Patent for “Human monoclonal antibody that binds CXCR4 and its uses”

Biopharmaceuticals – Michelle R. Kuhne, Peter Brams, Dawn M. Tanamachi, Alan J. Korman, Josephine M. Cardarelli, Bristol Myers Squibb Co

Search Patent for “Human monoclonal antibody that binds CXCR4 and its uses”

Abstract for “Human monoclonal antibody that binds CXCR4 and its uses”

“The present disclosure contains isolated monoclonal monoclonal antibody that binds to CXCR4 with high affinity. This is particularly true for monoclonal human antibodies. The disclosure also provides nucleic acid molecules that encode the antibodies, expression vectors and methods to express the antibodies. This disclosure also provides immunoconjugates, bispecific molecule and pharmaceutical compositions containing the antibodies. This disclosure provides methods to detect CXCR4 and methods to treat various cancers, inflammation disorders, HIV infection with an anti-CXCR4 antibodies.

Background for “Human monoclonal antibody that binds CXCR4 and its uses”

“Chemokines, a group of approximately 50 small proteins, regulate cell trafficking and angiogenesis, and play a significant part in the tumor microenvironment” (Vicari A. P., and Caux C., 2002 Cytokine Growth Factor Rev. 13:143-154). 13:143-154 These chemokines are classified by receptors as either members of the CCR or CXCR families. CXCR4, which is a seven-transmembrane G protein coupled receptor, is one member of the CXCR Family. It activates chemotaxis and is primarily expressed on lymphocytes. CXCR4 binds to the chemokine CXCL12, (SDF-1).

“CXCR4 is involved in embryogenesis, homeostasis, and inflammation. CXCR4/SDF-1 pathways were implicated in organ vascularization and immune system function in mice with CXCR4/SDF-1 deficiency (Tachibana K. et. al. Nature 393:591-594 (1998) CXCR4 was also shown to be a coreceptor of T lymphotrophic HIV-1 strains (Feng, Y. et. al. Science 272(1996):872-877. CXCR4 has also been demonstrated to be expressed in a variety of cancer cells. The CXCR4/SDF-1 pathway was also shown to stimulate the metastatic process of many different neoplasms. Murphy, P. M. (2001). N. Engl. J. Med. 345:833-835). CXCR4 or SDF-1, for example, have been shown to mediate organ specific metastasis through creating a chemotactic slope between the primary tumor site, and the metastatic site (Muller A. et. al. (2001) Nature 410:50-56; Murakami, T. et al. Cancer Res. 62:7328-7334; Hanahan, D. et al. Cancer Res. 63:3005-3008).”

The present disclosure contains isolated monoclonal monoclonal antibody, particularly human monoclonal antibody, that binds to human CXCR4 and has many desirable properties. These properties include the ability of the monoclonal antibodies to bind to the native human CXCR4 on the cell surface and the ability of SDF-1 to inhibit CXCR4-induced calcium flux in cells expressing CXCR4. The ability of SDF-1 to inhibit CXCR4-induced migration in cells expressing CXCR4. The ability of CXCR4+ cells to inhibit CXCR4+ cells from forming capillary tubes by human umbilical vein endingothelial cells (HuVECs) the ability of CXCR4+ cancer cells to reduce their survival time to increase the CXCR4+ subject’s CXCR4+ CXCR4+ to decrease the ability of CXCR4+ to prevent metastases of CXCR4+ cells.

The instant disclosure relates to an isolated human monoclonal antibodies or an antigen binding portion thereof. In one aspect, the antibody binds native human CXCR4 on a cell’s surface. The antibody can also inhibit binding of SDF-1to human CXCR4, preferably with a EC50 of 50 nM, 30 nM, 15 nM, 10 nM, or 5 nM, or 3 nM (e.g., an EC50 of inhibition of 28.60 nM, 12.51 nM, or 2.256 nM) Another embodiment of the antibody binds to human CXCR4 on a cell surface, but not to SDF-1. Another embodiment of the antibody blocks SDF-1-induced calcium flux in cells expressing human CXCR4, preferably with a EC50, which inhibits 3 nM, 2 nM, 1 nM, 0.9 nM, or 0.7 nM, or 0.6 nM, or 0.5 nM, or 0.4 nM (e.g. 0.9046 nM, 0.5684, or 0.3219 nM) The antibody can also be used to inhibit SDF-1-induced cell migration in cells expressing human CXCR4. Preferably, an EC50 is used for inhibitions of 50 nM, 30 nM, 20 nM, 15 nM, and 0.3219 nM. Other embodiments of the antibody inhibit capillary tube formation using HuVECs. It also induces apoptosis and tumor cell proliferation in vitro.

