)). G protein-coupled receptors (GPCRs) are integral membrane proteins containing seven putative transmembrane domains (TMs). These proteins mediate signals into the cell interior through activation of heterotrimeric G proteins, which in turn activate various effector proteins, ultimately producing physiological responses.

CXCR4 plays a role in embryogenesis, homeostasis, and inflammation. In addition, CXCR4 has been shown to act as a co-receptor for T lymphotropic HIV-1 isolates (Feng, Y. et al. Science 272:872 (1996)). CXCR4 also plays a pleiotropic role in human cancers. Its expression is upregulated in many tumor types, including breast cancer, lung cancer, colon cancer, pancreatic cancer, brain cancer, prostate cancer, ovarian cancer, and hematopoietic cancer. Some literature reports show that SDF-1 can act as a growth and/or survival factor for some tumors via CXCR4. CXCR4 is expressed on stem cell-like or tumor-initiating subpopulations of many tumors and can mediate the ability of these cells to support cancer recurrence and metastatic spread. In addition, CXCR4 is expressed on endothelial progenitor cells (EPCs), and its activity is required for EPCs to enter functional blood vessels during angiogenesis. This can significantly promote angiogenesis and tumor survival. CXCR4 signaling also induces the production of pro-angiogenic cytokines (such as VEGF), as well as integrins, adhesion molecules, and matrix-degrading enzymes that mediate tumor cell invasion. Furthermore, CXCR4 expression has been detected on tumor-infiltrating lymphocytes and fibroblasts, as well as tumor-associated macrophages. These cells tend to suppress immune recognition and attack on tumors and reshape the tumor microenvironment to promote tumor growth and metastasis.

The multiple roles of CXCR4 in tumor growth and metastasis and its widespread expression in many common tumor types make this receptor an attractive target for therapeutic intervention using inhibitors. Although peptide and small molecule inhibitors of CXCR4 and anti-CXCR antibodies have been identified or entered the clinic, their effectiveness is limited by pharmacokinetic properties and toxicology. Agents such as antibodies or antibody-drug conjugates with selectivity, long half-life, improved efficacy and safety profiles would be desirable agents for cancer treatment.

Although various agents targeting CXCR4 are under development, there is a need for other therapeutic agents targeting CXCR4, such as antibodies or antibody-drug conjugates, with improved efficacy and safety profiles and suitable for use in human patients. The antibodies and antibody-drug conjugates of the present invention are therapeutically useful anti-CXCR4 antibodies that possess various desirable properties, such as reducing tumor formation, tumor growth, angiogenesis, and metastasis. In addition, the antibodies and antibody-drug conjugates of the present invention induce apoptosis in tumor cells.

Summary of the Invention

The present invention provides isolated antibodies, antigen-binding fragments and derivatives thereof, and antibody-drug conjugates that bind to chemokine receptor 4 (CXCR4). The present invention includes the amino acid sequences of the variable heavy and light chains of the antibodies and their corresponding nucleic acid sequences.

In one aspect, the invention encompasses complementarity determining region (CDR) sequences of antibodies to obtain binding molecules comprising one or more CDR regions or CDR-derived regions that retain the CXCR4 binding ability of the parent molecule from which the CDRs were derived.

In another aspect, the invention provides antibody-drug conjugates comprising an anti-CXCR4 antibody disclosed herein.

In another aspect, the present invention comprises the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, and antibody-drug conjugates and corresponding methods for detecting and diagnosing disorders associated with CXCR4 expression or function. In one aspect, the disorder is cancer associated with increased CXCR4 expression relative to normal or any other pathology associated with overexpression of CXCR4.

In another aspect, the present invention comprises a product and/or composition or kit containing at least such an antibody, antigen-binding fragment or antibody-drug conjugate for use in the prognosis or diagnosis or treatment monitoring of certain cancers.

In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) and comprises: a) a heavy chain variable (VH) region comprising: (i) a VH CDR1 selected from the group consisting of SEQ ID NOs: 107, 113, 114, 108, 109, 115, 116, 117, 121, and 122, (ii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156, and 130, and (iii) a VH CDR3 selected from the group consisting of SEQ ID NOs: 1 and/or b) a light chain variable region (VL) comprising: (i) a VL CDR1 selected from the group consisting of SEQ ID NOs: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150, and 151, (ii) a VL CDR2 selected from the group consisting of 145, 132, 136, and 152, and (iii) a VL CDR3 selected from the group consisting of SEQ ID NOs: 139, 133, 137, 140, and 153.

In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) and comprises: a) a heavy chain variable (VH) region comprising a complementarity determining region selected from the group consisting of: (i) a VH CDR1 comprising a sequence set forth in SEQ ID NO: 107, 113, 114, 108, 109, 115, 116, 117, 121, or 122; (ii) a VH CDR2 comprising a sequence set forth in SEQ ID NO: 107, 113, 114, 108, 109, 115, 116, 117, 121, or 122; NO: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156 or 130; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 112 or 120; and/or b) a light chain variable (VL) region comprising a complementarity determining region selected from the group consisting of: (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: NO:144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150 or 151; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO:145, 132, 136 or 152; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO:139, 133, 137, 140 or 153.

In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) and comprises: a) a light chain variable (VL) region comprising: (i) a VL CDR1 comprising the sequence _{XiX2X3SLFNSX4X5RKNYLX6} , wherein _Xi is _R or _K ; _X2 is S or _A ; _X3 is _W , N or Q; _X4 is H or R; _{X5 is} T or F, and/or _X6 is A, L, N or M (SEQ ID NO: 151); (ii) a VL CDR2 comprising the sequence _WASARX1S , wherein _Xi is G or E (SEQ ID NO: 152); and (iii) a VL CDR3 comprising the sequence _KQSFX1LRT , wherein _Xi is N or R (SEQ ID NO: 153). NO: 153); and/or b) a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising the sequence of SEQ ID NO: 107, 108, 109, 113 or 114; (ii) a VH CDR2 comprising the sequence of SEQ ID NO: 157, and (iii) a VH CDR3 comprising the sequence of SEQ ID NO: 112.

In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to CXCR4 and comprises: a heavy chain variable (VH) region sequence comprising: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVX ₁ FIRHKX ₂ NX ₃ X ₄ TX ₅ EYSTX ₆ X ₇ X ₈ GRFTISRDX ₉ SKNX ₁₀ LYLQMNSLX ₁₁ X ₁₂ EDTAVYYCAX ₁₃ DLPGFAYWGQGTLVTVSS (SEQ ID NO: 106), wherein X ₁ is G or S; X ₂ is V or A; X ₃ is G, F, K, V, T, L, or I; X ₄ is E or Y; X ₅ is T or R; X ₆ is W or S; X ₇ is D or V; X ₈ is K, T, or R; X _X9 is D or N; _X10 is T or S; _X11 is R or K; _X12 is A or T, and/or _X13 is K or R.

In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) selected from: a) a heavy chain variable (VH) region having a VH CDR1 of SEQ ID NO: 107, 113, or 114, a VH CDR2 of SEQ ID NO: 162 or 128, and a VH CDR3 of SEQ ID NO: 112, and a light chain variable (VL) region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 115, 116, 117, 121, or 122, a VH CDR2 of SEQ ID NO: 118, or 119, and a VH CDR3 of SEQ ID NO: 120. CDR3, and a VL region having a VL CDR1 of SEQ ID NO: 135, a VL CDR2 of SEQ ID NO: 136, and a VL CDR3 of SEQ ID NO: 137; c) a VH region having a VH CDR1 of SEQ ID NO: 107, 108, 109, 113, or 114, a VH CDR2 of SEQ ID NO: 110, or 111, and a VH CDR3 of SEQ ID NO: 112, and a VL region having a VL CDR1 of SEQ ID NO: 131, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 133; d) a VH region having a VH CDR1 of SEQ ID NO: 107, 108, 109, 113, or 114, a VH CDR2 of SEQ ID NO: 154, or 123, and a VH CDR3 of SEQ ID NO: 1 NO: 112, and a VL domain having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139; and e) a VH domain having a VH CDR1 of SEQ ID NO: 107, 108, 109, 113 or 114, a VH CDR2 of SEQ ID NO: 157, and a VH CDR3 of SEQ ID NO: 112, and a VL domain having a VLCDR1 of SEQ ID NO: 151, a VL CDR2 of SEQ ID NO: 152, and a VL CDR3 of SEQ ID NO: 153.

In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof selected from: a) a heavy chain variable (VH) region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and a light chain variable (VL) region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 115, a VH CDR2 of SEQ ID NO: 118, and a VH CDR3 of SEQ ID NO: 120, and a VL region having a VL CDR1 of SEQ ID NO: 135, a VL CDR2 of SEQ ID NO: 136, and a VL CDR3 of SEQ ID NO: 137. NO: 137 VLCDR3; c) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 110, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 131, a VLCDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 133; d) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 154, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139; and e) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107 The VL region has a VL CDR1 of SEQ ID NO: 151, a VL CDR2 of SEQ ID NO: 152, and a VL CDR3 of SEQ ID NO: 153.

In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof selected from: a) a heavy chain variable (VH) region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and a light chain variable (VL) region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and a VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139. NO: 140 VLCDR3; c) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 150, a VLCDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; d) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VLCDR1 of SEQ ID NO: 141, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; e) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107 f) a VH and VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140; g) a VH and VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 147, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140 : h) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 160, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; i) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 161, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140 CDR3; j) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; k) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; l) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140. NO: 164 VH CDR2 and SEQ ID NO: 112 VH CDR3, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; m) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 165, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; o) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 166, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VH CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140 NO: 138, a VL CDR1 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; p) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 167, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; q) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140 CDR3; r) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; s) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140; t) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140. NO: 158, and a VH CDR2 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; u) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; v) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, the VL region having a NO: 144, a VL CDR1 of SEQ ID NO: 145, a VL CDR2 of SEQ ID NO: 139, and w) a VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and a VL region having a VLCDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140.

In a specific aspect of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising the three CDRs set forth in SEQ ID NOs: 107, 162, and 112. In some aspects, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising the three CDRs set forth in SEQ ID NOs: 144, 145, and 139. In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising the three CDRs set forth in SEQ ID NOs: 107, 162, and 112, and a light chain variable (VL) region comprising the three CDRs set forth in SEQ ID NOs: 144, 145, and 139.

In some aspects of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 from the VH region of SEQ ID NO: 33. In other aspects, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 from the VL region of SEQ ID NO: 73.

In some aspects of the invention, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 from the VH region of SEQ ID NO: 33, and a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 from the VL region of SEQ ID NO: 73.

In some aspects of the invention, the isolated antibody or antigen-binding fragment thereof is selected from: a) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 33 and a VL region of SEQ ID NO: 73; b) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 13 and a VL region of SEQ ID NO: 15; c) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 5 and a VL region of SEQ ID NO: 7; d) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 21 and a VL region of SEQ ID NO: 47; and e) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 106, and a VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139.

In some aspects of the invention, the isolated antibody or antigenic fragment thereof comprises a heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:33, and a light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:73.

In a specific aspect of the invention, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable (VH) region of SEQ ID NO: 33; and/or b) a light chain variable (VL) region of SEQ ID NO: 73.

In some aspects of the invention, the antibody or antigen-binding fragment comprises a heavy chain variable (VH) region produced by an expression vector having ATCC Accession No. PTA- 121353. In some aspects of the invention, the antibody or antigen-binding fragment comprises a light chain variable (VL) region produced by an expression vector having ATCC Accession No. PTA-121354.

In some aspects of the invention, the isolated antibody or antigen-binding fragment thereof is selected from:

a) a heavy chain variable (VH) region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and a light chain variable (VL) region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139;

b) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; c) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 150, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; d) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 150, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140 CDR2 and VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 141, a VL CDR2 of SEQ ID NO: 132, and a VLCDR3 of SEQ ID NO: 140; e) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VLCDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140; f) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 147, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112 NO: 132 and VL CDR3 of SEQ ID NO: 140; g) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 159, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; h) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 160, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140. CDR3; i) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 161, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; j) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; k) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140. NO: 163 VH CDR2 and SEQ ID NO: 112 VH CDR3, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; l) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 164 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; m) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 165 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 165 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: NO: 138, a VL CDR1 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; n) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 166, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; o) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 167, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140 CDR3; p) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; q) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; r) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140. NO: 163, and a VH CDR2 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140; s) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; t) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: NO: 144, a VL CDR1 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; u) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; and v) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: NO:140 VLCDR3.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to CXCR4 competes for binding to CXCR4 and/or binds to the same epitope of CXCR4 as an antibody or antigen-binding fragment thereof disclosed herein.

In some aspects of the invention, the isolated antibody or antigen-binding fragment thereof comprises a VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and a VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VLCDR3 of SEQ ID NO: 139.

In some aspects of the invention, the antibody or antigen-binding fragment thereof is a humanized, chimeric, CDR-grafted or recombinant human antibody. In other aspects of the invention, the antibody or antigen-binding fragment thereof is not caninized or felineized.

In some aspects of the invention, the antibody or antigen-binding fragment thereof comprises a tag containing the acyl donor glutamine engineered at a specific site.

In some aspects of the present invention, anti-CXCR4 antibody-drug conjugates are provided. The antibody-drug conjugates of the present invention generally have the formula: Ab-(T-L-D), wherein: Ab is an antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4); T is an optional tag containing an acyl donor glutamine; L is a linker; and D is a drug.

In a specific aspect, the antibody drug conjugate of the present invention is selected from: a) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: 107, 162, and 112, and the VL region comprises the CDRs of SEQ ID NOs: 144, 145, and 139; b) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: 115, 118, and 120, and the VL region comprises the CDRs of SEQ ID NOs: 135, 136, and 137; c) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: 107, 110, and 112, and the VL region comprises the CDRs of SEQ ID NOs: 131, 132, and 133; d) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: NO: 107, 157 and 112, the VH region containing the CDRs of SEQ ID NO: 138, 132 and 139; e) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, the VH region containing the CDRs of SEQ ID NO: 107, 157 and 112, the VH region containing the CDRs of SEQ ID NO: 151, 152 and 153; f) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 33 and a VL region of SEQ ID NO: 73; g) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 13 and a VL region of SEQ ID NO: 15; h) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 5 and a VL region of SEQ ID NO: 7; and i) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 21 and a VL region of SEQ ID NO: 47.

In some antibody-drug conjugates of the present invention, Ab is a humanized, chimeric, CDR-grafted or recombinant human antibody or antigen-binding fragment thereof. In some antibody-drug conjugates of the present invention, T is selected from SEQ ID NO: 171, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 172, 173, 102, 103 and LLQ. In some antibody-drug conjugates of the present invention, L is selected from acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (vc) and maleimidocaproic acid (mc). In some antibody-drug conjugates of the present invention, D is selected from cytotoxic agents, immunomodulators, imaging agents, therapeutic proteins, biopolymers and oligonucleotides.

Any of the antibody-drug conjugates of the present invention can be used in the preparation of a medicament for a cytotoxic agent. The cytotoxic agent can be an anthracycline, an auristatin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a pyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and a calicheamicin. Any antibody-drug conjugate of the present invention can be prepared using the drug auristatin. In one aspect, auristatin can be 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide), MMAD (monomethyl auristatin D or monomethyl dolastatin 10 and 8261 (2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).

In a specific aspect, the antibody-drug conjugate of the invention is selected from: a) Ab-LLQGA (SEQ ID NO: 91)-(Acetyl-Lysine-Valine-Citrulline-p-Aminobenzyloxycarbonyl (AcLys-VC-PABC))-0101; Ab-LLQGA (SEQ ID NO: 91)-(AcLys-VC-PABC)-MMAD; c) Ab-LLQX ₁ X ₂ X ₃ X ₄ X ₅ (SEQ ID NO: 102)-(AcLys-VC-PABC)-0101; d) Ab-LLQX ₁ X ₂ X ₃ X ₄ X ₅ (SEQ ID NO: 102)-(AcLys-VC-PABC)-MMAD; e) Ab-GGLLQGA (SEQ ID NO: 92)-(AcLys-VC-PABC)-0101; and f) Ab-GGLLQGA (SEQ ID NO: 91)-(AcLys-VC-PABC)-0101. NO:92)-(AcLys-VC-PABC)-MMAD.

In some aspects of the invention, the CXCR4 antibody-drug conjugate comprises any of the antibodies or antigen-binding fragments thereof disclosed herein. In a specific aspect of the invention, the antibody-drug conjugate comprises an antibody, wherein the antibody comprises a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: 107, 162, and 112, and the VL region comprises the CDRs of SEQ ID NOs: 144, 145, and 139.

Further provided are pharmaceutical compositions comprising the CXCR4 antibodies, antigen-binding fragments thereof, or antibody-drug conjugates disclosed herein and a pharmaceutically acceptable carrier.

In some aspects of the present invention, isolated polynucleotides are provided, comprising a nucleotide sequence encoding any of the anti-CXCR4 antibodies or antigen-binding fragments thereof disclosed herein. In some aspects, host cells are provided that recombinantly produce the antibodies or antigen-binding fragments thereof disclosed herein.

In other aspects, methods are provided for treating a disorder associated with CXCR4 function or expression in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of the invention. In a particular aspect of the invention, the disorder is cancer.

In other aspects, methods are provided for reducing metastasis of CXCR4-expressing cancer cells in a subject, comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition disclosed herein. In other aspects, methods are provided for inducing tumor regression in a subject having a tumor that expresses CXCR4, comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition disclosed herein.

These and other aspects of the invention will be apparent from a review of the application as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an alignment of h3G10 heavy chain variable regions (VH).

Figure 2 shows an h3G10 light chain variable region (VL) alignment.

Figure 3A shows the cross-reactivity of anti-human CXCR4 Ab h3G10.1.91.A58B with cynomolgus monkey CXCR4 as assessed by flow cytometry using serial dilutions (0.007-267 nM) on HPB-ALL (human T-cell leukemia) and cynomolgus monkey-CXCR4 transfected CHO cells.

Figure 3B shows the cross-reactivity of anti-human CXCR4 Ab h3G10.1.91.A58B with macaque CXCR4 as determined by flow cytometry using serial dilutions (0.007-267 nM) on Raji (NHL; human non-Hodgkin Lymphoma) and HSC-F (macaque T cell line).

Figure 4 shows the ADCC (A-C) and CDC (D-F) activities of anti-human CXCR4 antibodies against human cell lines expressing CXCR4.

FIG4A shows the ADCC activities of 12A11, 6B6 and 3G10 anti-human CXCR4 antibodies against Ramos (NHL) cells.

FIG4B shows a comparison of the ADCC activities of m3G10-hIgG1 and m3G10-hIgG4 anti-human CXCR4 antibodies against MOLT-4 (T acute lymphoblastic leukemia) cells.

FIG4C shows the ADCC activity of mouse (m3G10-) and humanized (h3G10-) anti-human CXCR4 antibodies against MOLT-4 (T acute lymphoblastic leukemia) cells.

FIG4D shows the CDC activities of 12A11, 6B6, and 3G10 anti-human CXCR4 antibodies on Ramos (NHL) cells.

FIG4E shows a comparison of the CDC activities of m3G10-hIgG1 and m3G10-hIgG4 anti-human CXCR4 antibodies on Daudi (NHL) cells.

FIG4F shows the CDC activity of mouse (m3G10-) and humanized (h3G10-) anti-human CXCR4 antibodies on Daudi (NHL) cells.

FIG5 shows the inhibition of calcium flux by 3G10, 6B6 and 12A11 anti-human CXCR4 antibodies.

FIG6A shows that 3G10, 6B6, and 12A11 anti-human CXCR4 antibodies can induce cell death in Ramos cells in a dose-dependent manner.

FIG6B shows that the ability of anti-human CXCR4 antibody 3G10 to induce cell death is bivalency dependent.

Figure 7 shows that NHL3G10, 6B6, and 12A11 anti-human CXCR4 antibodies significantly inhibited tumor growth compared to an isotype control antibody in a non-Hodgkin's lymphoma (NHL) model (Ramos).

Figure 8A shows that anti-human CXCR4 antibodies 3G10, 6B6, and 12A11 have a significant effect on tumor burden in animals systemically implanted with Raji-LUC cells compared to an isotype control antibody.

Figure 8B shows that treatment with anti-human CXCR4 antibodies 3G10, 6B6, and 12A11 had comparable and significant tumor growth inhibitory (TGI) activity relative to an isotype control antibody in this model.

Figure 8C is a survival curve showing the significant effect of anti-human CXCR4 antibodies 3G10, 6B6, and 12A11 compared to an isotype control antibody on the survival of animals systemically implanted with Raji-LUC cells.

Figure 9A shows the effect of CXCR4Ab6B6 on tumor burden in an acute myeloid leukemia (AML) model (MV4-11-LUC). Representative bioluminescence images of 5 animals per treatment group from day 20 to day 41 are shown.

Figure 9B shows the significant effect of anti-CXCR4 antibody 6B6 compared to an isotype control antibody on the survival of animals systemically implanted with MV4-11-LUC AML cells.

Figure 9C shows that on study day 35, human AML tumor burden was significantly reduced in the peripheral blood (PB) of animals treated with anti-CXCR4 6B6 antibody compared to isotype control-treated animals.

FIG10A shows representative luciferase imaging in mice bearing systemic chronic lymphocytic leukemia tumors treated with CXCR4 3G10 antibody.

Figure 10B is a survival curve showing the significant effect of anti-human CXCR4 antibody 3G10, compared to an isotype control antibody, on the survival of animals systemically implanted with chronic lymphocytic leukemia cells.

Figure 11 shows that humanized anti-human CXCR4 antibodies significantly inhibited tumor growth in a non-Hodgkin's lymphoma (NHL) model (Ramos) compared to an isotype control antibody.

Figure 12A shows the effect of CXCR4Ab h3G10.1.91.A58B on tumor burden in a multiple myeloma (MM) model (OPM-2-LUC). Representative bioluminescence images of 5 animals per treatment group from day 8 to day 30 are shown.

Figure 12B shows quantification of bioluminescence (luciferase activity) in a systemic multiple myeloma (MM) model in mice treated with the anti-human CXCR4 antibody h3G10.1.91.A58B compared to animals treated with an isotype control antibody and Melphalan.

Figure 12C is a survival curve showing the significant effect of anti-CXCR4 antibody h3G10.1.91.A58B compared to isotype control antibody and melphalan in mice systemically implanted with OPM-2-Luc MM cells.

FIG. 13A shows the significant anti-tumor effects of CXCR4 ADCs 3G10-TG6-vc0101 and 6B6-TG6-vc0101 in the Ramos (NHL) tumor model.

FIG13B shows the significant anti-tumor effect of the CXCR4 ADC 3G10-TG6-vc0101 in the HPB-ALL (T-ALL) tumor model.

DETAILED DESCRIPTION

The present invention disclosed herein provides antibodies and antibody conjugates (e.g., antibody-drug conjugates) that bind to CXCR4 (e.g., human CXCR4). The present invention also provides polynucleotides encoding these antibodies, compositions comprising these antibodies, and methods for preparing and using these antibodies. In certain aspects, the antibodies of the present invention are derived from specific heavy and light chain germline sequences and/or contain specific structural features, such as CDR regions comprising specific amino acid sequences. The present invention provides isolated antibodies, methods for preparing such antibodies, their antigen-binding fragments, antibody-drug conjugates, and bispecific molecules comprising such antibodies, and pharmaceutical compositions containing the antibodies of the present invention, their antigen-binding fragments, antibody-drug conjugates, or bispecific molecules. The present invention also relates to methods for using antibodies, for example, to detect CXCR4, modulate CXCR4 activity, and/or for targeting cells expressing CXCR4 to destroy (e.g., ADCC, CDC, toxins) in conditions related to CXCR4 function or expression (e.g., cancer). The present invention also provides methods for prophylactically and/or therapeutically treating conditions related to CXCR4 function or expression, such as cancer (e.g., solid tumor cancer or hematological cancer) in a subject. Finally, the invention encompasses compositions containing such antibodies conjugated (e.g., antibody-drug conjugates) or combined with other anti-cancer compounds (such as antibodies, toxins, cytotoxins/cytostatics), and the use of such compositions for preventing and/or treating disorders associated with CXCR4 function or expression, such as cancer.

General techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. These techniques are fully explained in the following literature, such as: Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); and The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions and Abbreviations

As used herein, the terms "chemokine (C-X-C motif) receptor 4" and "CXCR4" refer to a G protein-coupled chemokine receptor with seven transmembrane domains that is typically embedded in the cell membrane. The term also includes variants, isoforms, homologs, orthologs, and paralogs. The nucleic acid and polypeptide sequences of human CXCR4 are disclosed in GenBank Accession Nos. NM_003467 and NP_003458, respectively. Further description of human CXCR4 can be found in Federsppiel, B. et al. Genomics 16(3):707-712 (1993); Herzog, H. et al. DNA Cell Biol. 12(6):465-471 (1993); Jazin, E.E. et al. Regul. Pept 47(3):247-258 (1993); Nomura, H. et al. Int. Immunol. 5(10):1239-1249 (1993); Loetscher, M. et al. J. Biol. Chem. 269(1):232-237 (1994); Moriuchi, M. et al. J. Immunol. 159(9):4322-4329 (1997); Caruz, A. et al. FEBS Lett. 426(2):271-278 (1998); and Wegner, S.A. et al. J. Biol. Chem. 273(8):4754-4760 (1998).

CXCR4 is also known in the art as, for example, LESTR, CD 184, CD184 antigen, C-X-C chemokine receptor type 4, CXCR-4, CXCL-12, CXCR-R4, D2S201E, FB22, Fusion, Fusin, HM89, HSY3RR, LAP3, LCR1, leukocyte-derived seven transmembrane domain receptor, NPY3R, NPYR, NPYRL, NPYY3R, SDF-1 receptor, or stromal cell-derived factor 1 receptor.

For the purposes of the present invention, the term "CXCR4 antigen" encompasses any CXCR4, including human CXCR4, CXCR4 from another mammal (such as mouse CXCR4, rat CXCR4, canine CXCR4, feline CXCR4 protein or primate CXCR4), and different forms of CXCR4 (e.g., glycosylated CXCR4).

As used herein, the terms "antibody" and "Ab" refer to an immunoglobulin molecule that is capable of recognizing and binding a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" may encompass any type of antibody, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and antigen-binding fragments of intact antibodies that retain the ability to specifically bind to a given antigen (e.g., CXCR4), bispecific antibodies, heteroconjugate antibodies, mutants thereof, fusion proteins with antibodies, single chain (ScFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1126-1136), humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the desired specificity, including glycosylation variants of the antibodies, amino acid sequence variants of the antibodies, and covalently modified antibodies. The antibodies may be of murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some aspects of the invention, the antibody or antigen-binding fragment thereof used in the methods of the invention is a chimeric, humanized or recombinant human antibody or CXCR4-binding fragment thereof.

There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

Native or naturally occurring antibodies are typically heterotetramers of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, with the number of disulfide bonds varying between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. The term "variable" refers to the fact that the sequence of certain portions of the variable domain differs widely between antibodies.

As used herein, the terms "antigen-binding fragment," "antigen-binding portion," and "antibody portion" refer to one or more fragments of an intact antibody that retain the ability to bind to a given antigen (e.g., CXCR4). The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, an Fd fragment consisting of the VH and CH1 domains, and an Fv fragment consisting of the VL and VH domains of a single arm of an antibody. Single-domain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1989), isolated complementarity-determining regions (CDRs), nanobodies, and heavy chain variable regions containing a single variable domain and two constant domains. In addition, although the two domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be connected using recombinant methods through a synthetic linker that enables them to be made into a single protein chain that pairs the VL and VH regions to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., Science 242: 423-426 (1988), and Huston et al., PNAS 85: 5879-5883 (1988)). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

As used herein, the term "CDR" refers to the region of an antibody variable domain that confers its binding specificity. There are three CDRs on each heavy and light chain of an antibody. The amino acid residues within the variable region that comprise the CDRs can be identified using methods known in the art, including, but not limited to, Kabat (Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), Chothia (Chothia et al., 1989, Nature 342:877-883), the cumulative of Kabat and Chothia, the AbM definition (which is a compromise between Kabat and Chothia and was generated using Oxford Molecular's AbM antibody modeling software (now)), contact definition (MacCallum et al., 1996, J. Mol. Biol., 262:732-745), and/or conformational definition (Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). As used herein, CDRs can refer to CDRs defined by any method known in the art, including a combination of methods. The methods used herein can utilize CDRs defined according to any of these methods.

As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from the amino acid residue at Pro230 to its carboxyl terminus. The residue numbering in the Fc region is that of the EU index of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

As used herein, the terms "monoclonal antibody" and "mAb" refer to antibodies obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" refers to the characteristic that an antibody is obtained from a substantially homogeneous antibody population and should not be understood as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be obtained by first using the hybridoma method described in Kohler and Milstein, 1975, Nature, 256:495, or can be obtained by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries produced using, for example, the techniques described in McCafferty et al., 1990, Nature, 348:552-554.