“In a preferred embodiment, this disclosure refers to an isolated human monoclonal antibodies or an antigen binding portion thereof. The antibody:

“In an alternative aspect, this disclosure refers to an isolated monoclonal antibody from a human being, or the antigen binding portion thereof. The antibody crosses-competes with CXCR4 binding proteins, and the reference antibody contains:

“In some embodiments, the disclosure provides an isolated monoclonal anti-body or an antigen binding portion of an antibody. This includes a heavy chain variable area that is either derived from or produced by a human VH-348 gene. The antibody specifically binds to human CXCR4. This disclosure also provides an isolated monoclonal anti-body, or an antigen binding portion, that includes a light chain variable area that is either derived or produced from the human VK L15 genes. The antibody specifically binds to human CXCR4. This disclosure also provides an isolated monoclonal anti-body, or an antigen binding portion, that includes a heavy chain variable area that is either derived or produced from the human VH-348 gene, and a light channel variable region that’s the product or derived form the human VK L15 genes. The antibody binds specifically to human CXCR4.

“In an alternative aspect, this disclosure provides an isolate monoclonal antibody or antigen binding section thereof comprising:

“(a) The heavy chain variable region CDR3 sequence contains an amino acid selection from the group consisting amino acid sequences SEQ ID NOs 9-12 and conservative modifications thereof.

“(b) The light chain variable region CDR3 sequence consists of an amino acid sequence chosen from the group consisting SEQ ID NOs 21-24 and conservative modifications thereof;

“(c) The antibody binds native human CXCR4 on a cell surface. This antibody may also have one or more of these characteristics. It inhibits the binding of SDF-1 and CXCR4 in cells that express CXCR4; it inhibits SDF-1 induced calcium flux in cells cells expressing CXCR4; it inhibits SDF-1 induced migration in cells expressing CXCR-4; it induces apoptosis (in vitro/in vivo); inhibits CXCR4+ cancer cells in vitro/in vivo; and/or metatases of CXCR4+ cells.

“Preferably the heavy chain variable area CDR2 sequence consists of an amino acid selection from the group consisting amino acid sequencings of SEQ ID NOs 5-8 and conservative modifications thereof. The light chain variable regions CDR2 and CDR2 respectively consist of an amino acids sequence chosen from the group consisting amino acid sequences from SEQ ID Nos 17-20 and conservative modifications thereof. The heavy chain variable CDR1 sequence consists of an amino sequence chosen from the group consisting only of SEQID NOs 1-4 and conservative modifications thereof. The light chain variable CDR1 series consists of an amino sequence selected from SEQID NOs 13-16 and conservative modifications thereof.

“A preferred combination includes:

“Another preferred combination includes:

“Alternative preferred antibodies, or antigen binding parts of this disclosure,” include: (a) A heavy chain variable area comprising an amino acid sequence chosen from the group consisting SEQ ID Nos: 25-28, 41-44, and (b) A light chain variable regional comprising an amino acid sequence chosen from the SEQ ID nos: 29-32, 45-48, where the antibody binds specifically to CXCR4.

“A preferred combination consists of: (a) A heavy chain variable area comprising the amino acids sequence of SEQID NO: 25 or 41; (b) A light chain variable regional comprising SEQ ID No: 29 or 45.”

“Another preferred combination includes: (a) A heavy chain variable area comprising the amino acids sequence of SEQID NO: 26 or 42; (b) A light chain variable regional comprising SEQID NO: 30 and 46.”

“Another preferred combination is: (a) A heavy chain variable area containing the amino acids sequence of SEQID NO: 27 or 43; (b) A light chain variable regional containing the amino acids sequence of SEQ ID NO: 31 and 47.”

“Another preferred combination includes: (a) A heavy chain variable area comprising the amino acids sequence of SEQID NO: 28 or 44; (b) A light chain variable regional comprising SEQID NO: 32 and 48.”

“Another aspect of this disclosure is that antibodies or antigen-binding parts thereof are provided that compete to bind to CXCR4 and any of the aforementioned antibody.”

“The antibodies disclosed in this disclosure could be full-length antibodies such as IgG1 and IgG4 isotypes. Alternately, antibodies could be fragments of antibody, such as Fab or Fab? You can also use Fab?2 fragments or single-chain antibodies.

“This disclosure also contains an immunoconjugate that includes an antibody, or antigen binding portion thereof, and linked to a therapeutic drug such as a radioactive or cytotoxic isotope or cytotoxin. This disclosure also includes a bispecific moiety that contains an antigen-binding or antibody portion of this disclosure and linked to another functional moiety with a different binding specificity.

“Compositions that include an antibody or antigen-binding component thereof or an immunoconjugate molecule or bispecific molecule are also provided.”

This disclosure also includes expression vectors comprising these nucleic acids as well as host cells comprising such expression Vectors. Methods of preparing anti-CXCR4 antibody using host cells that have such expression vectors are also described. These methods may include (i) expressing anti-CXCR4 antibody in the host cell, and (ii). isolating anti-CXCR4 antibody from the host.

“Another aspect is disclosed concerning methods to modulate CXCR4 activity within a cell. In this case, the cells are contacted using an antibody or antigen-binding component of this disclosure. You can contact the cells in vitro by growing the cells with the antibody, or you can contact them in vivo by giving the antibody to a subject. The preferred embodiment uses tumor cells that express CXCR4 and results in the inhibition of growth and/or metastasis. Another embodiment uses T cells that express CXCR4 to inhibit HIV entry into cells. Another embodiment involves lymphocytes with an inflammatory disorder. The methods inhibit inflammation. Another embodiment involves the cells in vascularization. The method results in modulation and angiogenesis.

“In another aspect, this disclosure refers to a method to stimulate mobilization CD34+-derived stem cells from bone and marrow to peripheral in a subject. The method comprises administering an antigen-binding portion of this disclosure to the subject so that CD34+ stem cell mobilization from bone marrow is stimulated. Further, the method may include the collection of CD34+ stem cells in the peripheral blood for autologous stem cell transplantation.

“A number of other features and benefits of the instant disclosure will become apparent from the detailed description and examples. These should not be taken as limiting. This application contains all references, Genbank entries and patents cited in this application. These are incorporated by reference.

The present disclosure concerns monoclonal monoclonal antibodies that bind to native human CXCR4 on cell surfaces. The antibodies described in this disclosure may be derived from specific heavy or light chain germline sequences or include particular structural features, such as CDR regions that contain particular amino acid sequences. This disclosure includes methods for making antibodies, immunoconjugates, bispecific molecules, and pharmaceutical compositions that contain the antibodies, immunoconjugates, or bispecific molecules. The disclosure also covers methods for using the antibodies to detect CXCR4 and modulate CXCR4 activities in diseases or disorders involving CXCR4/SDF-1, such as cancers. Tumor metastasis, HIV infection. inflammation, angiogenesis, and HIV infection. This disclosure provides methods for using anti-CXCR4 antibody of this disclosure to treat certain types of cancer. For example, it allows you to treat breast, ovarian and prostate cancers. This disclosure also provides methods for using anti-CXCR4 antibody of this disclosure to prevent tumor metastasis.

“The present disclosure is easier to understand if certain terms are defined first. The detailed description also contains additional definitions.