As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to CXCR4 is substantially free of antibodies that specifically bind to antigens other than CXCR4). However, an isolated antibody that specifically binds to CXCR4 may have cross-reactivity to other antigens, such as CXCR4 molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and/or chemicals.

As used herein, the terms "humanized antibody" and "CDR-grafted antibody" refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequence derived from a non-human immunoglobulin. Humanized antibodies are preferably human immunoglobulins (acceptor antibody) in which residues in the complementarity determining regions (CDRs) of the recipient are replaced with residues from the CDRs of an antibody (donor antibody) of a non-human species (such as mouse, rat, or rabbit) having the desired specificity, affinity, and capacity.

In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may include residues that are neither found in the recipient antibody nor in the introduced CDR or framework sequences, but are included to further improve and optimize the antibody's efficacy. In general, the humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions are FR regions of a human immunoglobulin consensus sequence. The humanized antibody will also preferably comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically a constant region or domain of a human immunoglobulin. Preferably, the antibody has a modified Fc region as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

Humanization can be essentially performed by substituting rodent or mutant rodent CDRs or CDR sequences for the corresponding sequences of a human antibody following the method of Winter and colleagues (Jones et al. Nature 321:522-525 (1986); Riechmann et al. Nature 332:323-327 (1988); Verhoeyen et al. Science 239:1534-1536 (1988)). See also U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference. In some cases, residues in the framework regions of one or more variable regions of a human immunoglobulin are replaced with corresponding non-human residues (see, e.g., U.S. Patents 5,585,089, 5,693,761, 5,693,762, and 6,180,370). In addition, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody potency (e.g., to obtain a desired affinity). In general, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. Nature 331:522-525 (1986); Riechmann et al. Nature 332:323-329 (1988); and Presta Curr. Op. Struct. Biol. 2:593-596 (1992); incorporated herein by reference. Thus, such "humanized" antibodies may include antibodies in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. See, e.g., U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. See also U.S. Patent No. 6,180,370 and International Publication No. WO 01/27160, which disclose humanized antibodies and techniques for generating humanized antibodies with improved affinity for a predetermined antigen.

As used herein, the term "human antibody" refers to an antibody whose amino acid sequence corresponds to an antibody produced by a human and/or has been prepared using any technique for preparing human antibodies known to those skilled in the art or disclosed herein. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a murine light chain and a human heavy chain polypeptide. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, (1996); Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, (1998); Hoogenboom and Winter, J. Mol. Biol., 227:381, (1991); Marks et al., J. Mol. Biol., 222:581, (1991)). Human antibodies can also be made by immunizing animals into which human immunoglobulin loci have been introduced by transgenic methods to replace endogenous loci, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patents 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies can be made by immortalizing human B lymphocytes that produce antibodies against the target antigen (such B lymphocytes can be recovered from a subject or from a single cell clone of cDNA or can be immunized in vitro). See, eg, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, (1985); Boerner et al., J. Immunol., 147(1):86-95, (1991); and US Pat. No. 5,750,373.

As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. For veterinary applications, the constant domains of a chimeric antibody may be derived from another species, such as a cat or dog.

As used herein, the terms "canized antibodies" and "feline antibodies" refer to chimeric antibodies suitable for use as therapeutic agents in canines and felines, respectively. In both cases, the antigen-binding domain from a donor antibody of a heterologous species is combined with the non-antigen-binding domain of an acceptor antibody of the same species.

As used herein, the terms "preferentially bind" or "specifically bind" are well understood terms in the art when applied to an antibody binding to a target (e.g., a CXCR4 protein). Methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or binds to a particular cell or substance more frequently, more rapidly, for a longer period of time, and/or with greater affinity than it does to other cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if its binding affinity or avidity is greater, more readily, and/or persists longer than the antibody's binding affinity or avidity to other substances. For example, an antibody that specifically binds or preferentially binds to a CXCR4 epitope is an antibody that binds to that epitope with greater affinity, avidity, more readily, and/or persists longer than the antibody's binding affinity, avidity to other CXCR4 epitopes or non-CXCR4 epitopes.

As used herein, the terms "binding affinity" and " _KD " refer to the equilibrium dissociation constant for a specific antigen-antibody interaction. _KD is the ratio of the dissociation rate (also referred to as the "off-rate" or " _kd ") to the association rate or "association rate" or " _ka ". Thus, _KD is equal to _kd / _ka and is expressed in molar concentration (M). Thus, the smaller the _KD , the stronger the binding affinity. Thus, a _KD of 1 μM indicates weak binding affinity compared to a _KD of 1 nM. _KD values for antibodies can be determined using methods well established in the art. One method for determining _KD for an antibody is by using surface plasmon resonance, typically using a biosensor system such as a .

As used herein, the term "compete" refers to a first antibody that binds to an epitope in a manner sufficiently similar to the binding of a second antibody such that the binding of the first antibody to its cognate epitope in the presence of the second antibody is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. The present invention encompasses antibodies that compete with an antibody of the present invention for binding to an epitope. Based on the teachings provided herein, one skilled in the art will appreciate that such competing antibodies can be used in the methods disclosed herein.

As used herein, the term "drug" refers to any substance with biological or detectable activity. The term drug is intended to encompass cytotoxic agents, therapeutic agents, immunomodulators, detectable labels, imaging agents, binding agents, prodrugs (which are metabolized into active agents in vivo), growth factors, hormones, cytokines, antihormones, xanthines, interleukins, interferons, and cytotoxic drugs. The drug can be a small molecule, a polypeptide, an oligonucleotide, or a biopolymer. The drug can be conjugated to the antibody of the present invention via a linker to form an antibody-drug conjugate.

As used herein, the term "effector function" refers to the biological activity attributable to the Fc region of an antibody. Examples of antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, complement-dependent cytotoxicity (CDC), phagocytosis, C1q binding, and downregulation of cell surface receptors (e.g., B cell receptor; BCR). See, for example, U.S. Patent No. 6,737,056. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art for assessing such antibody effector functions. An exemplary measurement of effector function is via Fcγ3 and/or C1q binding.

As used herein, the term "epitope" includes any protein determinant that can bind to an immunoglobulin or T cell receptor or otherwise interact with a molecule. Epitope determinants are generally composed of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and generally have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes can be "linear" or "conformational". In a linear epitope, all interaction points between a protein and an interacting molecule (such as an antibody) appear linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction points appear through amino acid residues separated from each other on the protein. Once the desired epitope on the antigen is determined, antibodies against the epitope can be generated, for example, using the technology described in the present invention. Alternatively, during the discovery process, the generation and characterization of antibodies can elucidate information about the desired epitope. Based on this information, antibodies that bind to the same epitope can be competitively screened. One method for achieving this is to conduct competition studies to find antibodies that competitively bind to each other, i.e., antibodies compete for binding epitopes. A high-throughput method for "binning" antibodies based on competition is described in International Patent Application No. WO 03/48731. The term "partitioning" as used herein refers to a method of grouping antibodies based on their antigen-binding characteristics.

As used herein, the terms "polynucleotide," "nucleic acid/nucleotide," and "oligonucleotide" refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length, their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, DNA, cDNA, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, the nucleotide structure can be modified before or after chain assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "capping," substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as polynucleotides with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), polynucleotides containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), polynucleotides with intercalators (e.g., acridine, psoralen, etc.), polynucleotides containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), polynucleotides containing alkylating agents, polynucleotides with modified bonds (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl group normally present in a sugar can be replaced, for example, with a phosphonate group, a phosphate group, protected with standard protecting groups, or activated to prepare additional bonds with other nucleotides, or can be bound to a solid support. The 5' and 3' terminal OH groups may be phosphorylated or partially substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyl groups may also be derived into standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose), acyclic analogs, and debasic nucleoside analogs, such as methyl ribonucleosides. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thiol ester"), P(S)S ("dithiol ester"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("methylal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl group optionally containing an ether (-O-) linkage. All linkages in a polynucleotide need not be identical. The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, the terms "polypeptide," "oligopeptide," "peptide," and "protein" refer to amino acid chains of any length, preferably relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, may comprise modified amino acids, and/or may be interspersed with non-amino acids. The terms also encompass amino acid sequences that have been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (such as conjugation with a labeling component). Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It will be understood that polypeptides may appear as single chains or in conjunction with chains.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical.

As used herein, the terms "amino acid" and "natural amino acid" refer to arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine.

As used herein, the term "amino acid derivative" refers to an amino acid that is substituted or modified by the covalent attachment of a parent amino acid, such as by alkylation, glycosylation, acetylation, phosphorylation, and the like. Further included within the definition of "derivative" are, for example, one or more analogs of amino acids having substituted linkages and other modifications known in the art.

As used herein, the term "vector" refers to a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (such as production cells).

As used herein, the term "percentage of sequence identity" refers to the degree of similarity between two sequences. In the case of nucleic acid sequences, it refers to the identical residues in two sequences when aligned for maximum correspondence. The length of sequence identity comparison can be at least about 9 nucleotides, generally at least about 18 nucleotides, more generally at least about 24 nucleotides, usually at least about 28 nucleotides, more generally at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides in length. A variety of different algorithms are known in the art that can be used to measure nucleotide sequence identity. For example, FASTA, Gap or Bestfit can be used to compare polynucleotide sequences, and the algorithm is a program in the Wisconsin Package 10.0 version, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, including, for example, the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the best overlapping regions between query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein by reference). Unless otherwise specified, the default parameters of the particular program or algorithm are used. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (font size 6 and NOPAM coefficients for the scoring matrix) as provided in GCG version 6.1 (incorporated herein by reference), or Gap with its default parameters.

Unless otherwise indicated, references to a nucleotide sequence encompass its complementary sequence. Thus, it is understood that references to a nucleic acid having a specific sequence encompass its complementary strand and its complementary sequence.

In the context of amino acid sequences, it means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT, using the default gap weights provided with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity. In some substantially similar amino acid sequences, residue positions that are not identical differ by conservative amino acid substitutions.

Substantially similar polypeptides also include conservatively substituted variants in which one or more residues have been conservatively substituted with functionally similar residues. Examples of conservative substitutions include substitution of a non-polar (hydrophobic) residue (such as isoleucine, valine, leucine, or methionine) with another non-polar (hydrophobic) residue; substitution of a polar (hydrophilic) residue with another polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; substitution of a basic residue (such as lysine, arginine, or histidine) with another basic residue; or substitution of an acidic residue (such as aspartic acid or glutamic acid) with another acidic residue.

Another indication that two proteins are substantially identical is that they share an overall three-dimensional structure or are biologically functional equivalents.

As used herein, the term "cytotoxic activity" refers to the cell-killing, cell growth inhibition, or anti-proliferative effects of an antibody, ADC, or intracellular metabolite of the ADC. Cytotoxic activity can be expressed as an IC50 value, which is the concentration (molar or mass) per unit volume at which half of the cells survive.

As used herein, the term "contacting" refers to bringing an antibody or antigen-binding portion thereof of the present invention into contact with a target CXCR4 or an epitope thereof in such a manner that the antibody can affect the biological activity of CXCR4. The "contacting" can be achieved "in vitro" (i.e., in a test tube, petri dish, or the like). In a test tube, contacting can involve only the antibody or antigen-binding portion thereof and CXCR4 or an epitope thereof, or it can involve whole cells. Cells can also be maintained or grown in a cell culture dish and contacted with the antibody or antigen-binding portion thereof in such an environment. In this case, the ability of a particular antibody or antigen-binding portion thereof to affect a CXCR4-related condition, i.e., the antibody's IC50 value, can be determined before attempting to use the antibody in vivo in a more complex living organism. For cells outside of a living organism, various methods exist and are well known to those skilled in the art for contacting CXCR4 with an antibody or antigen-binding fragment thereof. In another embodiment, the effector function produces a substantial effect. Immune effector functions that have been shown to contribute to antibody-mediated cytotoxicity include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

As used herein, the terms "tag containing the acyl donor glutamine" and "glutamine tag" refer to a polypeptide or protein containing one or more Gln residues that acts as an amine acceptor for transglutaminase. See, for example, WO2012059882.

As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" refer to any substance that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Standard pharmaceutical carriers include, but are not limited to, phosphate-buffered saline solutions, water, emulsions (such as oil/water emulsions), and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or saline (0.9%). Compositions containing these carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005). The carrier is preferably suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (e.g., monoclonal antibody, antigen-binding fragment thereof, antibody-drug conjugate) can be coated with a material that protects the compound from acids and other natural conditions that may inactivate the compound.

The present invention also provides compositions comprising or consisting of one, two, three, four, five, ten, fifteen, twenty or more antibodies of the present invention (including molecules comprising or consisting of antibody fragments or variants thereof). The compositions of the present invention may comprise or consist of one, two, three, four, five, ten, fifteen, twenty or more amino acid sequences of one or more antibodies or fragments or variants thereof. Alternatively, the compositions of the present invention may comprise or consist of nucleic acid molecules encoding one or more antibodies of the present invention.

The term "CXCR4-related disorder" is any condition in which the pathology is attributable, at least in part, to increased or inappropriate CXCR4 expression or inappropriate CXCR4 function. Examples of such disorders include, but are not limited to, cancers associated with increased CXCR4 expression relative to normal, inflammatory and immune disorders, allergic disorders, infections (such as HIV infection), autoimmune disorders (e.g., rheumatoid arthritis), fibrotic disorders (e.g., lung), and cardiovascular disorders.

The term "cancer" refers to the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. Hematological cancers refer to cancers of the blood and include, but are not limited to, leukemias, lymphomas, and myelomas. Solid cancers refer to cancers of body tissues other than the blood and include, but are not limited to, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, lung cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, and hepatic carcinoma, among others.

As used herein, the term "patient" refers to a subject suffering from a CXCR4-related disorder. Patients may include, but are not limited to, humans, non-human primates (e.g., monkeys), rats, mice, guinea pigs, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. In preferred embodiments, the patient to whom the antibody or antibody-drug conjugate of the present invention is administered is a human.

As used herein, the term "effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition required to achieve one or more beneficial or desired therapeutic results. For preventive use, beneficial or desired results include eliminating or reducing the risk of a condition (including biochemical, histological, and/or behavioral symptoms of the condition, its complications, and intermediate pathological expression patterns that appear during the onset of the condition), reducing its severity, or delaying its onset. For therapeutic use, beneficial or desired results include clinical results such as reducing the incidence of one or more symptoms of the condition or improving one or more symptoms of the condition, being able to reduce the dose of other agents required to treat the condition, enhancing the effect of another agent used to treat the condition, and/or delaying the progression of the condition.

For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation (or destruction) of neoplastic or cancerous cells in a CXCR4-associated disorder; reducing or inhibiting the metastasis of neoplastic cells in a CXCR4-associated disorder; shrinking or reducing the size of a tumor expressing CXCR4; alleviating a CXCR4-associated disorder; increasing the life expectancy of a subject suffering from a CXCR4-associated disorder; reducing the symptoms resulting from a CXCR4-associated disorder; improving the quality of life of a person suffering from a CXCR4-associated disorder; being able to reduce the dose of other agents required to treat a CXCR4-associated disorder without adverse effects; delaying the progression of a CXCR4-associated disorder; curing a CXCR4-associated disorder; and/or prolonging the survival of a patient suffering from a CXCR4-associated disorder.

Unless otherwise defined herein, technical and scientific terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. For example, the terms "comprises," "comprising," "containing," and "having" and their similar terms may have the meanings ascribed thereto in U.S. patent law and may refer to "includes," "including," and their similar terms; similarly, "consisting essentially of" or "consisting essentially of" have the meanings ascribed thereto in U.S. patent law and the terms are open ended, allowing for more than the stated terms as long as the basic or novel features of the stated terms are not altered by the presence of the more than the stated terms, but do not include prior art implementations.

Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

References herein to "about" a value or parameter include (and describe) embodiments directed to the value or parameter itself. For example, a description of "about X" includes a description of "X." Numerical ranges include the numbers defining the range. Where aspects or embodiments of the invention are described in terms of Markush or other alternative groupings, the invention encompasses not only the entire group listed as a whole, but also each member of the group and all possible subgroups of the main group, and also encompasses the main group lacking one or more group members. The invention also contemplates the explicit exclusion of one or more of any group members in the claimed invention.

Throughout this specification and claims, the word "comprise" or variations thereof (such as "comprises" or "comprising") will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Unless otherwise defined, all scientific and technological terms used herein have the same meaning as those generally understood by those skilled in the art. In the event of a conflict, the present specification (including definitions) shall prevail. Exemplary methods and materials are described herein, but methods and materials similar to or equivalent to the methods and materials described herein can also be used to implement or test the present invention. Materials, methods, and examples are illustrative only and are not intended to be limiting.

Anti-CXCR4 antibody and preparation method thereof

The present invention provides anti-CXCR4 antibodies or antigen-binding fragments that bind to human CXCR4. In this section of the specification, the functional and structural features of exemplary anti-CXCR4 antibodies or antigen-binding fragments of the present invention are described in detail. It should be understood that the antibodies or antigen-binding fragments of the present invention can be described based on any one or more (2, 3, 4, 5, 6, 7, 8, 9, etc.) of the structural and/or functional features described herein. Throughout this section of the present invention, when describing functional or structural features for the antibodies of the present invention, it should be understood that unless the context clearly indicates otherwise, such structural or functional features can be similarly used to describe the antigen-binding fragments of the present invention.

The nucleotide sequence of human CXCR4 cDNA and the predicted amino acid sequence of human CXCR4 protein are shown in SEQ ID NOs: 104 and 105, respectively (Table 1). The human CXCR4 gene, which is approximately 1679 nucleotides in length, encodes a full-length protein with a molecular weight of approximately 38.7 kD and a length of approximately 352 amino acid residues. Further descriptions of the human CXCR4 nucleic acid and polypeptide sequences can be found in GenBank accession numbers NM_003467 and NP_003458, respectively; and in Federsppiel, B. et al. Genomics 16(3):707-712 (1993); Herzog, H. et al. DNA Cell Biol. 12(6):465-471 (1993); Jazin, E.E. et al. Regul. Pept. 47(3):247-258 (1993); Nomura, H. et al. Int. Immunol. 5(10):1239-1249 (1993); Loetscher, M. et al. J. Biol. Chem. 269(1):232-237 (1994); Moriuchi, M. et al. J. Immunol. 159(9):4322-4329 (1997); Caruz, A. et al. FEBS Lett. 426(2):271-278 (1998); and Wegner, S.A. et al. J. Biol. Chem. 273(8):4754-4760 (1998).

Table 1

As used herein, the human CXCR4 sequence may differ from the human CXCR4 of SEQ ID NO: 105 in having, for example, conservative mutations or mutations in non-conserved regions, and the CXCR4 has substantially the same biological function as the human CXCR4 of SEQ ID NO: 105. For example, the biological function of human CXCR4 is having an epitope in the extracellular domain of CXCR4 that is bound by an antibody of the present invention, or the biological function of human CXCR4 is binding to a chemokine or participating in a metastatic process.

A specific human CXCR4 sequence will generally be at least 90% identical in amino acid sequence to the human CXCR4 of SEQ ID NO: 105 and contain amino acid residues that identify the amino acid sequence as human when compared to the amino acid sequence of CXCR4 of another species (e.g., murine). In certain instances, the human CXCR4 may be at least 95% or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the CXCR4 of SEQ ID NO: 105. In some aspects of the invention, the human CXCR4 sequence will exhibit no more than 10 amino acid differences from the CXCR4 of SEQ ID NO: 105. In certain aspects of the invention, the human CXCR4 may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the CXCR4 of SEQ ID NO: 105. Percent identity can be determined as described herein.

The antibodies, antigen-binding fragments thereof, and antibody-drug conjugates of the present invention are characterized by any one or more of the following characteristics: (a) binding to CXCR4; (b) reducing or downregulating CXCR4 protein expression; (c) treating, preventing, or ameliorating one or more symptoms of a condition associated with CXCR4 function or expression (e.g., cancer, such as NHL, AML, MM, CLL, T-ALL, gastric cancer, head and neck cancer, lung cancer, ovarian cancer, or pancreatic cancer) in a subject; (d) reducing or inhibiting tumor growth or progression in a subject having a tumor expressing CXCR4; (e) reducing or inhibiting metastasis of CXCR4-expressing cancer cells in a subject having one or more CXCR4-expressing cancer cells; (f) inducing regression (e.g., long-term regression) of a CXCR4-expressing tumor; (g) exerting cytotoxic activity in cells expressing CXCR4; (h) inactivating or downregulating the CXCR4 pathway; and (i) blocking the interaction of CXCR4 with an extracellular binding partner of CXCR4. For example, the antibodies, antigen-binding fragments thereof, and antibody-drug conjugates of the present invention block the function of SDF-1.

Antibodies suitable for use in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising antibody portions (e.g., domain antibodies), humanized antibodies, and any other modified configurations of immunoglobulin molecules comprising an antigen recognition site of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be of murine, rat, human, or any other origin (including chimeric or humanized antibodies).

In some aspects of the invention, the anti-CXCR4 antibody described herein is a monoclonal antibody. For example, the anti-CXCR4 antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody.

In some aspects of the invention, the antibodies of the invention cross-react with CXCR4 from non-human species, such as mouse, rat, canine, feline, or primate CXCR4, as well as different forms of CXCR4 (e.g., glycosylated CXCR4). In some aspects of the invention, antibodies specific for human CXCR4 may be completely specific for human CXCR4 and may not exhibit species or other types of cross-reactivity.

In some aspects of the invention, the anti-CXCR4 antibodies described herein are caninized or feline antibodies. Caninized or feline antibodies according to the invention are capable of binding to at least one of a canine CXCR4 protein or a feline CXCR4 protein. In one aspect, the monoclonal antibodies of the invention bind to a canine CXCR4 protein or a feline CXCR4 protein and prevent it from binding to stromal cell-derived factor-1 (SDF-1).

In some aspects of the invention, the antibodies comprise a modified constant region, such as, but not limited to, a constant region that has an increased likelihood of eliciting an immune response. For example, the constant region can be modified to have an increased affinity for an Fcγ receptor, such as FcγRI, FcγRIIA, or FcγIII.

In some aspects of the invention, the antibody comprises a modified constant region, such as one having increased affinity for human Fcγ receptors, or one that is immunologically inert, i.e., less likely to elicit an immune response. In some aspects of the invention, the constant region is modified as described in: Eur. J. Immunol., 29:2613-2624, 1999; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 98099518. The Fc can be human IgG1, human IgG2, human IgG3, or human IgG4. The Fc can be human IgG2 containing the A330P331 to S330S331 mutation (IgG2Δa), where the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur. J. Immunol., 29:2613-2624, 1999. In some aspects of the invention, the antibody comprises an IgG4 constant region containing the following mutation (Armour et al., Molecular Immunology 40, 585-593, 2003): E233F234L235 mutated to P233V234A235 (IgG4Δc), where numbering is relative to wild-type IgG4. In other aspects of the invention, the Fc is a human IgG4 with E233F234L235 mutated to P233V234A235 and a deletion of G236 (IgG4Δb). In another aspect of the invention, the Fc is any human IgG4 Fc containing a hinge-stabilizing mutation from S228 to P228 (IgG4, IgG4Δb, or IgG4Δc) (Aalberse et al., Immunology 105, 9-19, 2002). In a specific aspect of the invention, the Fc may be aglycosylated Fc.

In some aspects of the invention, the constant region is deglycosylated by mutating the oligosaccharide attachment residue (such as Asn297) and/or the flanking residues that are part of the glycosylation recognition sequence in the constant region. In some aspects of the invention, the constant region is deglycosylated enzymatically for N-linked glycosylation. The constant region can be deglycosylated for N-linked glycosylation enzymatically or by expression in a glycosylation-deficient host cell.

One way to determine the binding affinity of an antibody to CXCR4 is by measuring the binding affinity of a monofunctional Fab fragment of the antibody. To obtain a monofunctional Fab fragment, the antibody (e.g., IgG) can be cleaved with papain or recombinantly expressed. The affinity of the CXCR4 Fab fragment of the antibody can be determined by surface plasmon resonance (Biacore ^™ 3000 ^™ surface plasmon resonance (SPR) system, Biacore ^™ , INC, Piscataway, NJ) equipped with a pre-immobilized streptavidin sensor chip (SA) or anti-mouse Fc or anti-human Fc, using HBS-EP running buffer (0.01 M HEPES pH 7.4, 0.15% NaCl, 3 mM EDTA, 0.005% v/v surfactant P20). Biotinylated WGA can be coated on the SA chip to facilitate capture of lipid particles containing the CXCR4 protein. Serial dilutions of Fab (5 members, 3× dilution factor, highest concentration of 10 or 30 nM) were injected from low to high concentrations (each concentration with a 3 minute binding time) to perform kinetic analysis of the data using the “kinetic titration” method as described in Karlsson et al. (Karlsson, R., Katsamba, P. S., Nordin, H., Pol, E., and Myszka, D. G. Analyzing a kinetic titration series using affinity biosensors. Anal. Biochem. 349, 136-147 (2006). For some analysis cycles, buffer was injected instead of Fab on the captured particles to provide blank cycles for double referencing purposes (double referencing was performed as described in Myszka et al. (Myszka, D. G. Improving biosensor analysis. J. Mol. Recognit. 12, 279-284 (1999)).

The concentration of the Fab protein is determined by ELISA and/or SDS-PAGE electrophoresis using Fab of known concentration (as determined by amino acid analysis) as a standard. Using the BIAevaluation program, the data are globally fitted to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110) to simultaneously obtain kinetic association rates (k _on ) and dissociation rates (k _off ). The equilibrium dissociation constant (K _D ) value is calculated as k _off /k _on . This protocol is applicable to determining the binding affinity of an antibody for any CXCR4, including human CXCR4, another mammalian CXCR4 (such as mouse CXCR4, rat CXCR4, canine CXCR4, or primate CXCR4), and different forms of CXCR4 (e.g., glycosylated CXCR4). The binding affinity of the antibody is generally measured at 25°C, but can also be measured at 37°C.

In some aspects of the invention, the antibody or antigen-binding fragment thereof that binds to CXCR4 has an MFI of less than 10,000 μg/mL. In specific aspects of the invention, the antibody or antigen-binding fragment thereof that binds to CXCR4 has an MFI of less than 6000 μg/mL. In specific aspects of the invention, the antibody or antigen-binding fragment thereof that binds to CXCR4 has an MFI of less than 5000 μg/mL. In specific aspects of the invention, in some aspects of the invention, the antibody or antigen-binding fragment thereof that binds to CXCR4 has an MFI in the range of 2000 μg/mL to 5000 μg/mL.

As used herein, "MFI" refers to mean or median fluorescence intensity.

In some aspects of the invention, the antibody or antigen-binding fragment thereof that binds to CXCR4 has an EC50 of less than 3.00E-09 M. In some aspects of the invention, the antibody or antigen-binding fragment thereof that binds to CXCR4 has an EC50 in the range of about 1.00E-09 M to about 3.00E-09 M. For example, the antibody or antigen-binding fragment thereof that binds to CXCR4 has an EC50 of 2.00E-09 M.

In some aspects of the invention, an antibody or antigen-binding fragment thereof that binds to CXCR4 inhibits SDF-1-induced calcium mobilization. For example, the antibody or antigen-binding fragment thereof inhibits SDF-1α-induced calcium mobilization with an IC50 of inhibition ranging from about 1 nM to 50 nM. In one aspect, the antibody or antigen-binding fragment thereof inhibits SDF-1α-induced calcium mobilization with an IC50 of inhibition less than 30 nM. In one aspect, the antibody or antigen-binding fragment thereof inhibits SDF-1α-induced calcium mobilization with an IC50 of inhibition less than 2 nM.

In some aspects of the invention, the antibodies or antigen-binding fragments thereof significantly reduce the number of AML cells in AML cell models. For example, the number of human AML cells was significantly reduced in animals treated with the anti-CXCR4 6B6 antibody. In addition, the survival of animals treated with the anti-CXCR4 antibodies of the invention was significantly increased.

In some aspects, the antibodies of the invention increase survival time and reduce tumor burden in mouse systemic non-Hodgkin's lymphoma (NHL) and acute myeloid leukemia (AML) cell models. In one aspect, the antibodies of the invention increase survival in a mouse systemic chronic lymphocytic leukemia (CLL) tumor model. In one aspect, the antibodies of the invention inhibit NHL tumor growth in vivo. In some aspects, the antibodies of the invention increase survival time and reduce tumor burden in a mouse systemic multiple myeloma (MM) model.

The antibodies of the present invention can be produced using techniques well known in the art, such as recombinant technology, phage display technology, synthetic technology, or a combination of such technologies or other technologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50 (1999) and Fellouse, F.A. et al., J. Mol. Biol., 373(4): 924-40 (2007)).

The anti-CXCR4 antibodies or antigen-binding fragments thereof described herein can be prepared by any method known in the art. For example, to generate hybridoma cell lines, the route and schedule of immunization of the host animal are generally consistent with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for generating human and mouse antibodies are known in the art and/or described herein.