“CXCR4” is a term that refers to variants, orthologs and paralogs. “CXCR4” can refer to variants, isoforms as well as orthologs, paralogs, and homologs. In some cases, antibodies that are specific for CXCR4 might cross-react with CXCR4 of other species. Other embodiments of the antibody specific for human CXCR4 could be entirely specific for human CXCR4 without any other cross-reactivity. “Human CXCR4” is a term. Refers to the human sequence CXCR4, including the complete amino-acid sequence of CXCR4 with Genbank accession number (SEQ ID No.:51). CXCR4 can also be known in the arts as LESTR, Fusin, or CD184. Human CXCR4 may have a different sequence than the CXCR4 in SEQ ID NO. :51 may have, for example conserved mutations, or mutations in unconserved areas. The CXCR4 is substantially the same biological function that the human CXCR4 SEQ ID NO. 51. A biological function for human CXCR4 could be having an epitope within the extracellular domain CXCR4 that is bound specifically by the antibody of the immediate disclosure. Or, the biological function for human CXCR4 would be chemokine binding and involvement in the metastatic processes.

“A specific human CXCR4 sequence will usually be at least 90% identical in amino acid sequence to the human CXCR4 SEQ ID NO. :51 contains amino acid residues which identify the amino acids sequence as human, when compared with CXCR4 sequences from other species (e.g. murine). A human CXCR4 can be as high as 95% or higher, and even 96%, 97% or 98% more alike than CXCR4 of the same SEQ ID No.:51. A human CXCR4 sequence may not differ from the CXCR4 in SEQ ID NO. 51 in certain embodiments. The human CXCR4 sequence may not display more than 5, or even 4, 2, or 1 amino acids difference from the CXCR4 SEQ ID NO. 51. As described in this document, you can determine percent identity.

“The term ‘SDF-1?? The term stromal cell-derived factors 1 (SDF-1) is used to refer to CXCR4’s ligand. SDF-1 is the term. SDF-1 can be described as a variety of SDF-1 isoforms, including SDF-1? SDF-1?. What is the amino acid sequence for human SDF-1? Genbank Accession Number NP 954637. What is the amino acid sequence for human SDF-1 Genbank Accession Number NP 000600. U.S. Pat. No. 5,756,084. CXCL12 is also known for SDF-1. SDF-1 is also known as CXCL12.

“Immune response” is a term that refers to the body’s immune system. “Immune response” refers to the actions of lymphocytes and antigen-presenting cells, phagocytic and granulocytes as well as soluble macromolecules (including antibodies, complement, cytokines and complement) that cause selective damage to, destruction or elimination of the human body of pathogens, cells and tissues infected by pathogens, cancerous or normal cells and tissues.

“A signal transduction pathway” The biochemical relationship among a number of signal transduction molecules, which plays a role in the transmission and reception of signals from one part of a cell to the other. The expression “cell surface receptor” is used herein. This includes molecules and complexes that can receive a signal and transmit it across the plasma membrane of cells. One example of a “cell surface receptor”? The CXCR4 receptor is an example of a cell surface receptor.

“Antibody” is a generic term that refers to whole antibodies and antigen binding fragments. “Antibody” as used herein refers to whole antibodies and antigen binding fragments (i.e., the?antigen binding portion). or single chains. An “antibody” is a glycoprotein that contains at least two heavy (H) chains or one of the light (L) chains. An?antibody? is a glycoprotein that contains at least two heavy (H), and two light (L), chains interconnected by disulfide bond or an antigen binding section. Each heavy chain is composed of a heavy-chain variable region (abbreviated VH) as well as a heavy-chain constant region. The three domains that make up the heavy chain constant area are CH1, CH2 or CH3. Each light chain is composed of a light-chain variable region (abbreviated as VL) along with a light-chain constant region. One domain, CL, is the light chain constant region. You can further subdivide the VH and VL into regions of hypervariability (CDR), as well as regions that are conserved (FR). Each VH or VL is made up of four FRs and three CDRs. They are arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, CDR2, CDR2, CDR2, CDR3, CDR3, and FR4. Variable regions of heavy and light chains have a binding domain that interacts to an antigen. The binding domains of antibodies in the constant regions may facilitate the binding of immunoglobulins to host tissues and factors. This includes various cells of immune system (e.g. effector cells) as well as the first component (Clq), of the classical complement system.

“The term ‘antigen-binding part? An antibody (or simply the?antibody section) is a portion of an antibody. As used herein, an antibody fragment (or simply?antibody portion) is one or more fragments that can bind specifically to an antigen (e.g. CXCR4). A fragment of a full length antibody can perform the antigen-binding function. The term “antigen-binding section” includes examples of binding fragments. A Fab fragment is a monovalent fragment that includes the VL and VH domains, CL, CH1 domains, and CL domains. (iii) A F(ab?) Fab2 fragment is a bivalent fragment that consists of two Fab fragments linked together by a disulfide link at the hinge region. (iii). A Fab? fragment, which is essentially a Fab with a portion of the hinge area (see FUNDAMENTAL IMMUNOLOGY, Paul ed.). 1993); (iv), a Fd fragment containing the VH, CH1 and VH domains; and (vi) A Fv fragment containing the VL, VH, and CDR domains of an antibody. Although VL, and VH are distinct domains in the Fv fragment’s code, they can be combined using recombinant techniques to create a single protein chain. In this case, the VL-VH regions will pair to form monovalent molecules. This is known as single chain Fv (scFv); see Bird et. al. (1988) Science 242(4):423-426. Huston et. al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. These single-chain antibodies are also included in the term “antigen binding portion?” An antibody. These fragments of antibody are obtained using standard techniques that are well-known to those skilled in the art. They are then screened for utility in much the same way as intact antibodies.

An “isolated antibody” is an antibody that is substantially unbound by other antibodies with different antigenic specificities. (e.g. an isolated antibody that binds CXCR4 is significantly unbound from antibodies that bind other antigens than CXCR4) However, an isolated antibody that binds CXCR4 can cross-react with other antigens such as CXCR4 molecules of other species. An isolated antibody may also be free from other cellular material or chemicals.

“Monoclonal antibody” is a term that refers to monoclonal antibodies. Monoclonal antibody composition or monoclonal antibody? The term “monoclonal antibody composition” is used herein to refer to a preparation containing monoclonal antibodies molecules with a single molecular structure. Monoclonal antibody compositions have a single binding affinity and specificity for a particular epitope.

“Human antibody” is a term that refers to antibodies with variable regions. Both the CDR and framework regions are derived form human germline immunoglobulin sequencings. If the antibody has a constant area, that region is also derived from human germline immuneglobulin sequences. This disclosure could include amino acid residues that are not encoded in human germline immunoglobulin sequencings. For example, somatic mutations in vivo or random site-specific mutations in vitro. The term “human antibody” as used in this disclosure does not include antibodies in which CDR-derived sequences from another mammalian species (e.g., mice) have been grafted onto human frame sequences.

“Human monoclonal antibodies” is a term that refers to an antibody that is monoclonal. A single-binding specificity antibody is one that has variable regions. Both the CDR and framework regions are derived directly from human germline immunoglobulins sequences. One embodiment of the human monoclonal antibody is produced from a hybridoma that includes a human monoclonal antibody made from a human monoclonal antibody. This hybridoma may include a B cell derived from a transgenic nonhuman species, such as a transgenic mouse. It has a genome that contains a human heavy-chain transgene and light chain transgene, which are fused to immortalized cells.

Recombinant human antibodies, as they are called, include all antibodies that have been prepared, created, or expressed by recombinant methods. These recombinant human antibody have variable regions where the framework and CDR regions were derived from human germline immuneglobulin sequences. However, certain embodiments allow for in vitro mutation (or when an animal transgenic to human Ig sequences has been used, in-vivo somatic mutation) of such recombinant antibody recombinants. The VH and L regions of these recombinant antibodies contain sequences that are derived from the human germline VH or VL sequences.

“As used herein, ?isotype? The antibody class (e.g. IgM or IgG1) encoded by the heavy-chain constant region genes.

“The expressions?an antigen-recognizing antibody? An antibody that recognizes an antigen and an antibody specific for it? These terms are interchangeable with the term “an antibody that binds specifically for an antigen?”

“Human antibody derivatives” is a term that refers to any modified form of the human antibody. Any modified form of the human antibodies, such as a conjugate of the antibody with another agent or antibody, is called “human antibody derivatives”.