It is expected that any mammalian subject, including humans, or antibody-producing cells therefrom can be manipulated to serve as the basis for generating mammalian and hybridoma cell lines, including humans. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, plantarly, and/or intradermally with an amount of immunogen as described herein.

Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature 256:495-497 (1975), or modified as described by Buck, DW et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to _X63 -Ag8.653 and those obtained from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used for hybridization. Generally, the technique involves fusing myeloma cells with lymphocytes using a fusogenic agent such as polyethylene glycol or by electrical means familiar to those skilled in the art. Following fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to remove unhybridized parental cells. Any culture medium described herein, supplemented or not supplemented with serum, can be used to culture hybridomas that secrete monoclonal antibodies. As an alternative to cell fusion techniques, EBV-immortalized B cells can be used to produce the CXCR4 monoclonal antibodies of the present invention. If necessary, hybridomas are expanded and subcloned, and the supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

Hybridomas useful as a source of antibodies encompass all derivatives, progeny cells, or portions thereof of a parent hybridoma that produces a monoclonal antibody specific for CXCR4.

Hybridomas producing the antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture medium or body fluids, if necessary, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed, for example, by running the preparation over an adsorbent made of the immunogen attached to a solid phase and eluting or releasing the desired antibody from the immunogen. A population of antibodies (e.g., monoclonal antibodies) can be produced by ^immunizing a host animal with human CXCR4 or ^a fragment containing the target amino acid sequence conjugated to a protein immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, _bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing agent (e.g., maleimidobenzoylsulfonylsuccinimide ester (conjugated via cysteine residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl 2, or R 1 N═C═NR, where R and R 1 are different alkyl groups).

If necessary, the relevant anti-CXCR4 antibody (monoclonal or polyclonal) can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the relevant antibody can be maintained in a vector in the host cell and the host cells can then be expanded and frozen for future use. The production of recombinant monoclonal antibodies in cell culture can be carried out by cloning antibody genes from B cells using methods known in the art. See, for example, Tiller et al., J. Immunol. Methods 329, 112 (2008); U.S. Patent No. 7,314,622.

Alternatively, the polynucleotide sequence can be genetically manipulated to "humanize" the antibody or to improve its affinity or other characteristics. For example, the constant region can be modified to be more similar to a human constant region to avoid immune responses when the antibody is used in human clinical trials and therapeutics. It may be necessary to genetically manipulate the antibody sequence to obtain greater affinity for CXCR4 and/or greater efficacy in inhibiting CXCR4.

There are generally four steps to humanizing a monoclonal antibody. These steps are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework regions to use in the humanization process; (3) the actual humanization method/technique; and (4) transfecting and expressing the humanized antibody. See, e.g., U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

A variety of "humanized" antibody molecules comprising antigen-binding sites derived from non-human immunoglobulins have been described, including chimeric antibodies having rodent or modified rodent V regions and their associated CDRs fused to human constant regions. See, for example, Winter et al. Nature 349:293-299 (1991); Lobuglio et al. Proc. Nat. Acad. Sci. USA 86:4220-4224 (1989); Shaw et al. J Immunol. 138:4534-4538 (1987); Brown et al. Cancer Res. 47:3577-3583 (1987). Other references describe rodent CDRs transplanted into human supporting framework regions (FRs) before fusion with appropriate human antibody constant regions. See, e.g., Riechmann et al. Nature 332:323-327 (1988); Verhoeyen et al. Science 239:1534-1536 (1988), and Jones et al. Nature 321:522-525 (1986). Another reference describes rodent CDRs supported by recombinantly engineered rodent framework regions. See, e.g., European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize unwanted immune responses to rodent anti-human antibody molecules, which limit the duration and effectiveness of therapeutic applications of those portions in human recipients. For example, the antibody constant regions can be engineered to render them immunologically inert (e.g., do not trigger complement lysis). See, e.g., PCT Publication No. PCT/GB99/01441, UK Patent Application No. 9809951.8. Other methods of humanizing antibodies that may also be used are disclosed in Daugherty et al., Nucl. Acids Res. 19:2471-2476, 1991; and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO 01/27160.

The general principles related to the humanized antibodies discussed above are also applicable to antibodies customized for, for example, dogs, cats, primates, equines, and bovines. In addition, one or more aspects of antibody humanization described herein can be combined, such as CDR grafting, framework mutations, and CDR mutations.

In some aspects of the invention, fully human antibodies can be obtained by using commercially available mice that have been modified to express specific human immunoglobulins. Transgenic animals designed to produce more desirable (e.g., fully human antibodies) or more robust immune responses can also be used to produce humanized or human antibodies. Methods for obtaining human antibodies from transgenic mice are disclosed in Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994); and Taylor et al., Int. Immun. 6:519 (1994). A non-limiting example of such a system is the ® from Abgenix (Fremont, CA) (e.g., Green et al., J. Immunol. Methods 231:11-23 (1999), incorporated herein by reference). In ® and similar animals, mouse antibody genes have been inactivated and replaced with functional human antibody genes, while the rest of the mouse immune system remains intact.

Transformed with a YAC (yeast artificial chromosome) of a germline configuration containing portions of human IgH and Igκ loci, including most of the variable region sequences, along with additional genes and regulatory sequences. Human variable region lineages can be used to generate antibody-producing B cells, which can be processed into hybridomas by known techniques. Immunization with the target antigen will produce human antibodies by normal immune response, which can be collected and/or produced by the standard techniques discussed above. A variety of strains are available, each of which is capable of producing antibodies of different classes. Human antibodies produced in a transgenic manner have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al., J. Immunol. Methods 231: 11-23 (1999)). Those skilled in the art will recognize that the claimed compositions and methods are not limited to the use of systems and that any transgenic animal that has been genetically modified to produce human antibodies can also be utilized.

In some aspects of the invention, antibodies can be prepared using one or more of the VH and/or VL sequences disclosed herein as starting materials for modifying modified antibodies, which may have properties that are changed from the starting antibodies. Antibodies can be modified by modifying one or two variable regions (i.e., VH and/or VL), for example, one or more residues in one or more CDR regions and/or in one or more framework regions. Alternatively, antibodies can be modified by modifying residues in the constant region, for example, to change the effector function of the antibody. In certain aspects, CDR transplantation can be used to transform the variable region of an antibody. Antibodies interact with target antigens primarily through amino acid residues located within the six heavy and light chain complementary determining regions (CDRs). For this reason, between individual antibodies, the amino acid sequence within the CDRs differs more than the sequence outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific naturally occurring antibody can be expressed by constructing expression vectors that include the CDR sequences from a specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L et al., Nature 332 323-327 (1995); Jones, P et al., Nature 32 J. 522-525 (1986); Queen, C et al., Proc Natl Acad See U.S.A 86 10029-10033 (1989); U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

Such framework sequences can be obtained from public DNA databases or from the published literature that includes germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpecamac.uk/vbase) and in Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops"; Cox, JPL et al., J Mol. Biol. TTL 776-798 (1994) "A Directory of Human Germ-line Vn Segments Reveals a Strong Bias in their Usage" Ew. J. Immunol. 2A. 827-836, the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in HCo7HuMAb mice can be obtained with the accompanying Genbank accession numbers 1-69 (NG_0010109, NT_024637, and BC070333), 3-33 (NG_0010109 and NT_024637), and 3-7 (NG_0010109 and NT_024637). As another example, the following heavy chain germline sequences found in the HCoI 2 HuMAb mouse are available with the accompanying Genbank accession numbers 1-69 (NG_0010109, NT_024637, and BCO70333), 5-51 (NG_0010109 and NT_024637), 4-34 (NG_0010109 and NT_024637), 3-303 (CAJ556644), and 3-23 (AJ406678). Yet another source of human heavy and light chain germline sequences is the database of human immunoglobulin genes available from IMGT (http://imgt.cines.fr).

The preferred framework sequences for the antibodies of the present invention are framework sequences that are structurally similar to the framework sequences used by the selected antibodies of the present invention. The VH CDR1, CDR2 and CDR3 sequences and the VL CDR1, CDR2 and CDR3 sequences can be transplanted onto framework regions that are identical to the sequences found in the germline immunoglobulin genes from which the framework sequences are derived, or the CDR sequences can be transplanted onto framework regions that contain one or more mutations compared to the germline sequences. Another type of framework modification involves mutating one or more residues within the framework region or even within one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is further described in detail in U.S. Patent Publication No. 20030153043.

In addition to or instead of modifications made within the framework or CDR regions, the antibodies of the present invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the present invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter their glycosylation, thereby altering one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The residue numbering in the Fc region is that of the EU index of Kabat and/or Chothia. For example, it has been found that in some cases it is beneficial to mutate residues within the framework region to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Patents 5,530,101; 5,585,089; 5,693,762). In some aspects of the present invention, the hinge region of CH1 is modified so that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In some aspects of the invention, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment to weaken the Staphylococcal protein A (SpA) binding of the antibody relative to the native Fc hinge domain SpA binding. This method is further described in detail in U.S. Patent No. 6,165,745 to Ward et al.

In some aspects of the present invention, antibodies are modified to extend their biological half-life. A variety of methods are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375. Alternatively, to extend biological half-life, antibodies can be modified within the CH1 or CL regions to contain salvage receptor binding epitopes derived 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 to Presta et al.

In some aspects of the invention, antibodies can be recombinantly produced and expressed using any method known in the art. Alternatively, antibodies can be recombinantly produced using phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12: 433-455 (1994). Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires of unimmunized donors. According to this technology, the antibody V domain gene is cloned in frame into the major or minor coat protein gene of a filamentous phage (such as M13 or fd) and displayed on the surface of the phage particle as a functional antibody fragment. Because the filamentous particles contain single-stranded DNA copies of the phage genome, selection based on the functional properties of the antibodies also selects genes encoding antibodies that embody those properties. Thus, phages mimic some of the properties of B cells. Phage display can be performed in a variety of ways; for reviews, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Phage display can use V gene segments from several sources. Clackson et al., Nature, 352:624-628 (1991) isolated a series of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. V gene repertoires from unimmunized human donors can be constructed, and antibodies against a range of antigens (including self-antigens) can be isolated essentially according to the techniques described by Marks et al., J. Mol. Biol. 222: 581-597 (1991) or Griffith et al., EMBO J. 12: 725-734 (1993). In the natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the introduced changes will confer higher affinity, and B cells presenting high-affinity surface immunoglobulins will preferentially replicate and differentiate during subsequent antigenic attack. This natural process can be emulated using a technique called "chain shuffling" (Marks et al., Bio/Technol. 10: 779-783 (1992)). In this method, the affinity of the "primary" human antibodies obtained by phage display can then be improved by replacing the heavy and light chain V region genes with repertoires of naturally occurring variants (repertoires) of the V domain genes obtained from unimmunized donors. This technology produces antibodies and antibody fragments with affinities in the pM-nM range. The strategy for making very large phage antibody repertoires (also known as "the mother library of all libraries") has been described by Waterhouse et al., Nucl. Acids Res. 21: 2265-2266 (1993). Gene shuffling can also be used to derive human antibodies from rodent antibodies, wherein the human antibodies have affinities and specificities similar to those of the starting rodent antibodies. According to this method, also known as "epitope imprinting", the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with human V domain gene repertoires to produce rodent-human chimeras. The selection of antigens allows the isolation of human variable regions capable of restoring functional antigen binding sites, i.e., the selection of epitope-controlled (imprinted) partners. When the method is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT Publication No. WO 93/06213). Unlike traditional humanization of rodent antibodies by CDR grafting, this technology provides fully human antibodies that have no framework or CDR residues of rodent origin.

Antibodies can be recombinantly produced by first isolating antibodies and antibody-producing cells from a host animal, obtaining gene sequences, and using the gene sequences to recombinantly express the antibodies in host cells (e.g., CHO cells). Another method that can be used is to express the antibody sequences in plants (e.g., tobacco) or transgenic milk. Methods for recombinantly expressing antibodies in plants or milk have been disclosed. See, for example, Peeters et al., Vaccine 19:2756, (2001); Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65 (1995); and Pollock et al., J Immunol Methods 231:147 (1999). Methods for preparing antibody derivatives such as humanized and single-chain antibodies are known in the art.

Immunoassays and flow cytometric sorting techniques, such as fluorescence activated cell sorting (FACS), can also be used to isolate antibodies specific for CXCR4.

Antibodies as described herein can be bound to many different carriers. The carrier can be active and/or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, agarose, and magnetite. For the purposes of the present invention, the nature of the carrier can be soluble or insoluble. Those skilled in the art will know other suitable carriers for binding antibodies or will be able to determine such carriers using routine experimentation. In some aspects of the present invention, the carrier comprises a portion that targets the myocardium.

DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells are used as a preferred source of the DNA. Once isolated, the DNA can be placed in an expression vector (such as the expression vector disclosed in PCT Publication No. WO 87/04462), which is then transfected into a host cell that does not otherwise produce immunoglobulins (such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to achieve synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO 87/04462. DNA can also be modified, for example, by replacing the homologous murine sequences with the coding sequences of human heavy and light chain constant regions (Morrison et al., Proc. Nat. Acad. Sci. 81: 6851, 1984), or by covalently linking the coding sequences of all or a portion of non-immunoglobulin polypeptides to the immunoglobulin coding sequences. In this manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the CXCR4 monoclonal antibodies herein.

Methods for determining the binding specificity of anti-CXCR4 antibodies are well known to those skilled in the art. General methods are provided, for example, in Mole, "Epitope Mapping", METHODS IN MOLECULAR BIOLOGY, Vol. 10: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.), pp. 105-116 (The Humana Press, Inc. 1992).

Anti-CXCR4 antibodies or antigen-binding fragments thereof as described herein can be identified or characterized using methods known in the art, wherein a decrease in CXCR4 expression is detected and/or measured. In some aspects of the invention, anti-CXCR4 antibodies or antigen-binding fragments thereof are identified by incubating the candidate agent with CXCR4 and monitoring binding and/or the associated decrease in CXCR4 expression. Binding assays can be performed using purified CXCR4 polypeptides, or using cells that express the CXCR4 polypeptides naturally or through transfection. In one aspect, the binding assay is a competitive binding assay, wherein the ability of the candidate antibody to compete with a known anti-CXCR4 antibody or antigen-binding fragment thereof for binding to CXCR4 is assessed. The assay can be performed in a variety of ways, including ELISA.

After initial identification, the activity of the candidate anti-CXCR4 antibody or antigen-binding fragment thereof can be further confirmed and optimized by bioassays known to test the biological activity of interest. Alternatively, the candidate can be directly screened using bioassays. Several methods for identifying and characterizing anti-CXCR4 antibodies or antigen-binding fragments thereof are described in detail in the Examples.

Anti-CXCR4 antibodies or antigen-binding fragments thereof can be characterized using methods well known in the art. For example, one method is to identify the epitope to which they bind, or "epitope mapping." Many methods are known in the art for locating and characterizing the position of epitopes on proteins, including solving crystal structures of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Harlow and Lane, Using Antibodies, a Laboratory Manual, Chapter 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In another example, epitope mapping can be used to determine the sequence to which an anti-CXCR4 antibody or antigen-binding fragment thereof binds. Epitope mapping is commercially available from a variety of sources, such as Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). Epitopes can be linear epitopes, i.e., contained within a single stretch of amino acids, or conformational epitopes, formed by three-dimensional interactions of amino acids that are not necessarily contained within a single stretch. Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in binding assays with anti-CXCR4 antibodies or their antigen-binding fragments. In another example, the epitope bound by an anti-CXCR4 antibody or its antigen-binding fragment can be determined in a systematic screening by using overlapping peptides derived from the CXCR4 sequence and assaying the binding of the anti-CXCR4 antibody or its antigen-binding fragment. Based on gene fragment expression assays, the open reading frame encoding CXCR4 is segmented randomly or by specific genetic constructs, and the reactivity of the expressed fragments of CXCR4 with the antibody to be tested is determined. For example, gene fragments can be generated by PCR, then transcribed in vitro in the presence of radioactive amino acids and translated into protein. Binding of the antibody to the radiolabeled CXCR4 fragment is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, binding of a defined library of overlapping peptide fragments to a test antibody can be tested in a simple binding assay. In another example, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues that are required, sufficient, and/or essential for epitope binding. For example, domain swapping experiments can be performed using mutant CXCR4 (in which various fragments of the CXCR4 protein have been replaced (exchanged) with sequences from CXCR4 from another species (e.g., mouse), or closely related but antigenically distinct proteins (e.g., CXCR4). By evaluating antibody binding to mutant CXCR4, the importance of specific CXCR4 fragments for antibody binding can be assessed.

Another method that can be used to characterize anti-CXCR4 antibodies is to use competition assays with other antibodies known to bind to the same antigen (i.e., various fragments on CXCR4) to determine whether the anti-CXCR4 antibodies compete with and/or bind to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art.

In some aspects of the invention, the isolated antibody or antigen-binding fragment thereof that binds to CXCR4 competes for binding to CXCR4 and/or binds to the same epitope of CXCR4. Antibodies that compete for binding to CXCR4 and/or bind to the same epitope of CXCR4 include antibodies comprising at least a heavy chain variable (VH) region comprising (i) a VH CDR1 selected from the group consisting of SEQ ID NOs: 107, 113, 114, 108, 109, 115, 116, 117, 121, and 122, (ii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156, and 130, and (iii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 112 and 120. CDR3; and/or at least a light chain variable (VL) region comprising (i) a VL CDR1 selected from the group consisting of SEQ ID NOs: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150, and 151, (ii) a VLCDR2 selected from the group consisting of 145, 132, 136, and 152, and (iii) a VL CDR3 selected from the group consisting of SEQ ID NOs: 139, 133, 137, 140, and 153.

In some aspects of the invention, the antibody competes for CXCR4 binding with an antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region comprising the three CDRs set forth in SEQ ID NOs: 107, 162, and 112.

In some aspects of the invention, the antibody competes for CXCR4 binding with an antibody or antigen-binding fragment thereof comprising a light chain variable (VL) region comprising the three CDRs set forth in SEQ ID NOs: 144, 145, and 139.

In some aspects of the invention, the antibody competes for CXCR4 binding with an antibody or antigen-binding fragment thereof comprising a) a heavy chain variable (VH) region comprising the three CDRs set forth in SEQ ID NOs: 107, 162, and 112 and b) a light chain variable (VL) region comprising the three CDRs set forth in SEQ ID NOs: 144, 145, and 139.

In some aspects of the invention, the antibody competes for CXCR4 binding with an antibody or antigen-binding fragment thereof comprising a) a heavy chain variable (VH) region comprising a VH CDR1, VH CDR2, and VH CDR3 of a VH region from SEQ ID NO: 33, and b) a light chain variable (VL) region comprising a VL CDR1, VL CDR2, and VL CDR3 of a VL region from SEQ ID NO: 73.

In some aspects of the invention, the antibody competes for CXCR4 binding with an antibody or antigen-binding fragment thereof comprising a) a heavy chain variable (VH) region of SEQ ID NO: 33 and b) a light chain variable (VL) region of SEQ ID NO: 73.

Expression vectors can be used to directly express anti-CXCR4 antibodies or antigen-binding fragments thereof. Those skilled in the art are familiar with the administration of expression vectors to obtain expression of exogenous proteins in vivo. See, for example, U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471. Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheter administration, and topical administration. In another aspect of the invention, the expression vector is administered directly to the sympathetic trunk or ganglion, or to the coronary arteries, atria, ventricles, or pericardium.

Targeted delivery of therapeutic compositions containing expression vectors or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery technology is described in, for example, Findeis et al., Trends Biotechnol. 11: 202 (1993); Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, J.A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 263: 621 (1988); Wu et al., J. Biol. Chem., 269: 542 (1994); Zenke et al., Proc. Natl. Acad. Sci. USA, 87: 3655 (1990); and Wu et al., J. Biol. Chem., 266: 338 (1991). For local administration in gene therapy protocols, therapeutic compositions containing polynucleotides are administered in a range of about 100 ng to about 200 mg of DNA. During the gene therapy protocol, a DNA concentration range of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg can also be used. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vectors. Gene delivery vectors can be derived from viruses or non-viruses (see generally Jolly et al., Cancer Gene Therapy, 1:51 (1994); Kimura, Human Gene Therapy, 5:845 (1994); Connelly et al., Human Gene Therapy, 1:185 (1995); and Kaplitt, Nature Genetics, 6:148 (1994)). Endogenous mammalian or heterologous promoters can be used to induce expression of these coding sequences. The expression of the coding sequence can be constitutive or regulated.

Viral-based vectors for delivery of desired polynucleotides and expression in desired cells are well known in the art. Exemplary viral-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; British Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbisvirus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1247), and alphavirus-based vectors. VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984; and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther., 1992, 3:147 can also be used.

Non-viral delivery vectors and methods can also be used, including but not limited to polycation-condensed DNA linked or not to adenovirus that has been killed alone (see, e.g., Curiel et al., Hum. Gene Ther., 3:147 (1992)); ligand-linked DNA (see, e.g., Wu, J. et al., Biol. Chem., 264:16985 (1989)); eukaryotic cell delivery vectors (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338), and charge neutralization or fusion of the nucleus and cell membrane. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Other methods are described in Philip et al., Mol. Cell Biol., 14:2411 (1994) and Woffendin et al., Proc. Natl. Acad. Sci., 91:1581 (1994).

In some aspects of the present invention, the present invention encompasses compositions, including pharmaceutical compositions, comprising the antibodies described herein or produced by the methods described herein and having the characteristics described herein. As used herein, compositions comprise one or more antibodies that bind to CXCR4 and/or one or more polynucleotides comprising sequences encoding one or more of these antibodies. These compositions may further comprise suitable excipients well known in the art, such as pharmaceutically acceptable excipients, including buffers.

Thus, the present invention provides any of the following, or a composition (including a pharmaceutical composition) comprising any of the following sequences:

Table 2

In Table 2, the underlined sequences are CDR sequences according to Kabat, and the sequences in bold are CDR sequences according to Chothia.

The present invention also provides CDR portions of CXCR4 antibodies (including Chothia, Kabat CDRs, and CDR contact regions). Those skilled in the art are fully capable of determining CDR regions. It should be understood that in some aspects of the present invention, CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, CDRs are Kabat CDRs. In other embodiments, CDRs are Chothia CDRs. In other words, in embodiments having more than one CDR, the CDRs may be any one of Kabat, Chothia, combined CDRs, or a combination thereof. Tables 3 and 4 provide examples of CDR sequences provided herein.

Table 3

Table 4

In one aspect, the invention provides an antibody that binds to CXCR4 and/or to an antibody such as m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G10.2.25, h3G10.A59, h3G10.A62, or h3G10.L94D.

In another aspect, the antibody is expressed in combination with antibodies m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10.A18A, h3G10.A19A, h3G10.A58A, h3G10.A65A and h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G10.2.25, h3G10.A59, h3G10.A62 or h3G10.L94D compete for binding to CXCR4.

In some aspects of the invention, the antibodies compete for binding to CXCR4 with an antibody of the invention and have a monovalent antibody binding affinity ( _KD ) of about or less than about any of 6.5 nM, 6.0 nM, 5.5 nM, 5.0 nM, 4.5 nM, 4.0 nM, 3.5 nM, 3.0 nM, 2.5 nM, 2.0 nM, 1.5 nM, 1.0 nM, 0.5 nM, or 0.25 nM, as measured by surface plasmon resonance. In some aspects of the invention, the antibodies compete for binding to CXCR4 with antibody h3G10 and have a monovalent antibody binding affinity ( _KD ) of about or less than about any of 30 nM, 25 nM, 22 nM, 20 nM, 15 nM, or 10 nM.

In some aspects, the present invention also provides antibody CDR portions of anti-CXCR4 antibodies based on CDR contact regions. CDR contact regions are regions of an antibody that confer specificity to an antigen. Generally, CDR contact regions include residue positions in the CDRs and Vernier zone, which are defined to maintain the appropriate loop structure for the antibody to bind to a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. One skilled in the art is well-versed in determining CDR contact regions.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds chemokine receptor 4 (CXCR4) comprises: a heavy chain variable (VH) region comprising (i) a VH CDR1 selected from the group consisting of SEQ ID NOs: 107, 113, 114, 108, 109, 115, 116, 117, 121, and 122, (ii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156, and 130, and (iii) a VH CDR3 selected from the group consisting of SEQ ID NOs: 1 and/or b) a light chain variable (VL) region comprising (i) a VL CDR1 selected from the group consisting of SEQ ID NOs: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150, and 151, (ii) a VL CDR2 selected from the group consisting of 145, 132, 136, and 152, and (iii) a VL CDR3 selected from the group consisting of SEQ ID NOs: 139, 133, 137, 140, and 153.

In one aspect, an antibody or antigen-binding fragment thereof that binds to CXCR4 comprises a heavy chain variable (VH) CDR region comprising an amino acid sequence that is at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 107, 113, 114, 108, 109, 115, 116, 117, 121, 122; 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156, 130, 112 and 120. In one aspect, the antibody or antigen-binding fragment thereof that binds to CXCR4 comprises a light chain variable (VL) CDR region comprising an amino acid sequence that is at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150, 151, 145, 132, 136 and 152, 139, 133, 137, 140 and 153.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to CXCR4 comprises: a) a heavy chain variable (VH) region comprising a complementarity determining region selected from the group consisting of: (i) a VH CDR1 comprising a sequence set forth in SEQ ID NOs: 107, 113, 114, 108, 109, 115, 116, 117, 121, and 122; (ii) a VH CDR2 comprising a sequence set forth in SEQ ID NOs: NO: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156 and 130; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 112 or 120; and/or b) a light chain variable (VL) region comprising a complementarity determining region selected from the group consisting of: (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: NO:144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150 or 151; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO:145, 132, 136 or 152; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO:139, 133, 137, 140 or 153. In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to CXCR4 comprises: _a ) a light chain variable (VL) region comprising ₍ i) a VL CDR1 comprising the _{sequence XiSX2X3SLFNSX4X5RKNYLX6} , wherein _Xi is R or K; X2 is S or A; _X3 is W, N or Q; _X4 is H or R; _X5 is T or F, and/ _{or X6 is} A, L, N or M ₍ SEQ ID NO: 151); (ii) a VL CDR2 comprising the sequence _WASARX1S , wherein _Xi is G or E (SEQ ID NO: 152); and (iii) a VL CDR3 comprising the sequence _KQSFX1LRT , wherein Xi is N or R (SEQ ID NO: ₁₅₃ ). NO: 153); and/or b) a heavy chain variable (VH) region comprising (i) a VH CDR1 comprising the sequence of SEQ ID NO: 107, 108, 109, 113 or 114; (ii) a VH CDR2 comprising the sequence of SEQ ID NO: 157, and (iii) a VH CDR3 comprising the sequence of SEQ ID NO: 112.

In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) and comprises a heavy chain variable (VH) region sequence comprising: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVX ₁ FIRHKX ₂ NX ₃ X ₄ TX ₅ EYSTX ₆ X ₇ X ₈ GRFTISRDX ₉ SKNX ₁₀ LYLQMNSLX ₁₁ X ₁₂ EDTAVYYCAX ₁₃ DLPGFAYWGQGTLVTVSS (SEQ ID NO: 106), wherein X ₁ is G or S; X ₂ is V or A; X ₃ is G, F, K, V, T, L, or I; X ₄ is E or Y; X ₅ is T or R; X ₆ is W or S; X ₇ is D or V; X _X8 is K, T or R; _X9 is D or N; _X10 is T or S; _X11 is R or K; _X12 is A or T; and _X13 is K or R.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from: a) a heavy chain variable (VH) region having a VH CDR1 of SEQ ID NO: 107, 113, or 114, a VH CDR2 of SEQ ID NO: 162 or 128, and a VH CDR3 of SEQ ID NO: 112, and a light chain variable (VL) region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 115, 116, 117, 121, or 122, a VH CDR2 of SEQ ID NO: 118, or 119, and a VH CDR3 of SEQ ID NO: 120, and a VL region having a VH CDR1 of SEQ ID NO: 116, 117, 121, or 122, a VH CDR2 of SEQ ID NO: 118, or 119, and a VH CDR3 of SEQ ID NO: 120. c) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, 108, 109, 113 or 114, a VH CDR2 of SEQ ID NO: 110 or 111, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 131, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 133; d) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, 108, 109, 113 or 114, a VH CDR2 of SEQ ID NO: 154 or 123, and a VH CDR3 of SEQ ID NO: 112 CDR3 region, the VL region has a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VLCDR3 of SEQ ID NO: 139; and e) a VH region and a VL region, the VH region has a VH CDR1 of SEQ ID NO: 107, 108, 109, 113 or 114, a VH CDR2 of SEQ ID NO: 157, and a VH CDR3 of SEQ ID NO: 112, the VL region has a VLCDR1 of SEQ ID NO: 151, a VL CDR2 of SEQ ID NO: 152, and a VL CDR3 of SEQ ID NO: 153.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from: a) a heavy chain variable (VH) region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and a light chain variable (VL) region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VLCDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 115, a VH CDR2 of SEQ ID NO: 118, and a VH CDR3 of SEQ ID NO: 120, and a VL region having a VL CDR1 of SEQ ID NO: 135, a VL CDR2 of SEQ ID NO: 136, and a VLCDR3 of SEQ ID NO: 137. NO: 137; c) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 110, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VLCDR1 of SEQ ID NO: 131, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 133; d) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 154, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139; and e) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107 The VL region has a VL CDR1 of SEQ ID NO: 151, a VL CDR2 of SEQ ID NO: 152, and a VL CDR3 of SEQ ID NO: 153.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from: a) a heavy chain variable (VH) region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and a light chain variable (VL) region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and a VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139. : c) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 150, a VL CDR2 of SEQ ID NO: 132, and a VLCDR3 of SEQ ID NO: 140; d) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 141, a VL CDR2 of SEQ ID NO: 132, and a VLCDR3 of SEQ ID NO: 140. CDR3; e) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VLCDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140; f) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 147, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; g) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 147, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140. NO: 159 VH CDR2 and SEQ ID NO: 112 VH CDR3, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; h) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 160 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; i) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 161 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 161 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: 1 NO: 138 VL CDR1, SEQ ID NO: 132 VL CDR2 and SEQ ID NO: 140 VL CDR3; j) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; k) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140 CDR3; l) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 164, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; m) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 165, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; o) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 165, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140. NO: 166 VH CDR2 and SEQ ID NO: 112 VH CDR3, the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; p) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 167 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; q) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168 and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168 and a VH CDR3 of SEQ ID NO: 112, the VL region having a NO: 138 VL CDR1, SEQ ID NO: 132 VL CDR3 and SEQ ID NO: 140 VL CDR3; r) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VLCDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; s) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140 CDR3; t) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; u) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VLCDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; v) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VLCDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139. NO: 163, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; and w) a VH and VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising the three CDRs set forth in SEQ ID NOs: 107, 162, and 112. In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising the three CDRs set forth in SEQ ID NOs: 144, 145, and 139. In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable (VH) region comprising the three CDRs set forth in SEQ ID NOs: 107, 162, and 112; and b) a light chain variable (VL) region comprising the three CDRs set forth in SEQ ID NOs: 144, 145, and 139.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 from the VH region of SEQ ID NO: 33. In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 from the VL region of SEQ ID NO: 73. In other aspects of the invention, an isolated antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 from the VH region of SEQ ID NO: 33; and b) a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 from the VL region of SEQ ID NO: 73.

In some aspects of the invention, the isolated antibody or antigen-binding fragment thereof is selected from: a) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 33 and a VL region of SEQ ID NO: 73; b) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 13 and a VL region of SEQ ID NO: 15; c) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 5 and a VL region of SEQ ID NO: 7; d) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 21 and a VL region of SEQ ID NO: 47; and e) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 106, and a VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139.

In one aspect of the invention, the isolated antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable (VH) region of SEQ ID NO: 33; and/or b) a light chain variable (VL) region of SEQ ID NO: 73.

In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to CXCR4, wherein the antibody comprises a heavy chain variable (VH) region comprising the sequence shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 85, 87 or 106; and/or a light chain variable (VL) region comprising the sequence shown in SEQ ID NO: 3, 7, 11, 15, 19, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or 169. In one aspect, the antibody or antigen-binding fragment thereof that binds to CXCR4 comprises a heavy chain variable (VH) region comprising an amino acid sequence that is at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 85, 87 or 106. In one aspect, the antibody or antigen-binding fragment thereof that binds to CXCR4 comprises a light chain variable (VL) region comprising an amino acid sequence that is at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 7, 11, 15, 19, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or 169.

In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof of claim 6 is provided, which is selected from: a) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 33 and a VL region of SEQ ID NO: 73; b) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 13 and a VL region of SEQ ID NO: 15; c) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 5 and a VL region of SEQ ID NO: 7; d) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 21 and a VL region of SEQ ID NO: 47; and e) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 106, and a VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139.

In some aspects of the invention, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable (VH) region of SEQ ID NO: 33; and/or b) a light chain variable (VL) region of SEQ ID NO: 73. In a specific aspect, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region produced by an expression vector having ATCC Accession No. PTA-121353. In other aspects, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region produced by an expression vector having ATCC Accession No. PTA-121354.

In some aspects of the invention, an antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from: a) a heavy chain variable (VH) region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and a light chain variable (VL) region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and a VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 139. NO: 140 VL CDR3; c) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 150, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; d) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 141, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; e) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107 : f) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140; g) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 158, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 147, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140. : h) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 160, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; i) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 161, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140 CDR3; j) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; k) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; l) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140. NO: 164 VH CDR2 and SEQ ID NO: 112 VH CDR3 region, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; m) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 165, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; n) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 166, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 166, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR1 of SEQ ID NO: NO: 138, a VL CDR1 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; o) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 167, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; p) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140 CDR3; q) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 168, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 138, a VL CDR2 of SEQ ID NO: 132, and a VL CDR3 of SEQ ID NO: 140; r) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140; s) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, and the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140. NO: 158, and a VH CDR2 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; t) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139; u) a VH region and a VL region, the VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 163, and a VH CDR3 of SEQ ID NO: 112, the VL region having a VH CDR3 of SEQ ID NO: 1 NO: 144, a VL CDR1 of SEQ ID NO: 145, a VL CDR2 of SEQ ID NO: 139, and v) a VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and a VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 140.

In a specific aspect of the invention, the antibody or antigen-binding fragment thereof comprises a VH region having a VH CDR1 of SEQ ID NO: 107, a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NO: 112, and a VL region having a VL CDR1 of SEQ ID NO: 144, a VL CDR2 of SEQ ID NO: 145, and a VL CDR3 of SEQ ID NO: 139.

The anti-CXCR4 antibodies or antigen-binding fragments thereof as described herein have a binding affinity ( _KD ) for CXCR4, such as human CXCR4, of about 0.002 to about 200 nM. In some aspects of the invention, the binding affinity is any of about 200 nM, about 100 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 2 pM. In some aspects of the invention, the binding affinity is less than any of about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM, or about 2 pM.

In some aspects of the invention, the binding affinity of the antibodies as described herein (e.g., monovalent antibody binding) as measured by surface plasmon resonance is about 35 nM or less. In some aspects of the invention, the binding affinity of the antibodies as described herein (e.g., monovalent antibody binding) as measured by surface plasmon resonance is about 6.5 nM or less.

The present invention also provides methods for preparing any of these antibodies. The antibodies of the present invention can be manufactured by procedures known in the art. The polypeptides can be produced by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides), or by chemical synthesis. Polypeptides of antibodies, especially shorter polypeptides of up to about 50 amino acids, are conveniently manufactured by chemical synthesis. Chemical synthesis methods are known in the art and can be provided commercially. For example, antibodies can be produced by an automated polypeptide synthesizer using a solid phase method. See also U.S. Patent Nos. 5,807,715; 4,816,567; and 6,331,415.

In another aspect of the invention, antibodies can be recombinantly produced using procedures well known in the art. In another aspect, the polynucleotides comprising the polynucleotides encoding antibodies m6B6, h6B6, m12A11, h12A11, m3G10, h3G10(VH), h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10.2.54, h3G10.A59, h3G10.A62, h3G10(VL), h3G10. The sequences encoding the heavy and/or light chain variable regions of h3G10.2.72, h3G10.A11A, h3G10.A18A, h3G10.A19A, h3G10.A58A, h3G10.A65A, and h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G10.2.25, or h3G10.L94D can be maintained in vectors in host cells, which can then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

The present invention also encompasses scFvs of the antibodies of the present invention. Single-chain variable region fragments are prepared by connecting the light chain variable region and/or the heavy chain variable region using a short connecting peptide (Bird et al., Science 242:423-426, 1988). An example of a connecting peptide is (GGGGS)3 (SEQ ID NO:89), which bridges approximately 3.5 nm between the carboxyl terminus of one variable region and the amino terminus of the other variable region. Linkers with other sequences have been designed and used (Bird et al., 1988, supra). The linker should be a short, flexible polypeptide and preferably contains less than about 20 amino acid residues. The linker can then be modified for additional functions, such as attaching drugs or attaching to a solid support. Single-chain variants can be produced recombinantly or synthetically. To synthetically produce scFv, an automated synthesizer can be used. To recombinantly produce scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into the amino terminus of the other variable region in a suitable host cell (eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic). Other sequence joints have been designed and used (Bird et al., 1988). The joint can be modified to have additional functions, such as attachment of drugs or attachment to solid supports. Single-chain variants can be produced recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For the recombinant production of scFv, an appropriate plasmid containing a polynucleotide encoding the scFv can be introduced into an appropriate host cell, which is a eukaryotic cell, such as a yeast, plant, insect or mammalian cell, or a prokaryotic cell such as Escherichia coli. The polynucleotide encoding the relevant scFv can be prepared by conventional manipulations such as polynucleotide ligation. The obtained scFv can be isolated using standard protein purification techniques known in the art.

Other forms of single-chain antibodies are also encompassed, such as diabodies or minibodies. Diabodies are bivalent bispecific antibodies in which the heavy chain variable (VH) and light chain variable (VL) domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and generate two antigen-binding sites (see, e.g., Holliger, P. et al., Proc. Natl. Acad Sci. USA 90: 6444-6448 (1993); Poljak, R.J. et al., Structure 2: 1121-1123 (1994)). Minibodies include the VL and VH domains of a natural antibody fused to the hinge region and CH3 domain of an immunoglobulin molecule. See, e.g., U.S. Patent No. 5,837,821.

Bispecific antibodies are antibodies that can simultaneously bind to two different targets. Bispecific antibodies (bsAbs) and bispecific antibody fragments (bsFabs) can have at least one arm that binds, for example, a tumor-associated antigen and at least one other arm that binds a targetable conjugate carrying a therapeutic or diagnostic agent. A variety of bispecific fusion proteins can be produced using molecular engineering.

For example, the antibodies disclosed herein can be used to prepare bispecific antibodies, which are monoclonal antibodies that have binding specificities for at least two different antigens. Methods for making bispecific antibodies are known in the art (see, for example, Suresh et al., 1986, Methods in Enzymology 121:210). Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two heavy chains have different specificities (Millstein and Cuello, Nature 305, 537-539 (1983)).

According to a method for making bispecific antibodies, the antibody variable domain with the desired binding specificity (antibody-antigen combination site) is fused with an immunoglobulin constant region sequence. The fusion preferably has an immunoglobulin heavy chain constant region comprising at least a portion of a hinge, CH2, and CH3 region. Preferably, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusions and, if necessary, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual ratio of the three polypeptide fragments in the embodiment where the unequal ratios of the three polypeptide chains used in the construction provide the best yield. However, when the expression of at least two polypeptide chains of equal ratios produces high yields or when the ratio is not particularly important, the coding sequences of two or all three polypeptide chains can be inserted into one expression vector.

In some aspects of the invention, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure, with an immunoglobulin light chain in only half of the bispecific molecule, facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This approach is described in PCT Publication WO 1994/004690.

In another aspect, bispecific antibodies are constructed by modifying the amino acids in the first hinge region of one arm, wherein the substituted/replaced amino acids in the first hinge region have a charge opposite to that of the corresponding amino acids in the second hinge region of the other arm. This approach is described in PCT Publication No. WO 2011/143545.

In another aspect, bispecific antibodies can be produced in the presence of transglutaminase using a glutamine-containing peptide tag engineered into an antibody against an epitope (e.g., CXCR4) in one arm and another peptide tag (e.g., a Lys-containing peptide tag or reactive endogenous Lys) engineered into a second antibody against a second epitope in the other arm. This approach is described in PCT Publication No. WO 2012/059882.

Heteroconjugate antibodies comprising two covalently linked antibodies are also within the scope of the present invention. Such antibodies have been used to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and for treating HIV infection (PCT Publication No. 1991/000360). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are described in U.S. Patent No. 4,676,980.

Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

The present invention encompasses antibodies and polypeptides of the variants of the present invention shown in Table 2, including functionally equivalent antibodies that do not significantly affect their characteristics, and variants with enhanced or reduced activity and/or affinity. For example, the amino acid sequence may be mutated to obtain an antibody with a desired binding affinity to CXCR4. Polypeptide modification is a routine operation in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative sequence modifications. As used herein, the term "conservative sequence modification" means an amino acid modification that does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions that do not significantly adversely change the functional activity or mature (enhance) the affinity of the polypeptide for its ligand, or the use of chemical analogs.

Standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, can be utilized to introduce modifications into the antibodies of the present invention. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. The family includes amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for retained function (ie, the function described above) using the functional assays described herein.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to epitope tags. Other insertional variants of antibody molecules include fusions of the N- or C-terminus of antibodies to enzymes or polypeptides, which increase the half-life of the antibody in the blood circulation.

Substitution variants remove at least one amino acid residue in the antibody molecule and insert different residues in its place. The most notable substitution mutagenesis sites include hypervariable regions, but also encompass FR changes. Conservative substitutions are shown under the heading "conservative substitutions" in Table 5. If the substitutions result in changes in biological activity, more substantial changes (named "exemplary substitutions" in Table 5, or as further described below with reference to amino acid classes) can be introduced, and the product screened.

Table 5

Amino acid substitution

Substantial modification of the biological properties of antibodies can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, e.g., a sheet or helical conformation; (b) charge or hydrophobicity at the molecular target site; or (c) side chain bulk. Naturally occurring amino acid residues are grouped based on common side chain properties:

(1) Non-polar: norleucine, Met, Ala, Val, Leu, Ile;

(2) Polarity, no charge: Cys, Ser, Thr, Asn, Gln;

(3) Acidic (negatively charged): Asp, Glu;

(4) Basic (positively charged): Lys, Arg;

(5) Residues that affect chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

Any cysteine residues not involved in maintaining the proper conformation of the antibody may also be substituted, typically with serine, to increase the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bond(s) may be added to the antibody to increase its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one or more amino acids to completely redesigning regions such as variable regions. Changes in variable regions can alter binding affinity and/or specificity. In some aspects of the invention, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other aspects of the invention, no more than one to three conservative amino acid substitutions are made within a CDR domain.

Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, Chem. Immunol. 65: 111-128 (1997); Wright and Morrison, TibTECH 15: 26-32 (1997)). The oligosaccharide side chains of immunoglobulins influence protein function (Boyd et al., Mol. Immunol. 32: 1311-1318 (1996); Wittwe and Howard, Biochem. 29: 4175-4180 (1990)) and intramolecular interactions between glycoprotein moieties, which can affect the conformation and three-dimensional surface presentation of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech. 7: 409-416 (1996)). Oligosaccharides can also be used to target a given glycoprotein to certain molecules based on specific recognition structures. Glycosylation of antibodies has also been reported to influence antibody-dependent cellular cytotoxicity (ADCC). Specifically, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisecting GlcNAc, have been reported to have enhanced ADCC activity (Umana et al., Mature Biotech 17:176-180 (1999)).

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars sucrose N-acetylgalactamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be accomplished by adding one or more serine or threonine residues to the sequence of the original antibody, or substituting one or more serine or threonine residues for one or more serine or threonine residues (for O-linked glycosylation sites).

The glycosylation pattern of an antibody can also be altered without changing the underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Because the cell types used to express recombinant glycoproteins (e.g., antibodies) as potential therapeutic agents are rarely native cells, variations in the glycosylation pattern of antibodies can be expected (see, e.g., Hse et al., J. Biol. Chem. 272:9062-9070 (1997)).

In addition to the choice of host cells, factors that affect glycosylation during recombinant antibody production include growth mode, medium formulation, culture density, oxygenation, pH, purification schemes, and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. Patents 5,047,335; 5,510,261 and 5,278,299). Glycosylation or certain types of glycosylation can be enzymatically removed from glycoproteins, for example, using endoglycosidase H (EndoH), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, recombinant host cells can be genetically engineered to be defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

Another modification of the antibodies herein encompassed by the present invention is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or its fragment is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is performed via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similarly reactive water-soluble polymer). The term "polyethylene glycol" as used herein is intended to encompass any form of PEG that has been used to derive other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain aspects of the present invention, the antibody to be pegylated is a deglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EP 0154316 to Nishimura et al. and EP 0401384 to Ishikawa et al.

The present invention is not limited to traditional antibodies and includes the use of antibody fragments and antibody mimics. Various antibody fragments and antibody mimicking technologies have been developed and are known in the art. Although many of these technologies, such as domain antibodies, nanobodies, and monoclonal antibodies, utilize fragments or other modifications of traditional antibody structures, there are alternative technologies, such as affibodies, DARPins, high-affinity multimers, anticalins, and universal antibodies that employ binding structures that mimic traditional antibody binding but are produced and act via unique mechanisms.

Domain antibodies (dAbs) are the smallest functional binding units of antibodies, corresponding to the variable regions of the heavy (VH) or light (VL) chains of human antibodies. Domantis has developed a series of large and highly functional libraries of fully human VH and VL dAbs (over 10 billion different sequences in each library) and uses these libraries to select dAbs specific for therapeutic targets. Compared to many conventional antibodies, domain antibodies express well in bacterial, yeast, and mammalian cell systems. Further details of domain antibodies and methods for their production can be found in U.S. Patent Nos. 6,291,158, 6,582,915, 6,593,081, 6,172,197, and 6,696,245; U.S. Patent Application No. 2004/0110941; European Patent Application No. 1433846 and European Patents 0368684 and 0616640, WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019, and WO03/002609, each of which is incorporated herein by reference in its entirety.

Nanobodies are therapeutic proteins derived from antibodies that contain the unique structure and functional properties of naturally occurring heavy chain antibodies. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). The cloned and isolated VHH domains are very stable polypeptides with the complete antigen binding ability of the original heavy chain antibody. Nanobodies have a high degree of homology to the VH domains of human antibodies and can be further humanized without any loss of activity. In addition, nanobodies combine the advantages of conventional antibodies with the important features of small molecule drugs similar to conventional antibodies. Nanobodies display high target specificity, high affinity for their targets, and low inherent toxicity. Nanobodies are encoded by a single gene and are efficiently produced in nearly all prokaryotic and eukaryotic hosts, such as Escherichia coli (see, e.g., U.S. Patent No. 6,765,087, incorporated herein by reference in its entirety), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyce, Kluyveromyce, Hansenula, or Pichia) (see, e.g., U.S. Patent No. 6,838,254, incorporated herein by reference in its entirety).

In some aspects, the present invention provides the use of monoclonal antibodies. Monoclonal antibodies are another antibody fragment technology based on the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region will produce a molecule that is substantially half the size of a traditional IgG4 antibody and has a monovalent binding region rather than the bivalent binding region of an IgG4 antibody. It is also known that IgG4 antibodies are inert and thus do not interact with the immune system, which is beneficial for treating diseases that do not want an immune response, and this benefit is passed on to monoclonal antibodies. For example, monoclonal antibodies can be used to inhibit or silence but do not kill the cells to which they bind. In addition, monoclonal antibodies bind to cancer cells without stimulating their proliferation. In addition, because monoclonal antibodies are half the size of traditional IgG4 antibodies, they can be shown to have a better distribution on larger solid tumors, with potentially beneficial effects. Monoclonal antibodies are cleared from the body at a rate similar to that of full IgG4 antibodies and can bind to their antigens with an affinity similar to that of full antibodies. Further details of monoclonal antibodies can be obtained with reference to PCT Publication WO 2007/059782, which is incorporated herein by reference in its entirety.

In some aspects, the present invention encompasses affibody molecules. Affibody molecules are new affinity protein classes, based on a protein domain of 58 amino acid residues derived from an IgG binding domain of staphylococcal protein A. These three helical bundle domains have been used as the skeleton for combinatorial phagemid library construction, from which phage display technology can be used to select affibody variants targeting desired molecules (Nord K, Gunnettsson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA, Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain, Nat Biotechnol 15 772-7 (1997); Ronmark J, Gronlund H, Uhlen M, Nygren PA, Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A, Eur J Biochem., 269 2647-55 (2002)). The simple, robust structure of the affibody molecule, coupled with its low molecular weight (6 kDa), makes it suitable for a variety of applications, such as as a detection reagent (Ronmark J, Hansson M, Nguyen T et al., Construction and characterization of affibody-Fc chimeras produced in Escherichia coli, J Immunol Methods, 261 199-211 (2002)), and inhibition of receptor interactions (Sandstorm K, Xu Z, Forsberg G, Nygren PA, Inhibition of the CD28-CD80 co-stimulation signal by aCD28-binding Affibody ligand developed by combinatorial protein engineering, Protein Eng., 16 691-7 (2003)). Further details of affibodies and methods for their production can be obtained by reference to U.S. Pat. No. 5,831,012, which is incorporated herein by reference in its entirety.

Another antibody mimetic technology suitable for use in the context of the present invention is high-affinity multimers. High-affinity multimers are derived from a large family of human extracellular receptor domains, generated through in vitro exon shuffling and phage display to produce multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains has been shown to generate avidity and improve affinity and specificity compared to conventional single epitope binding proteins. Other potential advantages include simple and efficient production of multi-target specific molecules in E. coli, improved thermal stability, and resistance to proteases. High-affinity multimers with sub-nanomolar affinities have been obtained for a variety of targets. Additional information related to high-affinity multimers can be found in U.S. Patent Application Publication Nos. 2006/0286603, 2006/0234299, 2006/0223114, 2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932, 2005/0053973, 2005/0048512, and 2004/0175756, all of which are incorporated herein by reference in their entireties.

Universal antibodies are another antibody mimetic technology that can be used in the context of the present invention. Universal antibodies are small proteins with >15% cysteine of 3-5kDa, which form a high disulfide density skeleton, replacing the hydrophobic core possessed by typical proteins. Replacing the large amount of hydrophobic amino acids that constitute the hydrophobic core with a few disulfides makes the protein smaller, more hydrophilic (less aggregation and non-specific binding), more resistant to proteases and heat, and has a lower T cell epitope density, because the residues that are most conducive to MHC presentation are hydrophobic. It is well known that all of these characteristics affect immunogenicity, and together, it is expected that they cause a significant reduction in immunogenicity. Other information related to universal antibodies can be found in U.S. Patent Application Publication No. 2007/0191272, which is incorporated herein by reference in its entirety.

The detailed description of antibody fragment and antibody mimetic technology provided above is not intended to be a comprehensive list of all technologies that can be used in the context of the present specification. For example, and without limitation, a variety of other technologies can be used in the context of the present invention, including alternative polypeptide-based technologies, such as fusions of complementary determining regions as summarized in Qui et al., Nature Biotechnology, 25(8)921-929 (2007) (which is incorporated herein by reference); and nucleic acid-based technologies, such as RNA aptamer technology described in U.S. Patents Nos. 5,789,157; 5,864,026; 5,712,375; 5,763,566; 6,013,443; 6,376,474; 6,613,526; 6,114,120; 6,261,774 and 6,387,620 (all incorporated herein by reference).

Other modification methods include the use of coupling techniques known in the art, including but not limited to enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, for the attachment of labels for immunoassays. Modified polypeptides are prepared using established procedures in the art and can be screened using standard assays known in the art, some of which are described below and in the Examples.

In some aspects of the invention, the antibodies comprise modified constant regions, such as those with increased affinity for human Fcγ receptors, immunologically inert or partially inert, for example, constant regions that do not trigger complement-mediated lysis, do not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or inactivate macrophages; or constant regions that have reduced activity in any one or more of the following (compared to unmodified antibodies): triggering complement-mediated lysis, stimulating antibody-dependent cell-mediated cytotoxicity (ADCC), or activating microglia. Different modifications of the constant region can be used to achieve the best level and/or combination of effector functions. See, e.g., Morgan et al., Immunology 86:319-324 (1995); Lund et al., J. Immunology 157:4963-4969 (1996); Idusogie et al., J. Immunology 164:4178-4184, (2000); Tao et al., J. Immunology 143:2595-2601 (1989); and Jefferis et al., Immunological Reviews 163:59-76 (1998). In some aspects of the invention, the constant region is modified as described in Eur. J. Immunol., 29:2613-2624 (1999); PCT Publication No. WO 1999/058572. In other aspects of the invention, the antibody comprises a human heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild-type IgG2 sequence). Eur. J. Immunol., 29: 2613-2624 (1999). In other aspects of the invention, the constant region is deglycosylated for N-linked glycosylation. In some aspects of the invention, the constant region is deglycosylated for N-linked glycosylation by mutating the glycosylated amino acid residue or the flanking residues that are part of the N-glycosylation recognition sequence in the constant region. For example, N-glycosylation site N297 can be mutated to A, Q, K, or H. See Tao et al., J. Immunology 143: 2595-2601 (1989); and Jefferis et al., Immunological Reviews 163: 59-76 (1998). In some aspects of the invention, the constant region is deglycosylated for N-linked glycosylation. The constant region can be deglycosylated for N-linked glycosylation enzymatically (such as by removal of carbohydrates by the enzyme PNGase) or by expression in a glycosylation-deficient host cell.

Other antibody modifications include antibodies modified as described in PCT Publication No. WO 99/58572. In addition to the binding domain for the target molecule, these antibodies also include an effector domain having an amino acid sequence substantially homologous to all or part of the constant domain of a human immunoglobulin heavy chain. These antibodies are able to bind to the target molecule without triggering significant complement-dependent lysis or cell-mediated target destruction. In some aspects of the invention, the effector domain is able to specifically bind to FcRn and/or FcγRIIb. These effector domains are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this way are particularly suitable for use in chronic antibody therapy to avoid inflammation and other adverse reactions of conventional antibody therapy.

The present invention includes affinity matured antibodies. For example, affinity matured antibodies can be produced by procedures known in the art (Marks et al., Bio/Technology, 10:779-783 (1992); Barbas et al., Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-9 (1995), Hawkins et al., J. Mol. Biol., 226:889-896 (1992); and PCT Publication No. WO2004/058184).

The following methods can be used to regulate the affinity of antibodies and to characterize CDRs. A method for characterizing the binding affinity of a CDR and/or a polypeptide such as an antibody, such as a change (such as improving) is referred to as "library scanning mutagenesis". Generally speaking, library scanning mutagenesis works as follows: using methods recognized in the art, one or more amino acid positions in a CDR are replaced by two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids. This produces a small library of clones (in some aspects of the invention, one amino acid position for each analysis), each library having the complexity of two or more members (if each position is replaced by two or more amino acids). Generally speaking, the library also includes clones comprising natural (unsubstituted) amino acids. A small number of clones (e.g., about 20-80 clones (depending on the complexity of the library)) from each library are screened for their binding affinity to the target polypeptide (or other binding target), and candidates with increased, equal, decreased, or no binding are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using Biacore ^™ surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or greater. Biacore ^™ is particularly useful when the starting antibody already binds with relatively high affinity, e.g., a _KD of about 10 nM or less. Screening using Biacore ^™ surface plasmon resonance is described in the Examples herein.

In another aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CXCR4, wherein the antibody or antigen-binding fragment thereof has an EC50 of less than 100 nM. In yet another aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CXCR4, wherein the antibody or antigen-binding fragment thereof has an EC50 of less than 50 nM. In yet another aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CXCR4, wherein the antibody or antigen-binding fragment thereof has an EC50 of less than 1 nM. The term "EC50" refers to the concentration required to inhibit 50% of the maximal effect of a test antibody, antigen-binding fragment thereof, or antibody-drug conjugate in vitro. The term "IC50" meaning is defined as "50% inhibitory concentration."

Binding affinity can be determined using Kinexa Biocensor, scintillation proximity assay, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinity can also be screened using a suitable bioassay.

In some aspects of the invention, each amino acid position in a CDR is replaced (in some embodiments, one at a time) with all 20 naturally occurring amino acids using art-recognized mutagenesis methods (some of which are described herein). This generates small libraries of clones (in some aspects of the invention, one for each amino acid position analyzed), each library having a complexity of 20 members (if each position is replaced with all 20 amino acids).

In some aspects of the invention, the library to be screened comprises substitutions at two or more positions that may be in the same CDR or in two or more CDRs. Thus, the library may comprise substitutions at two or more positions in one CDR. The library may comprise substitutions at two or more positions in two or more CDRs. The library may comprise substitutions at 3, 4, 5 or more positions, wherein the positions are present in two, three, four, five or six CDRs. The substitutions are prepared using low redundancy codons. See, for example, Table 2 of Balint et al., Gene 137(1):109-18 (1993).

The CDR may be CDRH3 and/or CDRL3. The CDR may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR may be a Kabat CDR, a Chothia CDR, or an extended CDR.

Candidates with improved binding can be sequenced, thereby identifying CDR substitution mutants that result in improved affinity (also referred to as substitution "improvement"). Candidates that bind can also be sequenced, thereby identifying CDR substitutions that retain binding.

Multiple rounds of screening can be performed. For example, candidates with improved binding (each comprising an amino acid substitution at one or more positions in one or more CDRs) are also suitable for designing a second library containing at least the original and substituted amino acids at each improved CDR position (i.e., an amino acid position in a CDR for which the substitution mutant exhibits improved binding). Preparation and screening or selection of such libraries is discussed further below.

Library scanning mutagenesis also provides a way to characterize CDRs, and in terms of the frequency of clones with improved binding, identical binding, reduced binding, or no binding, information related to the importance of each amino acid position to the stability of the antibody-antigen complex is also provided. For example, if the position of a CDR retains binding when changed to all 20 kinds of amino acids, the position is identified as being unlikely to be the position required for antigen binding. On the contrary, if the position of a CDR retains binding in only a small percentage of substitutions, the position is identified as a position important to CDR function. Therefore, the library scanning mutagenesis method generates information related to positions in the CDR that can be changed to a variety of different amino acids (including all 20 kinds of amino acids) and positions in the CDR that cannot be changed or can only be changed to a few amino acids.

Candidates with improved affinity can be combined in a second library comprising the improved amino acid, the original amino acid at that position, and, depending on the complexity of the library desired or permitted by the screening or selection method to be used, further comprising additional substitutions at that position. Additionally, if desired, adjacent amino acid positions can be randomized to at least two or more amino acids. Randomization of adjacent amino acids can allow for additional conformational flexibility in the mutant CDRs, which in turn can allow or facilitate the introduction of a larger number of improving mutations. The library can also comprise substitutions at positions that did not demonstrate improved affinity in the first round of screening.

The second library is screened or selected for library members with improved and/or altered binding affinity using any method known in the art, including screening using Biacore ^™ surface plasmon resonance analysis and selection using any method known in the art for selection techniques, including phage display, yeast display, and ribosome display.

The present invention also encompasses fusion proteins comprising one or more fragments or regions from an antibody of the present invention. In some aspects of the present invention, a fusion polypeptide is provided comprising at least 10 contiguous amino acids of the VH region set forth in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 85, 87, and 106; and/or at least 10 amino acids of the VL region set forth in SEQ ID NOs: 3, 7, 11, 15, 19, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, and 169. In various aspects of the invention, fusion polypeptides are provided that comprise at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of a variable light chain region and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of a variable heavy chain region. In another aspect, the fusion polypeptide comprises a light chain variable region and/or a heavy chain variable region as shown in any of the sequence pairs in Table 2. For example, SEQ ID NOs: 3 and 5; 3 and 9; 7 and 5; 11 and 13; 15 and 13; 19 and 17; 69 and 31; 49 and 37; and 33 and 73. In some aspects, the fusion polypeptide comprises one or more CDRs. In other aspects, the fusion polypeptide comprises H3 (VH CDR3) and/or CDRL3 (VL CDR3). For the purposes of the present invention, a fusion protein contains one or more antibodies and another amino acid sequence to which they are not attached in the native molecule, such as a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags," such as FLAG tags or 6His tags. Tags are well known in the art.

Fusion polypeptides can be produced by methods known in the art, for example, by synthesis or recombinant means. Although fusion proteins of the present invention can also be prepared by other means known in the art, for example, by chemical synthesis, fusion proteins of the present invention are typically manufactured by recombinant methods as described herein, by preparing and expressing polynucleotides encoding them.

The present invention also provides compositions comprising an antibody bound (e.g., linked) to an agent that facilitates coupling to a solid support, such as biotin or antibiotic protein. For simplicity, when referring generally to antibodies, it is generally understood that these methods are applicable to anti-CXCR4 antibodies or antigen-binding fragments thereof, or any of the embodiments described herein. Binding generally refers to connecting these components as described herein. The connection (which generally fixes these components in close association, at least for administration) can be achieved in a variety of ways. For example, when each has a substituent capable of reacting with the other, a direct reaction between the agent and the antibody is possible. For example, nucleophilic groups such as amino or sulfhydryl groups can react with carbonyl-containing groups such as anhydrides or acyl halides on the one hand, or with alkyl groups containing good leaving groups (e.g., halides) on the other hand.

The present invention also provides isolated polynucleotides encoding the antibodies of the present invention, as well as vectors and host cells comprising the polynucleotides.

Thus, the present invention provides polynucleotides (or compositions, including pharmaceutical compositions) comprising a polynucleotide encoding any of the following: m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10. 3G10.A18A, h3G10.A19A, h3G10.A58A, h3G10.A65A and h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G10.2.25, h3G10.A59, h3G10.A62, h3G10.L94D, or any fragment or portion thereof that is capable of binding to CXCR4.

In some aspects, the present invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides with reduced or enhanced effector function. Polynucleotides can be made and expressed by procedures known in the art.

In another aspect, the present invention provides compositions (such as pharmaceutical compositions) comprising any of the polynucleotides of the present invention. In some aspects, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies described herein. For example, the composition comprises one or two of the polynucleotides set forth in SEQ ID NOs: 2 and 4, or SEQ ID NOs: 6 and 8, or SEQ ID NOs: 86 and 68.

Administration of expression vectors and polynucleotide compositions is further described herein.

In other aspects, the invention provides a method of making any of the polynucleotides described herein.

The present invention also encompasses polynucleotides complementary to any of these sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner, and mRNA molecules that do not contain introns. Other coding or non-coding sequences may (but need not) be present within the polynucleotides of the present invention, and polynucleotides may (but need not) be linked to other molecules and/or support materials.

The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions, and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, yet more preferably at least about 90% identity, and optimally at least about 95% identity to a polynucleotide sequence encoding a native antibody or a portion thereof.

Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the sequences is the same when aligned for maximum identity as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions (generally 30 to about 75, 40 to about 50) over which two sequences can be compared after optimal alignment of a sequence with a reference sequence having the same number of contiguous positions.

Optimal alignment of sequences for comparison can be performed using the Megalign program of the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Suppl 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645; Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., CABIOS 5: 151-153 (1989); Myers, E.W. and Muller W., CABIOS 5: 151-153 (1989); 4:11-17(1988); Robinson, E.D., Comb.Theor.11:105(1971); Santou, N., Nes, M., Mol.Biol.Evol.4:406-425(1987); Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., Proc. Natl. Acad. Sci. USA 80:726-730 (1983).

Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, typically 5% to 15% or 10% to 12% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues appear in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

Variants may also be (or) substantially homologous to a native gene or a portion or complement thereof. These polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or complementary sequence).

Suitable "moderately stringent conditions" include a prewash in 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C-65°C, 5X SSC; followed by washing twice each with 2X, 0.5X and 0.2X SSC containing 0.1% SDS at 65°C for 20 minutes.

As used herein, "high stringency conditions" or "high stringency conditions" are those that (1) employ low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ a denaturing agent, such as formamide, e.g., 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer, pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate at 42°C during hybridization; or (3) employ 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution (pH 6.9). Solution), sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., and a wash in 0.2× SSC (sodium chloride/sodium citrate) and 50% formamide at 42° C., followed by a high stringency wash consisting of 0.1× SSC containing EDTA at 55° C. Those skilled in the art will know how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length and the like.

Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, there are a variety of nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that differ due to differences in codon usage are specifically encompassed in the present invention. In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that have changed due to one or more mutations such as nucleotide deletions, additions and/or substitutions. Gained mRNA and protein may (but not necessarily) have a changed structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and/or database sequence comparisons).

Polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods or PCR. The method of chemical polynucleotide synthesis is well known in the art and does not need to be described in detail herein. Those skilled in the art can use the sequence provided herein and commercial DNA synthesizer to produce the required DNA sequence.

To prepare polynucleotides using recombinant methods, polynucleotides comprising the desired sequence can be inserted into a suitable vector, and the vector can then be introduced into a suitable host cell for replication and amplification, as further discussed herein. The polynucleotides can be inserted into the host cell by any means known in the art. The cells are transformed by introducing exogenous polynucleotides using direct uptake, endocytosis, transfection, F-pairing, or electroporation. Once introduced, the exogenous polynucleotides can be maintained in the cell as an unintegrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotides thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

Alternatively, PCR allows for the replication of a DNA sequence. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and in PCR: The Polymerase Chain Reaction, ed. Mullis et al., Birkauswer Press, Boston, 1994.

RNA is obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. Once the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, such as those described in Sambrook et al., 1989, supra.

Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector can vary depending on the host cell to be used, suitable cloning vectors will generally be able to self-replicate, may have a single target for a specific restriction endonuclease, and/or may carry genes for markers that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBSSK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the present invention. This means that the expression vector must be replicable in the host cell as an episome or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, retroviruses), cosmids, and expression vectors disclosed in PCT Publication No. WO 87/04462. Vector components generally may include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons, are also generally required.

A vector containing a polynucleotide of interest can be introduced into a host cell by any of a number of appropriate means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide to be introduced will often depend on the characteristics of the host cell.

The present invention also provides host cells comprising any of the polynucleotides described herein. To achieve the purpose of isolating genes encoding the relevant antibodies, polypeptides, or proteins, any host cell capable of overexpressing heterologous DNA can be used. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisiae, S. pombe, or Kluyveromyces lactis). The host cells preferably express the cDNA at a level that is approximately 5-fold, more preferably 10-fold, and even more preferably 20-fold higher than the level of the corresponding endogenous antibody or protein (if present) in the host cell. Screening of host cells for specific binding to CXCR4 or CXCR4 domains (e.g., domains 1-4) is accomplished by immunoassay or FACS. Cells that overexpress the relevant antibody or protein can be identified.

Anti-CXCR4 antibody-drug conjugate

The present invention provides an antibody-drug conjugate of the formula Ab-(T-L-D), wherein a) Ab is an antibody or antigen-binding fragment thereof that binds to CXCR4, b) T is a tag that may optionally include an acyl donor glutamine; c) L is a linker; and d) D is a drug. Also provided are methods for preparing and manufacturing such antibody-drug conjugates, as well as their use in clinical applications. An "antibody-drug conjugate" or "ADC" refers to an antibody or antigen-binding fragment thereof that includes an antibody derivative that binds to CXCR4 and is conjugated to a drug.

In some aspects of the invention, Ab is selected from: a) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: 107, 162, and 112, and the VL region comprises the CDRs of SEQ ID NOs: 144, 145, and 139; b) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: 115, 118, and 120, and the VL region comprises the CDRs of SEQ ID NOs: 135, 136, and 137; c) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: 107, 110, and 112, and the VL region comprises the CDRs of SEQ ID NOs: 131, 132, and 133; d) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, wherein the VH region comprises the CDRs of SEQ ID NOs: NO: 107, 157 and 112, and the VL region contains the CDRs of SEQ ID NO: 138, 132 and 139; e) an antibody or antigen-binding fragment thereof comprising a VH region and a VL region, the VH region containing the CDRs of SEQ ID NO: 107, 157 and 112, and the VL region containing the CDRs of SEQ ID NO: 151, 152 and 153; f) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 33 and a VL region of SEQ ID NO: 73; g) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 13 and a VL region of SEQ ID NO: 15; h) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 5 and a VL region of SEQ ID NO: 7; and i) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO: 21 and a VL region of SEQ ID NO: 47.

Methods for conjugating cytotoxic agents or other therapeutic agents to antibodies have been described in various publications. For example, chemical modification can be performed in antibodies via lysine side chain amines or via cysteine sulfhydryl groups activated by reducing interchain disulfide bonds to allow conjugation reactions to occur. See, for example, Tanaka et al., FEBS Letters 579:2092-2096, (2005) and Gentle et al., Bioconjug. Chem. 15:658-663, (2004). Reactive cysteine residues modified at specific antibody sites for specific drug conjugation with defined stoichiometry have also been described. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, (2008). Conjugation using a tag containing the acyl donor glutamine and/or rendering reactive endogenous glutamine (i.e., capable of forming a covalent bond as an acyl donor) by polypeptide engineering in the presence of a transglutaminase and an amine (e.g., a cytotoxic agent comprising or attached to a reactive amine) is also described in International Patent Application No. PCT/IB2011/054899 (WO2012/059882); Strop et al., Chem. Biol. 20(2):161-167 (2013); and Farias et al., Bioconjug. Chem. 25(2):245-250 (2014).

One example of a suitable conjugation procedure relies on the conjugation of hydrazides and other nucleophiles to aldehydes generated by oxidation of naturally occurring carbohydrates on antibodies. Hydrazone-containing conjugates can be made with an introduced carbonyl group that provides the desired drug release characteristics. Conjugates can also be made with linkers that have a disulfide at one end, an alkyl chain in the middle, and a hydrazine derivative at the other end. Anthracyclines are an example of cytotoxins that can be conjugated to antibodies using this technology.

In other aspects of the invention, the anti-CXCR4 antibody or antigen-binding fragment thereof or conjugate as described herein comprises a tag (T) containing the acyl donor glutamine engineered at a specific site of the antibody (e.g., the carboxyl terminus, the amino terminus, or another site in the anti-CXCR4 antibody). In some aspects of the invention, the tag comprises the amino acid glutamine (Q) or the amino acid sequence LLQGG (SEQ ID NO: 171), GGLLQGG (SEQ ID NO: 90), LLQGA (SEQ ID NO: 91), GGLLQGA (SEQ ID NO: 92), LLQ, LLQGPGK (SEQ ID NO: 93), LLQGPG (SEQ ID NO: 94), LLQGPA (SEQ ID NO: 95), LLQGP (SEQ ID NO: 96), LLQP (SEQ ID NO: 97), LLQPGK (SEQ ID NO: 98), LLQGAPGK (SEQ ID NO: 99), LLQGAPG (SEQ ID NO: 100), LLQGAP (SEQ ID NO: 101), GGLLQGPP (SEQ ID NO: 172), LLQGPP (SEQ ID NO: 173), LLQX1X2X3X4X5 (wherein X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where _X1 is _{G or P} , where X1 is G or _P , where _X1 is G or P, where X1 is G or P, where X1 is G or wherein _X is A, G, P or absent, wherein _X is A, G, K, P or absent, wherein _X is K, G or absent, and wherein X is K or absent (SEQ ID NO: ₁₀₂ ) or LLQ _{X X X X X} (wherein _X is any naturally occurring amino acid and wherein _X , X , _{X ,} and _X are any naturally occurring amino acids or absent) (SEQ ID NO: 103). In specific aspects of the invention, T is selected from SEQ ID NOs: 91, 92, and 102.

In another aspect of the present invention, an isolated antibody is provided, comprising a tag comprising an acyl donor glutamine and an amino acid modification at position 222, 340, or 370 (EU numbering scheme) of the antibody, wherein the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof. For example, the amino acid modifications are K222R, K340R, and K370R.

In some aspects of the invention, the conjugate of the antibody or antigen-binding fragment is conjugated to a drug, wherein the drug is selected from the group consisting of: a cytotoxic agent, an immunomodulatory agent, an imaging agent, a therapeutic agent (eg, a therapeutic protein), a biopolymer, and an oligonucleotide.

In some aspects of the invention, the drug can be linked or combined with an anti-CXCR4 antibody or antigen-binding fragment thereof as described herein to provide targeted local delivery of the drug to a tumor (e.g., a tumor expressing CXCR4). In specific aspects of the invention, the drug is a cytotoxic agent. Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, camptothecins, combretastatins, dolastatins, duocarmycins, enediynes, geldanamycins, indoline-benzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, terpilacin, hemiesterin, scotomatins, pranololactone, calicheamicins, and stereoisomers, isosteres, analogs, or derivatives thereof. In some aspects of the invention, anthracyclines are derived from the bacterium Strepomycetes and have been used to treat various cancers, such as leukemias, lymphomas, breast cancer, uterine cancer, ovarian cancer, and lung cancer. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (ie, adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

Dolastatin and its peptide analogs and derivatives, auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, for example, U.S. Patent No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965 (1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (monomethyl auristatin D or monomethyl dolastatin 10), MMAF (monomethyl auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), 5-benzoylvalerate-AE ester (AEVB), and other novel auristatins such as those described in U.S. Publication No. 20130129753.

In a particular aspect of the invention, the auristatin is selected from: 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide ), MMAD (monomethyl auristatin D or monomethyl dolastatin 10) and 8261 (2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).

In some aspects of the invention, the auristatin is 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:

In other aspects, the auristatin is 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide) having the following structure:

In other aspects of the invention, the auristatin is 0131 (2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:

In other aspects, the auristatin is 0121 (2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:

In other aspects, the auristatin is 8261 (8261 2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:

Camptothecin is a cytotoxic quinoline alkaloid that inhibits the enzyme topoisomerase I. Examples of camptothecin and its derivatives include, but are not limited to, topotecan and irinotecan and their metabolites, such as SN-38.

Combretastatin is a natural phenol with vasculogenic properties in tumors. Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A-4 (CA-4) and ombrabulin.

Duocarmycins are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplary duocarmycins include, but are not limited to, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065. Enediynes are a class of antitumor bacterial products characterized by nine- and ten-membered rings or the presence of ring systems with conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin.

Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 (heat shock protein 90) and has been used as an anti-tumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

Maytansine or its derivatives, maytansine-like compounds, inhibit cell proliferation by inhibiting tubulin polymerization and inhibiting microtubule formation during mitosis. See Remillard et al., Science 189:1002-1005 (1975). Exemplary maytansine and maytansine-like compounds include, but are not limited to, mertansine (DM1) and its derivatives, and ansamitocin.

Pyrrolobenzodiazepine dimers (PBDs) and indolino-benzodiazepine dimers (IGNs) are anti-tumor agents containing one or more imine functional groups or their equivalents that bind to double-stranded DNA. PBD and IGN molecules are based on the natural product atramycin and interact with DNA in a sequence-selective manner, preferably with purine-guanine-purine sequences. Exemplary PBDs and their analogs include, but are not limited to, SJG-136.

Scolstatin and pranolactone are anti-tumor compounds that inhibit splicing and interact with the spliceosomal SF3b. Examples of scolstatins include, but are not limited to, scolstatin A and FR901464. Examples of pranolactones include, but are not limited to, pranolactone B, pranolactone D, and E7107.

Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., paclitaxel) and docetaxel (e.g.,

Vinca alkaloids are also anti-tubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

In some aspects of the invention, the drug is an immunomodulator. Examples of immunomodulators include, but are not limited to, gancyclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, , glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF)), interferons (e.g., interferon-α, interferon-β, and interferon-γ), a stem cell growth factor known as "S1 factor", erythropoietin, and thrombopoietin, or a combination thereof.

In some aspects of the invention, the drug is an imaging agent (e.g., a fluorophore or chelator) such as fluorescein, rhodamine, lanthanide phosphors, and derivatives thereof. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amide (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5,-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101). Examples of chelating agents include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N",N'-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-acetic acid (NODAGA), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).

In some aspects of the invention, therapeutic or diagnostic radioisotopes or other labels (eg, PET or SPECT labels) can be incorporated into the drug used to conjugate the anti-CXCR4 antibodies or antigen-binding fragments as described herein. ¹⁰⁷ Hg, ¹⁰⁸ Pd, 110 Ag, ¹¹¹ In, 113 In, ¹¹⁴ Te, ¹¹⁵ I, ¹¹⁶ I, ¹¹⁷ Pd, 118 Rh, ¹¹⁹ Pd, 120 Te, 121 I, 122 Te, 123 I, 124 I, ¹²⁵ Rh, ¹²⁶ Rh, ¹²⁷ Pd, ¹²⁸ Rh, ¹²⁹ Pd, 130 Rh, 131 Rh, ¹³² Rh, ¹³³ Pd, ¹³⁴ Rh, 135 Rh, ¹³⁶ Rh, ¹³⁷ Pd, ¹³⁸ Rh, 139 Pd, ¹⁴⁰ Rh, ¹⁴¹ Rh, ¹⁴² Rh, ¹⁴³ Pd, 144 Rh, ¹⁴⁵ Rh, 146 Rh, ¹⁴⁷ Pd, 148 Rh, ¹⁴⁹ Pd, ¹⁵⁰ Rh, ¹⁵¹ Rh, ¹⁵² Rh, ¹⁵³ Pd, 154 Rh, 155 Rh, 156 Rh, ¹⁵⁷ Pd, ¹⁵⁸ Rh, ¹⁵⁹ Pd, ¹⁶¹ Rh, ¹⁶² Rh, 163 Rh, 164 Rh, 165 Rh, ¹⁶⁶ Rh, 167 Pd, 168 Pd, ¹⁶⁹ Pd, ¹⁷⁰ Rh, ¹⁷¹ Rh, ¹⁷² Rh, ¹⁷³ Pd, 174 Rh, ^{175 125} I, ¹²⁵ Te, ¹²⁶ I, ¹³¹ I, ¹³¹ In, ¹³³ I, ¹⁴² Pr, ¹⁴³ Pr, ¹⁵³ Pb, ¹⁵³ Sm, ¹⁶¹ Tb, ¹⁶⁵ Tm, ¹⁶⁶ Dy, ¹⁶⁶ H, ¹⁶⁷ Tm, ¹⁶⁸ Tm, ¹⁶⁹ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁹ Re, ¹⁹⁷ Pt, ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰¹ Tl, ²⁰³ Hg, ²¹¹ At, ²¹² Bi, ²¹² Pb, ²¹³ Bi, ²²³ Ra, ²²⁴ Ac and ²²⁵ Ac.

In some aspects of the invention, the drug is a therapeutic protein, including but not limited to toxins, hormones, enzymes, and growth factors.

Examples of toxin proteins (or polypeptides) include, but are not limited to, diphtheria (e.g., diphtheria A chain), Pseudomonas aeruginosa exotoxin and endotoxin, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain), modicin (e.g., modicin A chain), α-sarcin, Aleurites fordii protein, dianthin protein, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, saponin inhibitor, mitogen, restrictocin, phenomycin, enomycin, tricothecene, inhibitor cystine knot (ICK) peptide (e.g., ceratotoxin), and conotoxin (e.g., KIIIA or SmIIIa).

In some aspects of the present invention, the drug is a biocompatible polymer. The anti-CXCR4 antibodies or antigen-binding fragments described herein can be conjugated to a biocompatible polymer to increase serum half-life and bioactivity, and/or extend in vivo half-life. Examples of biocompatible polymers include water-soluble polymers such as polyethylene glycol (PEG) or its derivatives, and biocompatible polymers containing zwitterions (e.g., polymers containing phosphorylcholine).

In some aspects of the invention, the drug is an oligonucleotide, such as an antisense oligonucleotide.

In another aspect, the present invention provides a conjugate of an antibody or antigen-binding fragment as described herein, wherein the antibody-drug conjugate comprises the formula: antibody-(tag containing acyl donor glutamine)-(linker)-(drug), wherein the tag containing acyl donor glutamine is engineered at a specific site of the antibody or antigen-binding fragment (e.g., at the carboxyl terminus of the heavy chain or light chain or at another site), wherein the tag is conjugated to a linker (e.g., a linker containing one or more reactive amines (e.g., a primary amine NH2)) and wherein the linker is bound to a cytotoxic agent (e.g., MMAD or other auristatins as described herein). In some aspects of the invention, the antibody-drug conjugate does not comprise a tag containing acyl donor glutamine.

Examples of linkers containing one or more reactive amines include, but are not limited to, acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC) or aminoPEG6-propionyl. See, for example, WO2012/059882.

In some aspects of the invention, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) linker, or a maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. In another aspect, the linker can be sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfonate-smcc conjugation occurs via reaction of the maleimido group with a sulfhydryl (thiol, -SH) group, while its sulfonate-NHS ester is reactive toward primary amines, such as those found in lysine and the N-terminus of proteins or peptides. Additionally, the linker can be maleimidocaproic acid (mc). In some aspects of the invention, tags containing the acyl donor glutamine include LLQGG (SEQ ID NO: 171), GGLLQGG (SEQ ID NO: 90), LLQGA (SEQ ID NO: 91), GGLLQGA (SEQ ID NO: 92), LLQ, LLQGPGK (SEQ ID NO: 93), LLQGPG (SEQ ID NO: 94), LLQGPA (SEQ ID NO: 95), LLQGP (SEQ ID NO: 96), LLQP (SEQ ID NO: 97), LLQPGK (SEQ ID NO: 98), LLQGAPGK (SEQ ID NO: 99), LLQGAPG (SEQ ID NO: 100), LLQGAP (SEQ ID NO: 101), GGLLQGPP (SEQ ID NO: 172), LLQGPP (SEQ ID NO: 173), LLQX1X2X3X4X5 (wherein X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, _where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where X1 is G or P, where _X1 is G or P, where X1 is G or P, where X1 is G or P, where _{X1 is G} or _P , where X1 is G or P, where _X1 is G or P, where X1 is G or wherein _X2 is A, G, P or absent, wherein _X3 is A, G, K, P or absent, wherein _X4 is K, G or absent, and wherein _X5 is K or absent) ( _SEQ ID NO: 102) or _{LLQX1X2X3X4X5} ( _{wherein X1} is any naturally occurring amino acid, and wherein _{X2 , X3 , X4} and _X5 are any naturally occurring amino acids or absent) (SEQ ID NO: 103).

In some aspects of the invention, an anti-CXCR4 antibody or conjugate as described herein comprises an amino acid substitution of asparagine (N) to glutamine (Q) or N to alanine (A) at position 297 of the anti-CXCR4 antibody.

In some aspects of the invention, a tag containing an acyl donor glutamine, such as LLQ, SEQ ID NO: 91, 90, 94, 95, 95, 97, 98, 99, 100, 171, 101, 172, or 173, is engineered at the C-terminus of the antibody heavy chain, wherein the C-terminal lysine residue is deleted. In other aspects, a tag containing an acyl donor glutamine, such as GGLLQGA (SEQ ID NO: 92), is engineered at the C-terminus of the antibody light chain. Examples of antibodies include, but are not limited to, m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10. A18A, h3G10.A19A, h3G10.A58A, h3G10.A65A and h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G10.2.25, h3G10.A59, h3G10.A62 or h3G10.L94D.

In other aspects of the invention, the conjugate comprises the formula: antibody-(tag containing the acyl donor glutamine)-(L)-(cytotoxic agent). In some aspects of the invention, the conjugates are a) Ab-LLQGA (SEQ ID NO: 91)-(Acetyl-Lysine-Valine-Citrulline-p-Aminobenzyloxycarbonyl (AcLys-VC-PABC))-0101; b) Ab-LLQGA (SEQ ID NO: 91)-(AcLys-VC-PABC)-MMAD; c) Ab-LLQX ₁ X ₂ X ₃ X ₄ X ₅ (SEQ ID NO: 102)-(AcLys-VC-PABC)-0101; d) Ab-LLQX ₁ X ₂ X ₃ X ₄ X ₅ (SEQ ID NO: 102)-(AcLys-VC-PABC)-MMAD; e) Ab-GGLLQGA (SEQ ID NO: 92)-(AcLys-VC-PABC)-0101; and f) Ab-GGLLQGA (SEQ ID NO: 91)-(AcLys-VC-PABC)-0101. NO:92)-(AcLys-VC-PABC)-MMAD.

Examples of antibodies include, but are not limited to, m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10.A18A, h3G10. 3G10.A19A, h3G10.A58A, h3G10.A65A and h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G10.2.25, h3G10.L94D, h3G10.A59, h3G10.A62 or h3G10.L94D.

In some aspects of the invention, Ab is an antibody or antigen-binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOs: 107, 162, and 112 and a VL region comprising CDRs of SEQ ID NOs: 144, 145, and 139.

In some aspects of the invention, the anti-CXCR4 antibody-drug conjugate causes tumor regression.

Methods of using anti-CXCR4 antibodies, antigen-binding fragments thereof, or antibody-drug conjugates thereof

The antibodies and antibody-drug conjugates of the invention are useful in a variety of applications, including, but not limited to, therapeutic treatment methods and diagnostic treatment methods.

In some aspects of the invention, when administered to a patient, the antibody or conjugate and the pharmaceutically acceptable carrier are sterile. When the compound or conjugate is administered intravenously, water is an exemplary carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. If desired, the compositions of the invention may also contain trace amounts of wetting agents or emulsifiers or pH buffers.

The compositions of the present invention may be in the form of solutions, pellets, powders, sustained release formulations or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described by E.W. Martin in "Remington's Pharmaceutical Sciences".

In some aspects, the antibodies of the present invention and/or their antibody-drug conjugates are formulated as pharmaceutical compositions suitable for intravenous administration to animals, especially humans, according to conventional procedures. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic buffered aqueous solution. If necessary, the composition may also include a solubilizing agent. The composition for intravenous administration may optionally contain a local anesthetic (such as lignocaine) to relieve pain at the injection site. In general, the components are supplied separately or mixed together in unit dosage form, for example, in the form of a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a sachet) indicating the amount of the active agent. In the case where the compound of the present invention and/or its antibody-drug conjugate is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case where the compound of the present invention and/or the antibody-drug conjugate is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

The composition may include various substances that modify the physical form of a solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be selected from, for example, sugar, comfrey, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

Whether in solid or liquid form, the compositions of the present invention may include agents for treating cancer. In one aspect, the present invention provides a method for treating a condition associated with CXCR4 expression in a subject. In some aspects of the present invention, the method for treating a condition associated with CXCR4 function or expression in a subject comprises administering to the subject in need thereof an effective amount of a composition (e.g., a pharmaceutical composition) comprising an anti-CXCR4 antibody, an antigen-binding fragment thereof, or an anti-CXCR4 antibody-drug conjugate as described herein. Conditions associated with CXCR4 expression include, but are not limited to, abnormal CXCR4 expression, altered or abnormal CXCR4 expression, overexpression of CXCR4, and proliferative conditions (e.g., cancer).

Thus, in some aspects, a method of treating cancer in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising an anti-CXCR4 antibody, an antigen-binding fragment thereof, or an anti-CXCR4 antibody-drug conjugate as described herein. As used herein, cancer includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, lung cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and blood cancer. In some aspects of the present invention, a method of reducing tumor growth or progression in a subject having a tumor that expresses CXCR4 is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising an anti-CXCR4 antibody, an antigen-binding fragment thereof, or an anti-CXCR4 antibody-drug conjugate as described herein. In other aspects, a method of reducing metastasis of CXCR4-expressing cancer cells in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising an anti-CXCR4 antibody, an antigen-binding fragment thereof, or an anti-CXCR4 antibody-drug conjugate as described herein. In other aspects, a method of inducing regression of a CXCR4-expressing tumor in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising an anti-CXCR4 antibody or antigen-binding fragment thereof or an anti-CXCR4 antibody-drug conjugate as described herein.

In some aspects, the present invention provides an isolated antibody, antigen-binding fragment, or antibody-drug conjugate of any of the antibodies described herein for use in detecting, diagnosing, and treating pathological conditions associated with CXCR4 function or expression. In one aspect, the condition is a cancer associated with increased CXCR4 expression relative to normal or any other pathology associated with overexpression of CXCR4.

In some aspects of the invention, a method of treating cancer in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising: a heavy chain variable (VH) region comprising (i) a VH CDR1 selected from the group consisting of SEQ ID NOs: 107, 113, 114, 108, 109, 115, 116, 117, 121, and 122, (ii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156, and 130, and (iii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 112 and 120. and/or b) a light chain variable (VL) region comprising (i) a VL CDR1 selected from the group consisting of SEQ ID NOs: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150, and 151, (ii) a VL CDR2 selected from the group consisting of 145, 132, 136, and 152, and (iii) a VL CDR3 selected from the group consisting of SEQ ID NOs: 139, 133, 137, 140, and 153.

In some aspects of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, wherein the antibody comprises: a heavy chain variable (VH) region comprising the three CDRs set forth in SEQ ID NOs: 107, 162, and 112. In some aspects of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, wherein the antibody comprises: a light chain variable (VL) region comprising the three CDRs set forth in SEQ ID NOs: 144, 145, and 139.

In some aspects of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described in any one of the foregoing technical solutions, wherein the antibody comprises: a heavy chain variable (VH) region comprising three CDRs shown in SEQ ID NOs: 107, 162, and 112; and a light chain variable (VL) region comprising three CDRs shown in SEQ ID NOs: 144, 145, and 139.

In some aspects, the present invention provides a method of treating cancer in a subject, comprising administering to the subject in need thereof an effective amount of a composition comprising an isolated antibody or antigen-binding fragment thereof that binds to CXCR4 and comprises: a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 from the VH region of SEQ ID NO: 33; and a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 from the VL region of SEQ ID NO: 73.

In other aspects, the present invention provides a method for treating cancer in a subject, comprising administering to the subject in need thereof an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described in any one of the foregoing technical solutions, wherein the antibody comprises: a) a heavy chain variable (VH) region of SEQ ID NO: 33; and b) a light chain variable (VL) region of SEQ ID NO: 73.

In some aspects, the present invention provides the use of an isolated antibody, antigen-binding fragment, or antibody-drug conjugate of any of the antibodies as described herein in the manufacture of a medicament for treating a condition associated with CXCR4 function or expression. In one aspect, the condition is a cancer associated with increased CXCR4 expression relative to normal or any other pathology associated with overexpression of CXCR4.

In other aspects of the invention, the anti-CXCR4 antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof having at least one heavy chain variable region and at least one light chain variable region, wherein the at least one heavy chain variable region comprises the three CDRs set forth in SEQ ID NOs: 107, 113, 114, 162, 128, and 112. In some aspects of the invention, the anti-CXCR4 antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof having at least one heavy chain variable region and at least one light chain variable region, wherein the at least one light chain variable region comprises the three CDRs set forth in SEQ ID NOs: 144, 145, and 139.

In some aspects of the invention, the antibody-drug conjugate that binds to CXCR4 comprises an antibody or antigen-binding fragment thereof having a heavy chain variable region set forth in any one of SEQ ID NOs: 33, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 85, or 87, and/or a light chain variable region set forth in any one of SEQ ID NOs: 73, 3, 7, 11, 15, 19, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 75, 77, 79, 81, 83, or 169. For example, an anti-CXCR4 antibody-drug conjugate of the present invention may include an antibody or an antigen-binding fragment thereof having a heavy chain variable region with an amino acid sequence at least 90% identical to SEQ ID NO: 33, and a light chain variable region with an amino acid sequence at least 90% identical to SEQ ID NO: 73; or having a heavy chain variable region set forth in SEQ ID NO: 33, and a light chain variable region with an amino acid sequence set forth in SEQ ID NO: 73.

In specific aspects of the invention, the antibody is not caninized or felineized.

Antibodies can also be modified, for example, in the variable domains of the heavy and/or light chains, e.g., to alter the binding properties of the antibody. For example, mutations can be made in one or more CDR regions to increase or decrease the _KD of an anti-CXCR4 antibody, increase or decrease _koff , or alter the binding specificity of the antibody. Techniques for site-directed mutagenesis are well known in the art. See, e.g., Sambrook et al. and Ausubel et al., supra.

In some aspects of the present invention, a method for detecting, diagnosing, and/or monitoring a condition associated with CXCR4 function or expression is provided. For example, the anti-CXCR4 antibodies described herein can be labeled with a detectable moiety such as an imaging agent and an enzyme-substrate label. The antibodies described herein can also be used in in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT) or staining reagents.

In one aspect of the present invention, a method for detecting, diagnosing, and/or monitoring a condition associated with CXCR4 function or expression is provided, comprising the steps of: (i) obtaining a biological sample from a patient suspected of having a condition associated with CXCR4 function or expression; (ii) contacting the sample to be tested with an antibody, or an antigen-binding portion thereof, under conditions that allow formation of a complex between the antibody and the protein antigen CXCR4; (iii) detecting the antibody-protein antigen complex, wherein the presence of the detected antibody-protein antigen complex indicates that the patient has a condition associated with CXCR4 function or expression. In a specific aspect of the present invention, the method further comprises administering to the patient a therapeutically effective amount of an isolated antibody, antigen-binding fragment, or antibody-drug conjugate of any of the antibodies described herein.

In some aspects of the invention, ADCs can be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, radiotoxins, immunosuppressants, etc.) to cells with CXCR4 cell surface receptors by linking such compounds to antibodies or fragments thereof. Thus, in some aspects of the invention, methods are provided for localizing cells expressing CXCR4 in vitro or in vivo (e.g., detectable labels such as radioisotopes, fluorescent compounds, and enzymes). Alternatively, ADCs can be used to kill cells with CXCR4 cell surface receptors by targeting cytotoxins or radiotoxins to CXCR4.

In some aspects of the invention, the methods described herein further comprise the step of treating the subject with other forms of therapy. In some aspects, other forms of therapy are other anti-cancer therapies, including but not limited to chemotherapy, radiation, surgery, hormone therapy, and/or other immunotherapies.

In some aspects of the present invention, the other forms of therapy comprise administering one or more therapeutic agents in addition to the anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates described herein. Such therapeutic agents include, but are not limited to, a second antibody (e.g., an anti-VEGF antibody, an anti-HER2 antibody, an anti-CD25 antibody, and/or an anti-CD20 antibody), an angiogenesis inhibitor, a cytotoxic agent, an anti-inflammatory agent (e.g., paclitaxel, docetaxel, cisplatin, doxorubicin, prednisone, mitomycin, progesterone, tamoxifen, or fluorouracil). In one aspect, the other forms of therapy may be administered simultaneously, sequentially, or concurrently with the anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates described herein.

The anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates of the present invention can be administered to a subject via any suitable route. Those skilled in the art will appreciate that the examples described herein are not intended to be limiting, but rather to illustrate available techniques. Thus, in some aspects of the present invention, anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates are administered to a subject according to known methods, such as intravenous administration, for example, rapid injection or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intracranial, transdermal, subcutaneous, intraarticular, sublingual, intrasynovial, via insufflation, intrathecal, oral, inhalation, or topical routes. Administration can be systemic (e.g., intravenous) or local. Commercially available sprayers for liquid formulations, including nozzle sprayers and ultrasonic sprayers, are suitable for administration. Liquid formulations can be directly formed into a spray, and lyophilized powders can be atomized after reconstitution. Alternatively, the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate can be nebulized using a fluorocarbon formulation and a metered dose inhaler or inhaled as a lyophilized and ground powder. In some aspects of the invention, the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate can be administered via inhalation, as described herein. In other aspects, the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate of the invention can be combined with a pharmaceutically acceptable vehicle (such as saline, Ringer's solution, dextrose solution, and the like). The specific dosing regimen (i.e., dosage, timing, and repetition) will depend on the specific subject and the subject's medical history.

In one aspect, the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate is administered via a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable reservoir sources or local delivery catheters of the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate, such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts, and stents or other implantable devices, site-specific carriers, direct injection, or direct coating. See, for example, PCT Publication No. WO 00/53211 and U.S. Patent No. 5,981,568.

Various formulations of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates can be used for administration. In some aspects of the present invention, anti-CXCR4 antibodies (antigen-binding fragments thereof or anti-CXCR4 antibody-drug conjugates) and pharmaceutically acceptable excipients can be in various formulations. Some formulations of the present invention include pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of pharmacologically effective substances. For example, excipients can impart shape or consistency or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts that change permeability, encapsulating agents, buffers, and skin penetration enhancers. Excipients and formulations for parenteral and enteral drug delivery are described in Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000.

In general, for administration of an anti-CXCR4 antibody, antigen-binding fragment thereof, and/or anti-CXCR4 antibody-drug conjugate, an initial candidate dose may be about 2 mg/kg. For the purposes of the present invention, a typical daily dose may be in the range of about 3 μg/kg to 30 μg/kg, to 300 μg/kg, to 3 mg/kg, to 30 mg/kg, to 100 mg/kg, or more, depending on the factors described above. For example, doses of about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be used. For repeated administration over several days or longer, depending on the condition, treatment is continued until the desired symptom suppression occurs or until a sufficient therapeutic level is achieved, such as inhibition or delay of tumor growth/progression or cancer cell metastasis. An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of an anti-CXCR4 antibody, an antigen-binding fragment thereof, or an anti-CXCR4 antibody-drug conjugate, or followed by a maintenance dose of about 1 mg/kg every other week. Other exemplary dosing regimens comprise administering escalating doses (e.g., an initial dose of 1 mg/kg and gradually increasing to one or more higher doses per week or longer). Other dosing regimens may also be applicable, depending on the pharmacokinetic decay pattern that the practitioner wishes to achieve. For example, in some aspects, dosing is contemplated one to four times a week. In other aspects, dosing is contemplated once a month, every other month, or every three months. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate used) may vary over time.

For the purposes of the present invention, the appropriate dosage of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody conjugate will depend on the anti-CXCR4 antibody, antigen-binding fragment thereof, or CXCR4 antibody-drug conjugate (or combination thereof) employed, the type and severity of the symptom to be treated, whether the agent is being administered for therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, the patient's clearance rate of the administered agent, and the discretion of the attending physician. Typically, the clinician will administer the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate until a dose that achieves the desired result is reached. The dosage and/or frequency may vary over the course of treatment. Empirical considerations such as half-life will generally help determine the dosage. For example, antibodies compatible with the human immune system (such as humanized or fully human antibodies) can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment and is generally (but not necessarily) based on the treatment and/or suppression and/or amelioration and/or delay of symptoms, such as tumor growth inhibition or delay. Alternatively, sustained continuous release formulations of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In some aspects of the present invention, the dosage of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate can be determined empirically in subjects who have received one or more doses of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate. Increasing doses of the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate can be administered to the subject. Efficacy can be assessed based on indicators of disease.

Administration of the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate according to the methods of the present invention can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. Administration of the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate can be substantially continuous over a preselected period of time, or can be in a series of spaced doses.

In some aspects of the present invention, there may be more than one anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate. There may be at least one, at least two, at least three, at least four, at least five, or more different anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates. Generally, those anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates may have complementary activities that do not adversely affect each other. For example, one or more of the following anti-CXCR4 antibodies may be used: a first anti-CXCR4 antibody directed against one epitope on CXCR4 and a second anti-CXCR4 antibody directed against a different epitope on CXCR4.

Therapeutic formulations of the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate used according to the present invention are prepared by mixing the antibody having the desired purity with optionally selected pharmaceutically acceptable carriers, excipients, or stabilizers (Remington, The Science and Practice of Pharmacy 21st edition, Mack Publishing, 2005) in the form of a lyophilized formulation or aqueous solution for storage. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins The present invention also includes a chelating agent such as EDTA; a saccharide such as sucrose, mannitol, trehalose or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and/or a non-ionic surfactant such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Liposomes containing anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly suitable liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a defined pore size to produce liposomes with the desired diameter.

The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005.

Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)), and poly-D-(-)-3-hydroxybutyric acid.

Formulations intended for in vivo administration must be sterile. This is readily achieved, for example, by filtration through a sterile filtration membrane. Therapeutic anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugate compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle.

The compositions according to the present invention may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories, for oral, parenteral or rectal administration or administration by inhalation or insufflation.

For the preparation of solid compositions (such as tablets), the main active ingredient is mixed with a pharmaceutical carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum) and other pharmaceutical diluents (e.g., water) to form a solid pre-formulated composition containing a homogeneous mixture of the compound of the invention or a pharmaceutically acceptable non-toxic salt thereof. When these pre-formulated compositions are referred to as homogeneous, it is meant that the active ingredient is evenly distributed throughout the composition, so that the composition can be easily subdivided into equivalent unit dosage forms, such as tablets, pills, and capsules. The solid pre-formulated composition is then subdivided into unit dosage forms of the above type containing 0.1 to about 500 mg of the active ingredient of the invention. The tablets or pills of the novel composition can be coated or otherwise mixed to provide a dosage form with the advantage of prolonged action. For example, the tablet or pill can include an inner dosage component and an outer dosage component, the latter being in the form of a film coating the former. The two components may be separated by an enteric coating layer that resists disintegration in the stomach and allows the inner component to pass intact into the duodenum or to be released later. A variety of materials can be used for the enteric coating layer or coating, including a variety of polymeric acids and mixtures of polymeric acids with materials such as lithospermum parkii, cetyl alcohol, and cellulose acetate.

Suitable surfactants include, among others, nonionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80, or 85). Compositions containing surfactants preferably contain between 0.05% and 5% surfactant, and the amount may be between 0.1% and 2.5%. It will be appreciated that other ingredients, such as mannitol, or other pharmaceutically acceptable vehicles may be added as necessary.

Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition, or it can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and in an emulsion formed after mixing with a phospholipid (e.g., lecithin, soy lecithin, or soy lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the emulsion tension. Suitable emulsions will typically contain up to 20% oil, for example, between 5% and 20%. The fat emulsion may comprise fat droplets between 0.1 and 1.0 μm, particularly between 0.1 and 0.5 μm, and have a pH value within the range of 5.5 to 8.0.

The emulsion composition can be prepared by mixing an anti-CXCR4 antibody, an antigen-binding fragment thereof, or an anti-CXCR4 antibody-drug conjugate with Intralipid™ or its components (soybean oil, lecithin, glycerol, and water).

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some aspects of the invention, the compositions are administered via the oral or nasal respiratory route for local or systemic effect. Compositions in preferably sterile pharmaceutically acceptable solvents can be aerosolized using a gas. Aerosolized solutions can be breathed directly from a nebulizer, or the nebulizer can be attached to a mask, hood, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered from a device that delivers the formulation in an appropriate manner, preferably orally or nasally.

Composition

The compositions used in the methods of the present invention comprise an effective amount of an anti-CXCR4 antibody, an antigen-binding fragment thereof, or an anti-CXCR4 antibody-drug conjugate as described herein. Examples of such compositions and how to formulate them are also described in the preceding sections and below. In some aspects of the present invention, the compositions comprise one or more anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates. For example, the anti-CXCR4 antibody recognizes human CXCR4. In some aspects, the CXCR4 antibody is a human antibody, a CDR-grafted antibody, a humanized antibody, or a chimeric antibody. In other aspects, the anti-CXCR4 antibody comprises a constant region capable of triggering a desired immune response (such as antibody-mediated lysis or ADCC). In other aspects, the anti-CXCR4 antibody comprises a constant region that does not trigger an unwanted or undesirable immune response (such as antibody-mediated lysis or ADCC). In some aspects of the present invention, the anti-CXCR4 antibody comprises one or more CDRs (such as one, two, three, four, five, or in some embodiments, all six CDRs) of an anti-CXCR4 antibody or an anti-CXCR4 or antigen-binding fragment thereof as described herein.

It should be understood that a composition may comprise more than one anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate (e.g., a mixture of anti-CXCR4 antibodies that recognize different epitopes of a CXCR4 epitope). Other exemplary compositions comprise more than one anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate that recognizes the same epitope, or an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate that binds to different epitopes of CXCR4 (e.g., human CXCR4).

The compositions used in the present invention may further comprise a pharmaceutically acceptable carrier, excipient or stabilizer (Remington: The Science and practice of Pharmacy 21st edition, 2005, Lippincott Williams and Wilkins, ed. KE Hoover) in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations and may include: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

Reagent test kit

The present invention also provides kits for use in the methods of the present invention. The kits of the present invention include one or more containers containing an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate as described herein, and instructions for use according to any of the methods of the present invention described herein. Generally, these instructions include descriptions of the administration of the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate for the therapeutic treatments described above.

Instructions for use of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate as described herein generally include information regarding dosage, dosing schedule, and route of administration for achieving the intended treatment. The container may be a unit dose, bulk (e.g., multi-dose packaging), or sub-unit dose. The instructions provided in the kits of the present invention are typically written on a label or package insert (e.g., a paper sheet included with the kit), but machine-readable instructions (e.g., instructions contained on a magnetic or optical storage disk) are also acceptable.

The kits of the present invention are provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packaging for use with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices (e.g., mini-pumps), is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-CXCR4 antibody. The container may further comprise a second pharmaceutically active agent.

The kit may optionally provide other components such as a buffer and interpretative information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

Mutation and modification

To express the anti-CXCR4 antibodies or antigen-binding fragments thereof of the present invention, DNA fragments encoding the VH and VL regions can first be obtained using any of the methods described above. Various modifications, such as mutations, substitutions, deletions, and/or additions, can also be introduced into the DNA sequence using standard methods known to those skilled in the art. For example, mutagenesis can be performed using standard methods, such as PCR-mediated mutagenesis, in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the desired mutation, or site-directed mutagenesis.

For example, one type of substitution that can be made is to change one or more chemically reactive cysteines in the antibody to another residue such as, but not limited to, alanine or serine. For example, substitutions of atypical cysteines can be made. Substitutions can be made in the CDRs or framework regions of the antibody variable domain or in the constant region. In some aspects of the invention, cysteines are typical.

Antibodies can also be modified, for example, in the variable domains of the heavy and/or light chains, e.g., to alter the binding properties of the antibody. For example, mutations can be made in one or more CDR regions to increase or decrease the _KD of the antibody for CXCR4, increase or decrease _koff , or alter the binding specificity of the antibody. Techniques for site-directed mutagenesis are well known in the art. See, e.g., Sambrook et al. and Ausubel et al., supra.

Modifications or mutations can also be made in the framework or constant regions to increase the half-life of anti-CXCR4 antibodies. See, for example, PCT Publication No. WO 00/09560. Mutations can also be made in the framework or constant regions to alter the immunogenicity of the antibody, provide sites for covalent or non-covalent binding to another molecule, or alter properties such as complement fixation, FcR binding, and antibody-dependent cell-mediated cytotoxicity. According to the present invention, a single antibody can have mutations in any one or more CDRs or framework regions of the variable domain or in the constant region.

In a process called "germlining", certain amino acids in the VH and VL sequences can be mutated to match amino acids naturally found in germline VH and VL sequences. Specifically, the amino acid sequences of the framework regions in the VH and VL sequences can be mutated to match the germline sequences, thereby reducing the risk of immunogenicity when the antibody is administered. The germline DNA sequences of human VH and VL genes are known in the art (see, e.g., the "Vbase" human germline sequence database; see also Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., J. Mol. Biol. 227:776-798 (1992); and Cox et al., Eur. J. Immunol. 24:827-836 (1994)).

Another type of amino acid substitution that can be performed is the removal of potential proteolytic sites in the antibody. Such sites can be in the CDRs or framework regions of the antibody variable domain or in the constant region. The replacement of cysteine residues and the removal of proteolytic sites can reduce the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is the removal of asparagine-glycine pairs that form potential deamidation sites by changing one or both of these residues. In another example, the C-terminal lysine of the heavy chain of the anti-CXCR4 antibody of the present invention can be cleaved. In various aspects of the present invention, the heavy and light chains of the anti-CXCR4 antibody can optionally include a signal sequence.

Once the DNA fragments encoding the VH and VL segments of the present invention are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein (such as an antibody constant region or a flexible linker). As used herein, the term "operably linked" refers to the joining of two DNA fragments so that the amino acid sequences encoded by the two DNA fragments remain in frame.

The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but preferably an IgG1 or IgG2 constant region. The IgG constant region sequence can be any of the various alleles or allotypes known to exist in different subjects, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes represent naturally occurring amino acid substitutions in the IgG1 constant region. For the Fab fragment heavy chain gene, the DNA encoding the VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region. The CH1 heavy chain constant region can be derived from any heavy chain gene.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. The kappa constant region can be any of the various alleles known to be present in different subjects, such as Inv (1), Inv (2) and Inv (3). The lambda constant region can be derived from any of the three lambda genes.

To generate scFv genes, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, such as another fragment encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 80), so that the VH and VL sequences can be expressed as a contiguous single-chain protein in which the VL and VH regions are joined by a flexible linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); McCafferty et al., Nature 348: 552-554 (1990)). Single-chain antibodies can be monovalent if only a single VH and VL are used, bivalent if two VH and VL are used, or multivalent if more than two VH and VL are used. Bispecific or multivalent antibodies that bind to CXCR4 and another molecule can be generated.

In some aspects of the invention, fusion antibodies or immunoadhesins can be made that comprise all or a portion of an anti-CXCR4 antibody of the invention linked to another polypeptide. In other aspects, only the variable domain of the anti-CXCR4 antibody is linked to a polypeptide. In some aspects of the invention, the VH domain of the anti-CXCR4 antibody is linked to a first polypeptide, while the VL domain of the anti-CXCR4 antibody is linked to a second polypeptide, which binds to the first polypeptide in a manner that allows the VH and VL domains to interact with each other to form an antigen binding site. In other aspects, the VH domain and the VL domain are separated by a linker that allows the VH and VL domains to interact with each other. The VH-linker-VL antibody is then linked to a polypeptide of interest. In addition, fusion antibodies can be produced in which two (or more) single-chain antibodies are linked to each other. This applies if one wishes to produce a bivalent or multivalent antibody on a single polypeptide chain or if one wishes to produce a bispecific antibody.

In some aspects of the present invention, other modified antibodies can be prepared using anti-CXCR4 antibody encoding nucleic acid molecules. For example, "κ bodies" (Ill et al., Protein Eng. 10:949-57 (1997)), "minibodies" (Martin et al., EMBO J., 13:5303-9 (1994)), "diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or "Janusins" (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) can be prepared using standard molecular biology techniques following the teachings of this specification.

Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods, including hybridoma fusion or linking of Fab' fragments. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992). In addition, bispecific antibodies can be formed as "bifunctional antibodies" or "Janusins." In some aspects of the present invention, bispecific antibodies bind to two different epitopes of CXCR4. In some aspects, the modified antibodies described above are prepared using one or more variable domains or CDR regions from the anti-CXCR4 antibodies provided herein.

In one aspect, the present invention encompasses the use of multispecific antibodies. Multispecific antibodies are antibodies that can simultaneously bind to at least two targets with different structures (e.g., two different antigens, two different epitopes or haptens on the same antigen, and/or antigens or epitopes). Multispecific, multivalent antibodies are constructs with more than one binding site, and the binding sites have different specificities.

Representative materials of the present invention were deposited with the American Type Culture Collection (ATCC) on June 19, 2014. The vector with ATCC accession number PTA-121353 contains a polynucleotide encoding the heavy chain variable region of a humanized anti-CXCR4 antibody, and the vector with ATCC accession number PTA-121354 contains a polynucleotide encoding the light chain variable region of a humanized anti-CXCR4 antibody. These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (Budapest Treaty). This ensures that the deposited material remains a viable culture for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty and will be subject to an agreement between Pfizer, Inc. and ATCC, which agreement ensures that progeny of the deposit will be available to the public in perpetuity and without restriction upon the filing of a related U.S. patent or the publication of any U.S. or foreign patent application, whichever comes first, and will be available to persons as determined by the Commissioner of the United States Patent and Trademark under 35 U.S.C. section 122 and the regulations thereunder (including 37 C.F.R. section 1.14, with particular reference to 886 OG 638).

The assignee of this application has agreed that if a culture of the deposited material dies or is lost or destroyed when cultured under suitable conditions, the material will be replaced with another of the same kind upon notice. The availability of the deposited material should not be construed as a license to practice the invention in violation of the rights granted by any governmental authority under its patent laws.

The present invention also relates to the use of these anti-CXCR4 antibodies (e.g., full-length antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates) and pharmaceutical compositions comprising CXCR4 receptor antibodies (e.g., full-length antibodies or antigen-binding fragments thereof) for treating diseases and conditions associated with CXCR4 modulation, such as bone marrow transplantation, chemosensitization, cancer, metastatic diseases (e.g., cancer), autoimmune diseases (e.g., rheumatoid arthritis), fibrotic diseases (e.g., lung), AIDS infection, cardiovascular disease, uveitis, inflammatory diseases, celiac disease, HIV infection, and stem cell-based regenerative medicine.

cancer

The CXCR4 receptor is overexpressed in a variety of cancers, including, but not limited to, breast cancer (Muller, A. et al. Nature 410:50-56 (2001)); ovarian cancer (Scotton, C. et al. Br. J. Cancer 85:891-897 (2001); prostate cancer (Taichman, R.S. et al. Cancer Res. 62:1832-1837 (2002); non-small cell lung cancer (Spano J.P. et al. Ann. Oncol. 15:613-617 (2004)); pancreatic cancer (Koshiba, T. et al. Clin. Cancer Res. 6:3530-3535 (2000)); thyroid (Hwang, J.H. et al. J. Clin. Endocrinol. Metab. 88:408-416 (2003)); nasopharyngeal carcinoma (Wang, N. et al. J. Transl. Med. 3:26-33 (2005)); melanoma (Scala, S. et al. Clin. Cancer Res. 11:1835-1841 (2005)); renal cell carcinoma (Staller, P. et al. Nature 425:307-311 (2003)); lymphoma (Bertolini, F. et al. Cancer Res. 62:3530-3535 (2002)); neuroblastoma (Geminder, H. et al. J. Immunol. 167:4747-4757 (2001)); glioblastoma (Rempel, S.A. et al. Clin. Cancer Res. 6:102-111 (2000)); rhabdomyosarcoma (Libura, J. et al. Blood 100:2597-2606 (2002)); colorectal cancer (Zeelenberg, I.S. et al. Cancer Res. 63:3833-3839 (2003)); renal cancer (Schrader, A.J. et al. Br. J. Cancer 86:1250-1256 (2002)); osteosarcoma (Laverdiere, C. et al. Clin. Cancer Res. 11:2561-2567 (2005)); acute lymphoblastic leukemia (Crazzolara, R. et al. Br. J. Haematol. 115:545-553 (2001)); and acute myeloid leukemia (Rombouts, E.J.C. et al. Blood 104:550-557 (2004)).

In view of the above, the anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates of the present invention can be used to treat cancers including, but not limited to, breast cancer, ovarian cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, melanoma, renal cell carcinoma, lymphoma, neuroblastoma, glioblastoma, rhabdomyosarcoma, colorectal cancer, renal cancer, osteosarcoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, mantle cell lymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulinemia (WM), and B-cell lymphoma and diffuse large B-cell lymphoma (DLBCL). The antibodies can be used alone or in combination with other cancer treatments, such as surgery and/or radiation, and/or in combination with other anti-tumor agents, such as those discussed and illustrated above, including chemotherapeutic drugs and other anti-tumor antigen antibodies, such as antibodies that bind CD20, Her2, PSMA, Campath-1, EGFR, and the like.

In some aspects, the anti-CXCR4 antibodies of the present invention can be used in combination with anti-CD22 or anti-CD33 antibodies, antibody-drug conjugates, or compositions comprising such antibodies. For example, the anti-CXCR4 antibodies of the present invention can be combined with inotuzumab ozogamicin or gemtuzumab ozogamicin.

"Combination therapy" or administration "in combination" with one or more other therapeutic agents includes simultaneous, concurrent and sequential administration in any order. Administration of the components of the combined preparation of the invention may be simultaneous, separate or sequential.

According to the present invention, a method for treating cancer is provided, comprising administering an anti-CXCR4 antibody of the present invention and inotuzumab simultaneously, concurrently, or sequentially. For example, the anti-CXCR4 antibody can be administered before, after, or concurrently with inotuzumab. Furthermore, the present invention provides a method for treating a cancer such as AML, comprising administering an anti-CXCR4 antibody of the present invention and Mylotarg simultaneously, concurrently, or sequentially. For example, the anti-CXCR4 antibody can be administered before, after, or concurrently with Mylotarg.

Anti-CXCR4 antibodies or fragments thereof can be conjugated to therapeutic moieties and/or diagnostic agents, such as cytotoxins, drugs (e.g., immunosuppressants), or radiotoxins. Such conjugates are referred to herein as antibody-drug conjugates. Antibody-drug conjugates can include one or more cytotoxins.

In some aspects of the invention, treatment comprises administering an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate of the invention in combination with one or more bioactive agents selected from antibodies, growth factors, hormones, cytokines, anti-hormones, xanthines, interleukins, interferons, and cytotoxic drugs. In a specific aspect of the invention, the bioactive agent is an antibody directed against a cell surface antigen expressed on a B-cell malignancy.

In some aspects of the invention, the antibodies directed against cell surface antigens expressed on B-cell malignancies are selected from anti-CD19, anti-CD20, and anti-CD33 antibodies. Such antibodies include the anti-CD20 antibody rituximab (Rituxan ^™ ).

In some aspects of the invention, the bioactive agent is a cytokine or growth factor and includes, but is not limited to, interleukin 2 (IL-2), TNF, CSF, GM-CSF, and G-CSF. In specific aspects of the invention, the bioactive agent is a hormone and includes estrogen, androgen, progesterone, and corticosteroids.

In some aspects of the invention, the drug is selected from the group consisting of doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, bendamustine, bevacizumab, bortesomib, lenalidomide, melphalan, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, and cytarabine. tatin, broxuridine, capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin, tegafur, tiazofurin, doxorubicin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, flurouracil, etoposide, taxol, a paclitaxel analog, and mitomycin.

In some aspects of the present invention, a therapeutically effective dose of an anti-CXCR4 antibody, fragment thereof, or anti-CXCR4 antibody-drug conjugate of the present invention is administered together with one or more combinations of tyrosine kinase inhibitors. Tyrosine kinase inhibitors include protein and non-protein moieties. Tyrosine kinase inhibitors can be, for example, antibodies, receptor ligands, or small molecule inhibitors. Examples of tyrosine kinase inhibitors suitable for use in the methods of the present invention include, but are not limited to, gefitinib, sunitinib, erlotinib, lapatinib, canertinib, semaxinib, vatalanib, sorafenib, imatinib, dasatinib, leflunomide, vandetanib, derivatives thereof, analogs thereof, and combinations thereof. Other tyrosine kinase inhibitors suitable for use in the present invention are described, for example, in U.S. Patent Nos. 5,618,829; 5,639,757; 5,728,868; 5,804,396; 6,100,254; 6,127,374; 6,245,759; 6,306,874; 6,313,138; 6,316,444; 6,329,380; ; No. 6,344,459; No. 6,420,382; No. 6,479,512; No. 6,498,165; No. 6,544,988; No. 6,562,818; No. 6,586,423; No. 6,586,424; No. 6,740,665; No. 6,794,393; No. 6,875,767, No. 6,927,293; and No. 6,958,340.

In some aspects, a therapeutically effective dose of an anti-CXCR4 antibody, fragment thereof, or anti-CXCR4 antibody-drug conjugate of the invention is administered with one or more drug combinations as part of a treatment regimen, wherein the cytotoxic agent combination is selected from: CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone, and procarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone); COP (cyclophosphamide, vincristine, and prednisone); CAP-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine, and prednisone); m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, and leucovorin); ProMACE-MOPP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine, and prednisone); m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, and leucovorin); (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin, nitrogen mustard, vincristine, prednisone, and procarbazine); ProMACE-CytaBOM (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin, cytarabine, bleomycin, and vincristine); MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, and leucovorin); MOPP (mechlorethamine, vincristine, prednisone, and procarbazine); ABVD (doxorubicin/doxorubicin, bleomycin, vinblastine, and dacarbazine); MOPP (mechlorethamine, vincristine, prednisone, and procarbazine) alternating with ABV (doxorubicin/doxorubicin, bleomycin, and vinblastine); MOPP (mechlorethamine, vincristine, prednisone, and procarbazine) , vincristine, prednisone, and procarbazine) alternating with ABVD (doxorubicin/doxorubicin, bleomycin, vinblastine, and dacarbazine); ChIVPP (chlorambucil, vinblastine, procarbazine, and prednisone); IMVP-16 (ifosfamide, methotrexate, and etoposide); MIME (methyl-gag, ifosfamide, methotrexate, and etoposide); DHAP (dexamethasone, high-dose cytarabine, and cisplatin); ESHAP (etoposide, methylprednisone, high-dose cytarabine, and cisplatin); CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone, and bleomycin); CAMP (lomustine) The 35-week trial included 15 patients with leukopenia (85%), 25 with leukopenia (1%), 25 with mitoxantrone, cytarabine, and prednisone; CVP-1 (cyclophosphamide, vincristine, and prednisone); ESHOP (etoposide, methylprednisone, high-dose cytarabine, vincristine, and cisplatin); EPOCH (etoposide, vincristine, and doxorubicin for 96 hours followed by a single dose of cyclophosphamide and oral prednisone); ICE (ifosfamide, cyclophosphamide, and etoposide); CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone, and bleomycin); CHOP-B (cyclophosphamide, doxorubicin, vincristine, prednisone, and bleomycin); CEPP-B (cyclophosphamide, etoposide, procarbazine, and bleomycin); and P/DOCE (epirubicin or doxorubicin, vincristine, cyclophosphamide, and prednisone).

In some aspects of the invention, a drug can be administered simultaneously, sequentially, or concurrently with an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate as described herein. For example, an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate can be administered prior to the combined administration of one or more cytotoxic agents as part of a therapeutic regimen. In some aspects of the invention, a therapeutically effective dose of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate is administered after the combined administration of one or more cytotoxic agents as part of a therapeutic regimen.

In some aspects of the invention, an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate may be administered in combination with one or more cytotoxic agents as part of a therapeutic regimen.

The anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates of the present invention may also be administered in conjunction with non-drug treatments such as surgery, radiotherapy, chemotherapy, immunotherapy, and diet/exercise regimens. Other treatments may be administered before, concurrently, or after treatment with the anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates of the present invention. There may also be a delay of hours, days, and in some cases weeks between the administration of different treatments, such that the anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates of the present invention may be administered before or after the other treatments.

Therapeutic uses of CXCR4

According to various aspects of the present invention, anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drugs can be used to treat a variety of conditions including the following or to produce medicaments for treating a variety of conditions including the following: various cancers, inflammatory conditions, allergic conditions, infections (such as HIV infection), autoimmune conditions (such as rheumatoid arthritis), fibrotic conditions (such as lung), and cardiovascular conditions. Cancers include solid tumor cancers (such as gastric cancer, head and neck cancer, lung cancer, ovarian cancer, and pancreatic cancer) and hematological cancers (such as myelodysplastic syndrome, myeloproliferative disorders, and acute leukemia). Examples of hematopoietic disorders include non-B lineage-derived disorders such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-B cell acute lymphoblastic leukemia (ALL), myelodysplastic disorders, myeloproliferative disorders, polycythemia vera, thrombocythemia, or non-B atypical immune lymphoproliferation. Examples of B-cell or B-cell lineage-derived disorders include chronic lymphocytic leukemia (CLL), B-lymphocytic lineage leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), B-cell prolymphocytic leukemia, precursor B-lymphoblastic leukemia, hairy cell leukemia, or plasma cell disorders, such as amyloidosis or Waldenstrom's macroglobulinemia.

Blood disorders

Hematologic disorders include diseases of the blood and all its components, as well as diseases of organs and tissues involved in the production or degradation of all components of the blood. In some aspects of the invention, hematologic disorders include, but are not limited to, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, mantle cell lymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulinemia (WM), B-cell lymphoma, and diffuse large B-cell lymphoma (DLBCL). NHL may include indolent non-Hodgkin's lymphoma (iNHL) or aggressive non-Hodgkin's lymphoma (aNHL). In certain aspects, the subject relapses or is refractory to other treatments. In certain aspects, the subject relapses or is refractory to at least two or more other treatments. In some aspects of the invention, the subject relapses or is refractory to at least three or more other treatments. In some aspects of the invention, the subject has relapsed or is refractory to at least five or more other treatments.

The present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising the anti-CXCR4 antibodies as primary therapeutic compositions for treating hematological disorders. The compositions may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies. Furthermore, the therapeutic compositions of the present invention may contain mixtures of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes, mixtures of anti-CXCR4 antibody-drug conjugates, or mixtures of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

chronic lymphocytic leukemia

CLL cells express high levels of CXCR4. (Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood. 94: 3658-3667 (1999)). The present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising anti-CXCR antibodies as primary therapeutic compositions for treating B-cell chronic lymphocytic leukemia (CLL). The compositions may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies, or mixtures of polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies. In addition, the therapeutic compositions of the present invention may contain mixtures of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes, or mixtures of anti-CXCR4 antibody-drug conjugates, or mixtures of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

Other B-cell lymphomas

CXCR4 expression has been shown in B cells (Burger et al., Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood. 94: 3658-3667 (1999)) and T cells (Trentin L, Agostini C, Facco M et al., The chemokine receptor CXCR4 is expressed on malignant B cells and mediates chemotaxis. J Clin Invest. 104: 115-121 (1999)), and non-Hodgkin lymphoma (NHL). Malignant B cells from B-NHL patients express functional CXCR4 receptors. Distinct patterns of chemokine receptor expression are thought to be associated with lymphoma cell migration and homing and may allow differentiation of different NHL subsets. (Jones D, Benjamin RJ, Shahsafaei A, Dorfman DM. The chemokine receptor CXCR4 is expressed in a subset of B-cell lymphomas and is a marker of B-cell chronic lymphocytic leukemia. Blood. 95: 627-632 (2000)). In one animal model, mice were challenged with T cell hybridoma cells engineered to retain cytoplasmic CXCR4. In another mouse model of human high-grade NHL, neutralization of CXCR4 by a monoclonal antibody inhibited the homing of circulating NHL cells and increased survival (Bertolini F, Dell'Agnola C, Mancuso P et al., CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin's lymphoma. Cancer Res. 62: 3106-3112 (2002)), thus proposing CXCR4 neutralization as a novel therapeutic approach for NHL. The present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising anti-CXCR4 antibodies as primary therapeutic compositions for treating B-cell chronic B-cell lymphoma. The compositions may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies. In addition, the therapeutic compositions of the present invention may contain mixtures of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes, mixtures of anti-CXCR4 antibody-drug conjugates, or mixtures of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

CXCR4 in multiple myeloma

Multiple myeloma (MM) is a large, incurable plasma cell neoplasm. The chemokine receptor CXCR4 is expressed by MM cells in most patients. It promotes myeloma cell migration and homing to the bone marrow (BM) compartment, supports tumor cell survival, and protects myeloma cells from chemotherapy-induced apoptosis. Therefore, the anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates (e.g., antagonistic antibodies) that inhibit CXCR4 activity of the present invention can be used to treat hematological disorders such as multiple myeloma. In addition, the therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes, a mixture of anti-CXCR4 antibody-drug conjugates, or a mixture of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

CXCR4 in acute leukemia

Because CXCR4 plays a key role in maintaining hematopoietic progenitor cells in the bone marrow, several groups have examined the role of CXCR4 in progenitor leukemias. Precursor B-cell acute lymphoblastic leukemia (ALL) expresses a functional CXCR4 receptor (Bradstock KF, Makrynikola V, Bianchi A, Shen W, Hewson J, Gottlieb DJ. Effects of the chemokine stromal cell-derived factor-1 on the migration and localization of precursor-B acute lymphoblastic leukemia cells within bone marrow stromal layers. Leukemia. 14:882-888 (2000)), which is associated with homing of leukemic cells to the bone marrow in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. (Shen W, Bendall LJ, Gottlieb DJ, Bradstock KF. The chemokine receptor CXCR4 enhances integrin-mediated in vitro adhesion and facilitates engraftment of leukemic precursor-B cells in the bone marrow. Exp Hematol. 29: 1439-1447 (2001)). In an ingenious animal model, Sipkins et al. (Sipkins DA, Wei X, Wu JW et al., In vivo imaging of specialized bone marrow endothelial microdomains for tumor engraftment. Nature. 435: 969-973 (2005)) directly demonstrated that functional CXCR4 is required for ALL cell homing to the bone marrow microenvironment. The present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising anti-CXCR antibodies as primary therapeutic compositions for the treatment of acute lymphoblastic leukemia (ALL). The composition may contain a polyclonal anti-CXCR4 antibody or a monoclonal anti-CXCR4 antibody.

Furthermore, the therapeutic compositions of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes or a mixture of anti-CXCR4 antibody-drug conjugates or a mixture of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

CXCR4 in acute myeloid leukemia (AML)

Despite general sensitivity to chemotherapy, long-term disease-free survival in AML remains low because most patients relapse with minimal residual disease (MRD). CXCR4 appears to be an important regulator of survival signals that contribute to resistance to anticancer drugs. This concept is supported by the finding that high levels of CXCR4 expression by leukemic cells are a poor prognostic indicator in AML. Spoo AC, Wierda WG, Burger JA. The CXCR4 score: a new prognostic marker in acute myelogenous leukemia [abstract]. Blood. 104: 304a (2004). The present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising anti-CXCR antibodies as primary therapeutic compositions for the treatment of acute myeloid leukemia (AML). The composition may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies.

Furthermore, the therapeutic compositions of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes or a mixture of anti-CXCR4 antibody-drug conjugates or a mixture of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

CXCR4 in non-hematopoietic cancers

One of the most interesting and potentially important roles of chemokines and chemokine receptors is in regulating the metastasis of solid tumors. CXCR4 is one of the best-studied chemokine receptors, selectively binding to the CXC chemokine stromal cell-derived factor 1 (SDF-1), also known as CXCL12 (Fredriksson et al., Mol Pharmacol. 63:1256-72 (2003)). To date, CXCR4 has been shown to be overexpressed in more than 20 human malignancies, including breast, prostate, kidney, colon, thyroid, and pancreatic cancers (Müller et al., Nature 410:50-6 (2001); Akashi et al. Cancer Sci. 99(3):539-542 (2008); Maréchal et al. Br J Cancer, 100, 1444-51 (2009); Wang et al., Clin Exp Metastasis, 26, 1049-54. (2009); He X et al., Pathol Res Pract, 206, 712-5. (2010)).

The present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising such antibodies as primary therapeutic compositions for treating non-hematopoietic cancers. Such compositions may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies. Furthermore, the therapeutic compositions of the present invention may contain mixtures of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes, mixtures of anti-CXCR4 antibody-drug conjugates, or mixtures of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

CXCR4 in breast cancer

High levels of CXCR4 expression on neoplastic cells are associated with relatively poor overall survival in breast cancer patients. (Li YM, Pan Y, Wei Y, et al., Up-regulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell. 6:459-469 (2004)). High levels of HER2/neu expression, observed in approximately 30% of all breast cancers, are also associated with relatively poor prognosis. Li et al. recently demonstrated that HER2/neu enhances CXCR4 expression and function by inhibiting CXCR4 degradation. (Li YM, Pan Y, Wei Y, et al., Up-regulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell. 6:459-469 (2004)). Therefore, the present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising anti-CXCR4 antibodies as primary therapeutic compositions for treating breast cancer. The composition may contain a polyclonal anti-CXCR4 antibody or a monoclonal anti-CXCR4 antibody. In addition, the therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes, a mixture of anti-CXCR4 antibody-drug conjugates, or a mixture of a monoclonal anti-CXCR4 antibody and an anti-CXCR4 antibody-drug conjugate.

CXCR4 in lung cancer

Small cell lung cancer (SCLC) is an aggressive, rapidly metastatic neoplasm with a high propensity to invade the bone marrow. Even with combined chemotherapy and radiation therapy, the 5-year survival rate is only approximately 5% due to the rapid development of drug resistance. In SCLC cells, CXCR4 activation induces migration and invasive responses and adhesion to bone marrow stromal cells in a CXCR4- and integrin-dependent manner. CXCR4 may drive the unique metastatic pattern observed in SCLC patients. Therefore, the present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising anti-CXCR4 antibodies as primary therapeutic compositions for treating lung cancer. The compositions may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies. In addition, the therapeutic compositions of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes, a mixture of anti-CXCR4 antibody-drug conjugates, or a mixture of a monoclonal anti-CXCR4 antibody and an anti-CXCR4 antibody-drug conjugate.

CXCR4 in renal cell carcinoma (RCC)

Recently, the role of CXCR4 in mRCC has been thoroughly elucidated. The results show that the high expression of CXCR4 is strongly correlated with the poor survival of mRCC patients (Wang et al., Clin Exp Metastasis, 26, 1049-54 (2009); Zhao et al., Mol Biol Rep, 38, 1039-45 (2011)). In addition, results were obtained in a murine model, in which the metastatic ability of RCC cells expressing CXCR4 was strongly correlated with CXCR4 protein content on cancer cells and SDF-1α expression in target organs (Motzer et al. The New England Journal of Medicine, Vol. 335, No. 12, pp. 865-875 (1996)). Therefore, CXCR4 may be an interesting therapeutic target in the multimodal therapy of renal clear cell carcinoma.

The present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates comprising anti-CXCR4 antibodies as primary therapeutic compositions for treating renal cell carcinoma (RCC). The compositions may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies.

Furthermore, the therapeutic compositions of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes or a mixture of anti-CXCR4 antibody-drug conjugates or a mixture of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

CXCR4 in non-oncology indications

Chemokine receptors are expressed on various specific cells and at specific times. They are primarily involved in the control of inflammatory and immune responses by mechanisms that cause effector cells to accumulate at sites where chemokines are produced. For example, SDF-1 has been shown to specifically inhibit T cell-directed ( _X4 ) HIV infection in vitro (Bleul et al., Nature, 382:829-833 (1996); Oberlin et al., Nature, 833-835 (1996)). This can be seen as SDF-1 binding to CXCR4 before HIV, thereby removing the backbone that allows cells to be infected with HIV, thereby inhibiting HIV infection. In addition, HIV infection inhibitors have been shown to be CXCR4 antagonists (Nat. Med., 4, 72 (1998)).

Therefore, the present invention encompasses the use of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates thereof as therapeutic compositions for treating inflammatory and immune diseases, allergic diseases, infections (such as HIV infection), and diseases associated with HIV infection (such as acquired immune deficiency syndrome). The compositions may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies.

Furthermore, the therapeutic compositions of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes or a mixture of anti-CXCR4 antibody-drug conjugates or a mixture of monoclonal anti-CXCR4 antibodies and anti-CXCR4 antibody-drug conjugates.

WHIM syndrome

WHIM syndrome is a rare congenital immunodeficiency disorder characterized by the following main clinical manifestations: warts, hypogammaglobulinemia, recurrent bacterial infections, and neutropenia. [McDermott, DH et al. Blood 118(18):4957-4962 (2011); McDermott DH et al. J. Cell. Mol. Med. 15(10):2071-2081 (2011)]. Neutropenia is further characterized by an unusual blood disorder in which mature neutrophils fail to leave the bone marrow and lack a significant number of B and T cells or their function (Zueler WW et al. N. Engl. J. Med. 270:699-704 (1964)). Hernandez PA et al. first described mutations in CXCR4 as being associated with WHIM syndrome, with CXCR4 ^R334X being the most common and best-studied variant (Hernandez PA et al. Nature Genetics 34:70-74 (2003)). CXCR4 ^R334X , as a gain-of-function mutation, exhibits enhanced signaling to the endogenous ligand CXCL12. Therefore, the increased signaling capacity of CXCR4 ^R334X could be exploited to treat WHIM syndrome.

In some aspects of the present invention, the present invention encompasses the use of antibody-drug conjugates comprising anti-CXCR4 antibodies as primary therapeutic compositions for treating WHIM syndrome, wherein the compositions may contain polyclonal anti-CXCR4 antibodies or monoclonal anti-CXCR4 antibodies. In addition, the therapeutic compositions of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed against different non-blocking CXCR4 epitopes, or a mixture of anti-CXCR4 antibody-drug conjugates, or a mixture of a monoclonal anti-CXCR4 antibody and an anti-CXCR4 antibody-drug conjugate. Furthermore, in some aspects, the therapeutic compositions disclosed herein may be administered alone or in combination with current treatments for WHIM syndrome, such as G-CSF (filgrastim (Neupogen; Amgen Inc.)), intravenous immunoglobulin, and the small molecule CXCR4 antagonist AMD3100 (plerixafor, trade name Mozobil (Genyzme Corporation)).

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In fact, various modifications of the present invention except as shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Example 1

Antibody binding affinity determination of chimeric anti-CXCR4 mouse antibodies

The binding of chimeric mouse anti-CXCR4 antibodies 12A11, 6B6, and 3G10 (expressed as hIgG1 subtype) was evaluated by flow cytometry on human non-Hodgkin's lymphoma (NHL) Ramos cells expressing CXCR4. 100,000 cells were incubated with serial dilutions of anti-CXCR4 antibody in 100 μL of binding buffer (PBS + 0.5% BSA), followed by incubation with a Dylight 488-conjugated goat anti-human Fc secondary antibody from Jackson Immunoresearch Laboratories. The MFI value for each dilution was normalized to the maximum value to derive the percentage. EC50 was calculated using PRISM software.

Table 6

μg/mL 12A11 6B6 3G10 50 100.0% 100.0% 100.0% 20 79.1% 97.5% 103.8% 5 38.0% 90.7% 91.1% 2 13.4% 87.8% 88.0% 0.5 5.9% 79.8% 77.8% 0.2 3.1% 67.3% 65.7% 0.05 1.6% 44.6% 25.4% 0.02 0.1% 12.8% 10.2% 0.005 0.1% 3.8% 3.2% 0.002 0.0% 3.0% 2.0% EC50 (μg/mL) 7.28 0.09 0.13 EC50 (nM) 48.51 0.63 0.89

Example 2

Binding of humanized 3G10 Fab to HPB-ALL cells expressing CXCR4

Binding of humanized 3G10 Fab was assessed by flow cytometry on HPB-ALL cells expressing CXCR4. 150,000 cells were incubated with 2.5 or 0.25 μg/mL of the different h3G10 Fabs in 100 μL binding buffer (PBS + 0.5% BSA), followed by incubation with an APC-conjugated goat anti-human Fab-specific secondary antibody from R&D systems.

Table 7

Example 3

CXCR4 Antibody Binding: Cell Binding and Affinity Measurements of CXCR4Ab Fab

The binding of CXCR4 Fab to cells expressing CXCR4 was measured on HPB-ALL (human T cell leukemia) cells by flow cytometry. 150,000 HPB-ALL cells were resuspended in 100 μL FACS buffer (1×PBS+0.5% BSA) on a 96-well plate. Anti-CXCR4 Fab was added to each well to a final concentration of 0.25 μg/mL and incubated at 4°C for 30 minutes. After removing the primary antibody, the cells were washed twice with FACS buffer, then resuspended in FACS buffer, and then 2 μL (3 μg) of secondary antibody (Alexa Fluor 647-conjugated goat anti-human IgG, F(ab')2 specific, Jackson ImmunoResearch Laboratories, West Grove PA) was added to each well. The plate was incubated for another 30 minutes at 4°C. Fluorescent signals were obtained by cell analyzer LSRII (BD Biosciences, San Jose CA). The table shows the MFI (mean fluorescence intensity) of each sample.

The binding affinity of each Fab was determined using lipid particles enriched with human CXCR4 (LEV101, Integral Molecular, Philadelphia, PA). Biotinylated WGA (Sigma Aldrich, St. Louis, MO) was coated onto an SA chip to facilitate capture of the lipid particles containing the CXCR4 protein. Serial dilutions of the Fab (five sets, 3× dilution factor, highest concentration of 10 or 30 nM) were injected from low to high concentrations (each concentration with a 3-minute binding time). The data were kinetically analyzed using the "kinetic titration" method described in Karlsson et al. (Karlsson, R., Katsamba, P.S., Nordin, H., Pol, E., and Myszka, D.G. Analyzing a kinetic titration series using affinity biosensors. Anal. Biochem. 349, 136-147 (2006)). For some analysis cycles, buffer was injected over the capture particles instead of Fab to provide blank cycles for double referencing purposes (double referencing was performed as described in Myszka et al. Improving biosensor analysis. J. Mol. Recognit. 12, 279-284 (1999)).

Table 8

Example 4

Binding of CXCR4Ab to macaque and human CXCR4

Cross-reactivity of anti-human CXCR4 Abh3G10.1.91.A58B with macaque CXCR4 was tested by flow cytometry in serial dilutions (0.007-267 nM) against 1) HPB-ALL (human T-cell leukemia) and macaque CXCR4-transfected CHO cells, and 2) Raji (human non-Hodgkin's lymphoma) and HSC-F (a macaque T-cell line). Binding was detected using a secondary antibody (AlexaFluor 647-conjugated goat anti-human IgG, Fcγ-specific, Jackson ImmunoResearch Laboratories, West Grove, PA) and acquired using an LSR Fortessa cell analyzer (BD Biosciences, San Jose, CA). Curve fitting and EC50 calculations were performed using Prism software (GraphPad Software, La Jolla, CA). Tables 9A and 9B and Figures 3A and 3B.

Table 9A

HPB-ALL CHO-cynomolgus monkey CXCR4 EC50 (nM) 0.27 0.16

Table 9B

Raji HSC-F EC50 (nM) 3.050 2.585

Example 5

Effector function of CXCR4 antibodies

Therapeutic naked antibodies rely on two types of functions to achieve clinical efficacy: target-specific binding by Fab (antigen binding fragment) domains, and immune-mediated effector functions via the interaction of the Fc domains of the antibody with the Fc receptors on various cell types, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the antibody Fc region binds to the Fc receptors (FcγR) on the surface of immune effector cells (such as natural killer cells and macrophages), resulting in phagocytosis or dissolution of the target cells. In CDC, antibodies kill the target cells by triggering the complement cascade on the cell surface. Therefore, the Fc portion of the therapeutic antibody can play an important role in its mechanism of action via the impact on ADCC or CDC.

Each subclass of human IgG (IgG1, IgG2, IgG3, and IgG4) exhibits unique properties of effector function, which are dictated by their distinct binding to each of the FcγRs and complement complex proteins. While IgG1 and IgG3 bind relatively strongly, IgG2 and IgG4 have much lower affinity for Fc receptors and do not induce high levels of ADCC. IgG1 also has a higher affinity for complement complex proteins, while IgG2, IgG3, and IgG4 induce much lower levels of CDC.

The ADCC (antibody-dependent cellular cytotoxicity) activity of anti-CXCR4 antibodies was determined using the cytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison WI). The day before the assay, human tumor cells expressing CXCR4 were seeded at 10,000 cells. Donor PBMC cells were separated via a Ficoll gradient and cultured overnight in x-vivo medium at 37°C. The next day, 10 or 20 μg/mL of antibody were added to the wells, with or without the addition of 1,000,000 PBMC cells in RPMI+5% FBS (E:T=100:1). The plates were then incubated at 37°C for 4 hours. At the 3.5 hour time point, 20 μL of lysate was added to the wells containing only target cells. After spinning the plate at 8000 rpm for 3 minutes, 50 μL of supernatant was transferred to another plate. 50 μL of substrate was then added to each well and the plate was incubated at room temperature in the dark for 30 minutes. The reaction was stopped by adding 50 μL of stop solution to each well. The plate was then read at 490 nm using a spectrophotometer (Molecular Devices). The specific solubility percentage (%) was calculated as follows:

Specific lysis % = (treated LDH release - spontaneous LDH release from target cells - spontaneous LDH release from effector cells) / (maximum LDH release from target cells - spontaneous LDH release from target cells) × 100

CDC (antibody-dependent cellular cytotoxicity) activity of anti-CXCR4 antibodies was determined using the cytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI). One day prior to the assay, human tumor cells expressing CXCR4 were seeded at 10,000 cells. The following day, 5-20 μg/mL of each Ab was added to cells alone or with 2.5%, 10%, or 20% donor AB complement (from Innovative Biotech or Sigma). Plates were processed similarly to the ADCC assay and specific lysis assays were performed.

ADCC assays were performed on CXCR4-expressing Ramos (NHL) or MOLT-4 (human T-cell leukemia) cells using 20 μg/mL (Figure 4A) or 10 μg/mL (Figure 4C) of antibody plus normal donor PBMCs at a 100:1 (effector:target) ratio. After 4 hours of incubation, ADCC activity was evident for anti-human CXCR4 mouse Abs 12A11, 6B6, and 3G10, raised as chimeric hIgG1 on Ramos (NHL) cells (Figure 4A) or humanized 3G10 antibody on MOLT-4 cells (Figure 4C). Approximately 80% cell lysis was observed with m3G10-hIgG1 treatment, compared to approximately 20% cell lysis with the m3G10-hIgG4 isoform on MOLT-4 cells (Figure 4B).

CDC activity was observed in CXCR4-expressing Ramos cells treated with 20 μg/mL chimeric mouse 12A11, 6B6, and 3G10-hIgG1 antibodies ( FIG4D ), and in Daudi (NHL) cells treated with 5 μg/mL humanized antibodies ( FIG4F ). 5 μg/mL of the IgG1-backbone anti-CXCR4 3G10 antibody detected a maximum specific lytic activity of 20%-30%, similar to the positive control antibody Rituxan on Daudi cells, while the hIgG4 form of 3G10 showed no CDC activity ( FIG4E ).

Example 6

Inhibition of SDF-1-induced calcium flux by anti-CXCR4 antibody

When SDF-1 (ligand) binds to the CXCR4 receptor, it triggers a calcium mobilization response. To confirm the functional antagonist activity of CXCR4 antibodies—that is, the ability of anti-human CXCR4 antibodies to inhibit SDF-1-induced calcium mobilization—human T-cell leukemia (Jurkat) cells were used in conjunction with the Fluo-NW Calcium Assay Kit (Molecular Probes/Life Technologies). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% glutamine at 37°C in a _CO2 incubator until subconfluent. On the day of the assay, cells were plated at 70,000 cells per well in a black, clear-bottomed 384-well plate in 25 μl of assay buffer. The assay dye was then added to the plate at 25 μl per well for 110 minutes at room temperature, protected from light. For each anti-human CXCR4 antibody, 11 serial 1:3 dilutions were prepared in the Fluo-NW Calcium Assay Kit buffer, yielding a concentration range of 500 nM to 8 pM. The cells were incubated at room temperature for 20 minutes with the antibody. The cells were then stimulated with SDF-1α (Invitrogen) at a final concentration of 8 nM. Calcium flow was then measured using a FLIPR Tetra (Molecular Devices) for 95 seconds. The positive control consisted of cells in the presence of SDF-1α and without antibody treatment. The baseline was measured by cells that did not contain SDF-1α and without antibody treatment. The SDF-1α induction of calcium was measured by the development of the fluorescence signal over time. GraphPad Prism software was used to output and plot data, and EC50 values were calculated using a nonlinear curve fit with an S-shaped dose-effect formula. The inhibition of calcium flow by the resulting anti-human CXCR4 antibodies is shown in Figure 5. Antibodies 3G10, 6B6, and 12A11 inhibited SDF-1α-induced calcium flow, with inhibition IC50 values of 1.555 nM, 1.418 nM, and 27.07 nM, respectively (Table 10). The IC50s of the humanized CXCR4 antibodies evaluated in the calcium flux functional assay are summarized in Table 11 (average of n=3 independent experiments/antibody). In summary, the potency of the humanized CXCR4 antibodies was similar to that of the chimeric m3G10 antibody in this assay.

Table 10: Calcium mobilization activity of chimeric CXCR4 antibodies

3G10 6B6 12A11 IC50 1.555 1.418 27.07

Table 11: Calcium mobilization activity of humanized CXCR4 antibodies

Heavy chain light chain Antibody name IC50(M) m3G10VH m3G10VL m3G10 1.25E-09 h3G10VH h3G10VL h3G10 2.00E-09 A57 h3G10VL h3G10.A57 1.69E-09 h3G10.2.37VH h3G10.2.72VL h3G10.2.37.2.72 1.36E-09 h3G10.A57VH h3G10.A58A VL h3G10.A57.A58A 1.31E-09 h3G10.1.91VH h3G10.A58A VL h3G10.1.91.A58A 2.35E-09 h3G10.1.91VH h3G10.A58B VL h3G10.1.91.A58B 2.17E-09

Example 7

CXCR4 Antibody Activity in Cell-Based Function Assays of Cyclic AMP (cAMP)

When CXCL12 (ligand) binds to the CXCR4 receptor, cAMP is known to be inhibited. To confirm the antagonistic activity of CXCR4 Abs, a cAMP assay was performed using CHO-K1 cells transfected with human CXCR4 (purchased from DiscoverRx, Fremont, CA). The cAMPHunter eXpress GPCR Assay Kit (DiscoveRx) was used to perform the assay. In the absence of CXCR4 antibodies, treatment with the CXCR4 ligand CXCL12 inhibited cAMP production. Upon treatment with CXCR4 antibodies, this inhibition was abolished, and cAMP production was increased. The EC50 for this reaction is shown in Table 12. In summary, all tested CXCR4 antibodies had similar potent activity in this assay, ranging from 24.3 to 45.6 nM. This study demonstrates that mouse (chimeric) 3G10 and humanized 3G10 CXCR4 antibodies can compete with and block CXCR4 ligand activity in a cAMP functional assay.

Table 12: Summary of EC50s of anti-CXCR4 Abs in cAMP assay

Testing antibodies EC50(M) Isotype control antibodies 9.80E+10 m3G10 4.56E-08 h3G10.1.91.A58B 3.92E-08 h3G10.2.37.2.72 3.41E-08 h3G10.A57.A58A 2.43E-08

Example 8

Induction of cell death by anti-CXCR4 antibodies

The ability of anti-CXCR4 antibodies to induce cell death in the human non-Hodgkin's lymphoma Ramos cell line was tested. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 2nM glutamine at 37°C in a _CO2 incubator to subconfluence. Cells were seeded in growth medium in 48-well plates at 2.5× ¹⁰⁶ cells/ml. Anti-CXCR4 antibodies diluted to the specified concentrations in PBS were added to the cells and incubated at 37°C for 24 hours. Cell death was measured by Annexin V-PE and propidium iodide (PI) fluorescence signals measured in an LSRII flow cytometer (BD Biosciences). Overall cell death was determined by adding the Annexin V+/PI+ (late) population to the Annexin V+/PI- (early) population.

The results in Figure 6A show that 6B6 and 3G10 anti-human CXCR4 antibodies were able to induce cell death in Ramos cells in a dose-dependent manner. At the highest tested concentration of 100 nM, 3G10 and 6B6 caused >70% cell death. This effect exceeded that of the positive control, staurosporin (STS), a known potent inducer of cell death (approximately 60%). Untreated cells showed approximately 30% cell death in this assay. The anti-CXCR4 12A11 antibody was unable to induce cell death under the conditions tested. Similar results were observed when the activity of these antibodies was tested on Raji non-Hodgkin's lymphoma cells.

In similar studies, single-chain (Fab) and bivalent F(ab)2' fragments were generated from the 3G10 antibody. Fab is a single-chain antibody (monovalent) that contains the immunoglobulin variable region as part of the antigen-binding site and the first immunoglobulin constant region. This fragment was obtained by digesting the 3G10 antibody with the proteolytic enzyme papain. Pepsin digestion removes most of the Fc region while leaving some of the hinge region intact to produce F(ab')2. The F(ab')2 fragment has two antigen-binding Fab portions linked by a disulfide bond and is therefore bivalent. Fab and F(ab')2 were tested alongside the bivalent, full-length 3G10 antibody for their ability to induce cell death in Ramos cells. The results, shown in Figure 6B, demonstrate that the ability to induce cell death is bivalency-dependent, with the full-length antibody (3G10) and F(ab')2 antibody able to induce cell death, while the 3G10 Fab had a very limited effect on cell death.

Example 9

Inhibition of non-Hodgkin's lymphoma solid tumor growth by anti-CXCR4 antibodies in vivo

SDF-1/CXCR4 signaling is believed to play an important role in multiple stages of tumor development, including tumor growth, angiogenesis, invasion, and metastasis. To evaluate the ability of anti-human CXCR4 Ab to inhibit tumor growth, a tumor xenograft model using female 4-6 week old CB17SCID Beige mice (Jackson Laboratories) and subcutaneously implanted human non-Hodgkin's lymphoma Ramos cells was used. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a 5% _CO2 incubator. In this study, 5× ¹⁰⁶ cells were implanted in the right flank of each mouse and allowed to grow as solid tumors to an average size of approximately 175 ^mm3 , with the volume calculated by the formula (volume = length × ^width2 )/2. Mice were then randomized into 4 different treatment groups, with n = 10 animals per group. Each group of mice was treated with an intravenous (iv) injection of the following antibodies in sterile PBS solution: (i) isotype control Ab (15 mg/kg); (ii) 3G10 (15 mg/kg); (iii) 6B6 (15 mg/kg); and (iv) 12A11 (15 mg/kg). The animals were given the antibodies once a week for 3 weeks, for a total of 3 doses. Tumor volume was measured using a caliper once to three times a week for the duration of the study. The results of the experiment are shown in Figure 7. The results show that all three anti-CXCR4 antibodies tested significantly inhibited tumor growth compared to the isotype control antibody. The results show that anti-CXCR4 antibodies are able to inhibit the growth of established solid tumors in vivo. After the antibody administration was stopped, the tumor growth inhibition effect lasted for a long time (42 days). The activity of all three anti-CXCR4 antibodies tested was comparable in this solid tumor model. Figure 7.

Example 10

Anti-CXCR4 antibodies increase survival and reduce tumor size in a mouse systemic non-Hodgkin's lymphoma cell model Tumor burden

The anti-tumor activity of anti-CXCR4 antibodies was further studied in a hematological lymphoma model using Raji non-Hodgkin's lymphoma cells. The Raji cells used in this study were transfected with the luciferase gene (LUC) to allow for in vivo monitoring of tumor burden over time. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a 5% _CO2 incubator. The model was established by injecting 1× ¹⁰⁶ Raji-LUC cells into female 6-8 week old SCID Beige mice (from Charles River Laoratories) via the tail vein. On day 0 (the day of implantation), Raji-LUC cells were present in the lungs, indicating accurate intravenous injection of tumor cells in all animals. One day after cell implantation, mice were assigned to treatment groups (10 animals per group) based on the presence of lung bioluminescence. Mice were treated intraperitoneally (ip) once a week with 10 mg/kg of isotype control antibodies 3G10, 6B6, and 12A11 in sterile PBS until day 67. Tumor burden was monitored by bioluminescence imaging using a Xenogen IVIS 200 imaging system. Mice were imaged every 5 days in the abdominal area, luciferin was delivered intraperitoneally at 15 mg/mL, 200 μl was injected, and whole body bioluminescence was measured using Xenogen Living Image. Mice were sacrificed when they began to show hind limb paralysis or reached other humane endpoints. Tumor burden (as measured by bioluminescence) and survival were both assessed as endpoints.

Figures 8A and 8B show the bioluminescence results of this study. The reduction in tumor burden is shown by a decrease in bioluminescence levels over time. Figure 8A shows representative images of 4-5 animals in each group on day 0 (implant baseline), day 15, and day 25 of the study. As measured by luminescence levels, all three anti-CXCR4 antibodies significantly reduced tumor burden. Figure 8B shows that in this model, treatment with 3G10, 6B6, and 12A11 antibodies had comparable TGI activity relative to the isotype control antibody.

The survival curves shown in Figure 8C demonstrate that anti-human CXCR4 antibodies 3G10, 6B6, and 12A11 had a highly significant effect on the survival of animals systemically implanted with Raji-LUC cells compared to isotype control antibodies. While isotype control-treated animals showed a median survival of 18 days, animals treated with anti-CXCR4 antibodies did not reach median survival before study termination. All three anti-CXCR4 antibodies tested had comparable effects, with no statistical differences between them. Survival of anti-CXCR4 antibody-treated animals persisted well beyond day 67, the time the last antibody dose was administered.

Example 11

Anti-CXCR4 antibodies increase survival and reduce leukemia in a mouse systemic acute myeloid leukemia (AML) cell model. Low tumor burden

The anti-CXCR4 antibody 6B6 was tested for its ability to increase survival and reduce tumor burden in NSG mice using a disseminated/systemic intravenous model of AML. The human AML cancer cell line MV4-11 was transduced with the luciferase gene (MV4-11-LUC) to allow for in vivo monitoring of tumor burden over time. Cells were cultured in IMDM medium containing 10% fetal bovine serum and 1 μg/mL puromycin for luciferase expression selection at 37°C in a 5% _CO2 incubator. 4-6 week old NSG female mice (from Jackson Laboratories) were injected intravenously with MV4-11-LUC AML cells (1× ₁₀₆ /animal).

On day 13 after cell implantation, mice were randomized into three treatment groups (10 animals per group) based on total body bioluminescence intensity. Weekly subcutaneous (s.c.) antibody treatment at 10 mg/kg in sterile PBS solution was started on day 13 (isotype control and anti-CXCR4 6B6 antibody) and day 20 (anti-CXCR4 6B6 antibody), as shown in the figure (day 13; day 20). Tumor burden was assessed using a Xenogen IVIS 200 imaging system. Mice were imaged in the abdominal position every 7 days and whole body bioluminescence was measured using Xenogen Living Image software. Mice were sacrificed when they began to show hind limb paralysis or reached other humane endpoints. Tumor burden (as measured by bioluminescence) and survival were assessed at the endpoint. Blood samples collected on day 35 of the study were monitored for the number of human AML cells circulating in the peripheral blood (PB) of mice (n=10 per group). AML CD45 and CD33 markers were used to determine the percentage of circulating human AML cells by flow cytometric staining with anti-human CD45 and anti-human CD33 antibodies (from BD Biosciences).

Figure 9A shows representative bioluminescence imaging of 5 animals/treatment group in this study (from day 20 to day 41). The reduction in tumor burden is shown by a decrease in bioluminescence levels over time. The inhibition of tumor burden was more pronounced when the anti-CXCR46B6 antibody was started earlier (day 13) than when it was started a week later (day 20). Compared to the isotype control antibody, the 6B6 anti-CXCR4 antibody significantly reduced tumor burden in both treatment groups, indicating that the anti-CXCR4 antibody effectively inhibits tumor burden in a staged disseminated mouse model of human AML.

The survival curves shown in Figure 9B demonstrate that the anti-human CXCR4 antibody 6B6 has a significant effect on the survival of animals systemically implanted with MV4-11-LUC AML cells compared to an isotype control antibody. While isotype control-treated animals showed a median survival of 40 days, animals treated with the anti-CXCR4 6B6 antibody on days 13 and 20 had a median survival of 53 days and 61 days, respectively. These differences were statistically significant (p < 0.05) by the Mantel-Cox test.

Figure 9C shows that on study day 35, the number of human AML cells was significantly reduced in animals treated with anti-CXCR4 6B6 antibody compared to isotype control-treated animals.

Example 12

Anti-CXCR4 antibodies increase survival in a systemic chronic lymphocytic leukemia tumor model in mice

The anti-tumor activity of CXCR4 antibodies was further studied in a disseminated intravenous chronic lymphocytic leukemia (CLL) model. Human JVM-13 tumor cells stably transfected to express the luciferase gene were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 0.25 mg/mL puromycin at 37°C in a 5% _CO2 incubator. The model was established by injecting 1× ¹⁰⁶ JVM-13-Luc cells into female 6-8 week old SCIDBeige mice via the tail vein. Based on bioluminescence (BLI) readings, mice were assigned to treatment groups (10 animals per group) on day 21 after cell implantation. Mice were treated with an isotype control antibody, 3G10, administered subcutaneously (sc) at 10 mg/kg in sterile PBS solution once a week. Rituximab (an anti-CD20 Ab approved for the treatment of CLL patients) was used as a positive control. Rituximab was administered subcutaneously at 10 mg/kg in sterile PBS once weekly. Tumor burden was assessed using a Xenogen IVIS 200 imaging system. Mice were imaged in the abdominal area every 7 days and whole-body bioluminescence was measured using Xenogen Living Image software. Mice were sacrificed when they began to show hindlimb paralysis or reached other humane endpoints. Tumor burden (as measured by bioluminescence) and survival were both assessed as endpoints.

Figure 10A shows representative luciferase activity imaging of mice in each treatment group on study days 28, 35, 42, 49, and 56. Treatment with 3G10 significantly reduced JVM-13-Luc tumor burden in the large bones compared to the isotype control antibody, as measured by luminescence levels over time. Treatment with rituximab also significantly reduced tumor burden compared to the isotype control antibody.

Figure 10B shows the Kaplan-Meyer survival curves for this study. A significant increase in survival was observed with 3G10 and rituximab compared to the isotype control Ab (p < 0.0001). While animals treated with the isotype control antibody showed a median survival of 32.5 days, animals treated with Rituxan showed a median survival of 45 days, and animals treated with 3G10 did not reach median survival before day 60. The results of this study demonstrate that anti-CXCR4 antibodies effectively inhibit tumor burden in a staged, disseminated mouse model of human CLL.

Example 13

Humanized anti-CXCR4 antibody inhibits tumor growth of Ramos non-Hodgkin's lymphoma in vivo

To evaluate the ability of humanized CXCR4Ab to inhibit tumor growth, a tumor xenograft model using female 4-6 week old CB17.cg-Prkdc SCID Beige mice (Charles River) and subcutaneously implanted human non-Hodgkin's lymphoma Ramos cells was used. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a 5% _CO2 incubator. In this study, 5× ¹⁰⁶ cells were implanted in the right flank of each mouse and allowed to grow as solid tumors to an average size of approximately 350 ^mm3 , with the volume calculated by the formula (volume = length × ^width2 )/2. Mice were then randomized in 7 different treatment groups, with n = 10 animals per group. Groups of mice were treated subcutaneously (sc) with 10 mg/kg of each antibody in sterile PBS: (isotype control antibody; m3G10; h3G10.A57.WT; h3G10.2.37.2.72; h3G10.A57.A58; h3G10.1.91.A58A; h3G10.1.91.A58B). The animals were dosed with antibodies once a week for two weeks, for a total of two doses. Tumor volumes were measured twice a week using calipers for the duration of the study. The results are shown in Figure 11. The results show that all tested anti-CXCR4 antibodies significantly inhibited tumor growth compared to the isotype control antibody. The results indicate that the tested humanized CXCR4 antibodies had efficacy similar to that of the chimeric m3G10 antibody. The tumor growth inhibition effect persisted for several days after cessation of antibody administration (day 7).

Example 14

Humanized anti-CXCR4 antibody increases survival and reduces tumor progression in a systemic multiple myeloma (MM) model in mice Tumor burden

The humanized anti-CXCR4 antibody h3G10.1.91.A58B was tested for its ability to increase survival and reduce tumor burden in NSG mice using a disseminated/systemic intravenous model of MM. The human MM cancer cell line OPM-2 was transduced with the luciferase gene (OPM-2-LUC) to allow for in vivo monitoring of tumor burden over time. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 0.5 μg/mL puromycin for luciferase expression selection at 37°C in a 5% _CO2 incubator. OPM-2-Luc cells (5× ¹⁰⁶ cells/animal) were injected intravenously into 4-6 week old female NSG mice.

On the 8th day after cell implantation, mice were randomized to three treatment groups (10 animals per group) based on total body bioluminescence intensity. Weekly subcutaneous (s.c.) antibody treatment of 10 mg/kg in sterile PBS solution was started and continued for 5 weeks. Melphalan (one of the drugs approved for the treatment of multiple myeloma patients) was given at 1 mg/kg twice a week for 3 weeks. Tumor burden was assessed using the Xenogen IVIS 200 imaging system. Mice were imaged in the abdominal position every 7 days and whole body bioluminescence was measured using Xenogen Living Image software. When mice began to show hind limb paralysis or reached other humane endpoints, they were sacrificed. Tumor burden (measured by bioluminescence) and survival were both assessed as endpoints.

Figures 12A and 12B show the bioluminescence (luciferase activity) results from this study. The reduction in tumor burden is shown by a decrease in bioluminescence levels over time. Figure 12A shows representative bioluminescence imaging of 5 animals/treatment groups from this study. Figure 12B shows that treatment with the CXCR4 antibody h3G10.1.91.A58B significantly inhibited tumor growth over time compared to the isotype control antibody (p < 0.0001 on day 30), indicating that anti-CXCR4 antibodies effectively inhibit tumor burden in a staged disseminated xenograft model of human multiple myeloma. The survival curves from this study, shown in Figure 12C, confirm that the anti-CXCR4 antibody h3G10.1.91.A58B had a significant effect on the survival of animals systemically implanted with OPM-2-Luc MM cells compared to the isotype control antibody. While isotype control and melphalan-treated animals showed a median survival of 33.5 and 36 days, respectively, animals treated with h3G10.1.91.A58B CXCR4 antibody had an indeterminate median survival with no deaths observed on study day 50. Table 13. These differences were statistically significant by Mantel-Cox test (p<0.0001).

Table 13

Isotype control h3G10.1.91.A58B Melphalan Median survival (days) 33.5 uncertain 36

Example 15

Cytotoxicity of anti-CXCR4 ADCs in CXCR4-positive cells

Anti-CXCR4 antibodies (e.g., 3G10) were expressed as human IgG1 subtype and transfected with a glutamine-containing transglutaminase ("Q") tag, TG6 (SEQ ID NO:91 (LLQGA)) was modified and conjugated with AcLys-vc-PABC-MMAD (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl-MMAD) and AcLys-vc-PABC-0101 ((2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide). A transglutaminase tag was engineered at the C-terminus of the heavy chain of the antibody. Subsequently, an anti-CXCR4 antibody carrying a glutamine-containing tag at a specific site (e.g., the carboxyl terminus of the antibody heavy chain) was conjugated with an effective negative The conjugation of anti-CXCR4 antibodies to the cytotoxic agent MMAD or 0101 is achieved by a transamidation reaction catalyzed by a microbial transglutaminase between an amine-containing derivative of a carrier (e.g., MMAD or 0101). In some cases, the wild-type amino acid lysine at the carboxyl terminus (position 447 according to the EU numbering scheme) is deleted and replaced by a Q-tag. In other cases, the wild-type amino acid lysine at position 222 (according to the EU numbering scheme) is replaced by the amino acid arginine ("K222R"). The K222R substitution provides a significant effect in producing more homogeneous antibody and payload conjugates, and/or allows better intermolecular cross-linking between the antibody and the payload. In the transamidation reaction, the glutamine on the glutamine-containing tag is used as an acyl donor, and the amine-containing compound is used as an acyl acceptor (amine donor). In Streptoverticillium Purified anti-CXCR4 antibodies were incubated with excess acyl acceptor in the presence of 150-900 mM NaCl and 25 ^mM MES, HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], or Tris HCl buffer at a pH range of 6.2-8.8 in the presence of α-mobaraense transglutaminase (ACTIVA™, Ajinomoto, Japan). Reaction conditions were tailored to the individual acyl acceptor derivatives. After incubation for 2.5 hours at room temperature, the antibody-drug conjugate was purified on MabSelect resin (GE Healthcare, Waukesha, WI) using standard affinity chromatography methods known to those skilled in the art, such as commercial affinity chromatography methods from GE Healthcare.

CXCR4-expressing cells were then seeded on white-walled, clear-bottomed plates at 4,000 to 8,000 cells per well 24 hours prior to treatment. Cells were then treated with 4-fold serial dilutions of the antibody-drug conjugate in triplicate. Cell viability was determined 96 hours after treatment using the Luminescent Cell Viability Assay 96 (Promega, Madison, WI). Relative cell viability was determined as a percentage of the untreated control. IC50 values were then calculated. Anti-CXCR4 antibodies conjugated to MMAD or 0101 via a transglutaminase tag exhibited effective cell-killing activity in cells expressing CXCR4.

Table 14: Cytotoxicity of CXCR4 ADCs on CXCR4-expressing cells

Example 16

In vivo antitumor activity of CXCR4-ADC

The in vivo anti-tumor efficacy of CXCR4 ADCs was evaluated in Ramos (NHL) and HPB-ALL (T-ALL) xenograft models. As described in Example 9, 5×10 ⁶ Ramos (NHL) cells were subcutaneously implanted into female 4-6 week old CB17 SCID mice (Jackson Laboratories) to an average tumor volume of approximately 500 mm ³ , calculated by the formula (volume = length × width ² ) / 2. 10×10 ⁶ HPB-ALL cells were implanted into female 4-6 week old CB17 SCID mice until the average tumor volume also reached approximately 500 mm ³ . In the Ramos model, groups of mice were treated with a single intravenous (IV) injection of either a 6.0 mg/kg negative control ADC (negative control-TG6-vc0101) or 1.5, 3.0, or 6.0 mg/kg of CXCR4 ADCs (3G10-TG6-vc0101 and 6B6-TG6-vc0101). Tumor volumes were measured weekly using a caliper for the duration of the study. The experimental results are shown in Figure 13A. The results demonstrate that both CXCR4 ADCs induced tumor regression at doses ≥ 3 mg/kg. The control ADC had no effect on tumor growth. In the HPB-ALL model, a single injection of 1.5 mg/kg of 3G10-TG6-vc0101 was sufficient to induce tumor regression (Figure 13B).

Claims (23)

1. An isolated anti-CXCR4 antibody or its antigen-binding fragment, comprising: The heavy chain variable (VH) region and the light chain variable (VL) region, wherein the heavy chain variable (VH) region has VHCDR1 of SEQ ID NO:107, VH CDR2 of SEQ ID NO:162 and VH CDR3 of SEQ ID NO:112, and the light chain variable (VL) region has VL CDR1 of SEQ ID NO:144, VL CDR2 of SEQ ID NO:145 and VL CDR3 of SEQ ID NO:139. 2. The antibody or antigen fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having at least 95% identical amino acid sequence to SEQ ID NO:33, and the light chain variable region having at least 95% identical amino acid sequence to SEQ ID NO:73. 3. The antibody of claim 1 or its antigen-binding fragment, wherein the antibody comprises: a) The heavy chain variable (VH) region of SEQ ID NO:33; and/or b) Light chain variable (VL) region of SEQ ID NO:73. 4. The antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a heavy chain variable (VH) region generated from an expression vector having ATCC accession number PTA-121353. 5. The antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a light chain variable (VL) region generated from an expression vector having ATCC accession number PTA-121354. 6. The antibody of claim 1 or its antigen-binding fragment, wherein the antibody is a humanized, chimeric, CDR-transplanted, or recombinant human antibody. 7. The antibody of claim 1 or 6 or its antigen-binding fragment, wherein the antibody is not canine or feline. 8. The antibody of claim 1 or 6 or an antigen-binding fragment thereof, wherein the antibody comprises a tag containing an acyl donor glutamine modified at a specific site. 9. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof of any one of claims 1-8 and a pharmaceutically acceptable carrier. 10. An isolated polynucleotide comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof of any one of claims 1-8. 11. A vector comprising the polynucleotide of claim 10. 12. An isolated host cell, which is recombined to produce an antibody or an antigen-binding fragment thereof of any one of claims 1-8. 13. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell of claim 12 under conditions that produce said antibody or an antigen-binding fragment thereof, and isolating said antibody or antigen-binding fragment thereof from said host cell or culture medium. 14. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for treating cancers associated with CXCR4 function or expression in a subject. 15. The use of claim 14, wherein the cancer is a blood cancer. 16. The use of claim 14, wherein the cancer is a B-cell or T-cell malignancy. 17. The use of claim 14, wherein the cancer is selected from: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, mantle cell lymphoma (MCL), follicular lymphoma, Waldenström macroglobulinemia (WM), B-cell lymphoma, and diffuse large B-cell lymphoma (DLBCL). 18. The use of claim 14, wherein the cancer is selected from: bladder cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, lung cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, and skin cancer. 19. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for slowing tumor growth or progression in a subject having a tumor expressing CXCR4. 20. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for reducing metastasis of cancer cells expressing CXCR4 in a subject. 21. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for inducing tumor regression in a subject having a tumor expressing CXCR4. 22. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-8 in the preparation of a medicament for detecting and/or diagnosing conditions associated with CXCR4 expression. 23. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-8 in the manufacture of a medicament for treating a condition associated with CXCR4 expression.