“Humanized antibody” is a term that refers to antibodies that have been grafted onto human framework sequences. This term refers to antibodies in which CDR-sequences derived from another mammalian species (e.g. a mouse) have been grafted onto human frame sequences. The human framework sequences may also contain additional modifications to the framework regions.

“Chimeric antibody” is a term that refers to an anti-microbial that can be chimed. “Chimeric antibodies” is an acronym that refers to antibodies in which variable region sequences come from one species and constant region sequences from another. For example, an antibody in the variable area sequences of a mouse antibody are derived while constant region sequences from a human antibody are derived.

“Subject” is defined as: Any human or non-human animal is included. “Nonhuman animal” is a broad term. All vertebrates are included, including mammals and non-mammals such as sheep, dogs, cats and horses.

“Various aspects of the disclosure are described in greater detail in these subsections.”

“Anti-CXCR4 Antibodies”

“In some embodiments, the antibody of this disclosure binds native human CXCR4 to a cell surface. However, it does not inhibit binding to SDF-1 to CXCR4 or inhibit SDF-1’s calcium flux in cells expressing CXCR4. It does not hinder SDF-1’s induced calcium flux in cells that express CXCR4 but does not inhibit SDF-1’s induced migration of CXCR4-expressing cells. Another embodiment of the disclosure binds native human CXCR4 to a cell surface. It does not inhibit binding of SDF-1 and CXCR4 and does not inhibit SDF-1’s induced calcium flux in cells that express CXCR4, but it does inhibit SDF-1’s induced migration of CXCR4-expressing cells. Another embodiment of the disclosure is that it binds to native CXCR4 of human beings on a cell surface. This inhibits binding of SDF-1 and CXCR4 and inhibits SDF-1’s calcium flux in cells expressing CXCR4. It also inhibits SDF-1’s migration of CXCR4-expressing cells. Another embodiment of the disclosure is that an antibody binds to native CXCR4 of human beings on a cell surface. This inhibits binding of SDF-1 and CXCR4, inhibits SDF-1-induced calcium flux of cells expressing CXCR4, inhibits SDF-1 induced migration in cells expressing CXCR4, inhibits SDF-1-induced capillary tube formation from HuVECs, and does not inhibit SDF-1 induced calcium flux in cells cells expressing CXCR4.

“Preferably, an antibody that inhibits binding to SDF-1 to human CXCR4 has an EC50 of inhibition of 50 nM, more preferably 15 nM, 10 nM, 5 nM, or 3 nM (e.g. an EC50 of inhibition of 28.60 nM, 12.51 nM, or 2.256 nM)

“Preferably, an anti-SDF-1-induced calcium flux is inhibited in cells expressing human CXCR4 with a EC50 of 3 nM, more preferably 1 nM, 0.9 nM, or 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, less (e.g. 0.9046 nM, 0.5684, 0.3219 nM, or 0.3219 nM)

“Preferably, an anti-serum of this disclosure inhibits SDF-1 induced migration in cells expressing human CXCR4 with a EC50 for inhibition at 50 nM, more preferably 20 nM, 30 nM, or 15 nM (e.g. 18.99 nM, 12.44, or less).

Standard assays are available to assess the binding ability of antibodies to native human CXCR4 on cell surfaces. These include flow cytometry analysis with a cell line that expresses native CXCR4 naturally or has been transfected to produce native CXCR4. The Examples explains in detail the various assays that are suitable. CEM T cells are a preferred cell line that express native CXCR4. The Examples include detailed descriptions of assays that can be used to evaluate inhibition of binding of SDF-1, inhibition SDF-1-induced calcium flux, inhibition SDF-1-induced cell migration, induction of Apoptosis in cells expressing native CXCR4 and inhibition of CXCR4+ cancer cells in vitro or in vivo, inhibiting growth of CXCR4+ cells in vitro/or in live, and/or inhibition metastases of CXCR4+ tumour cells. Standard methods such as Scatchard analysis can also be used to determine the binding affinity of antibodies.

“Monoclonal antibodies F7 and F9, D1 & E2”

“Preferred antibodies in this disclosure are the monoclonal antibodies F7 and F9 from human. They have been structurally characterized and isolated as described in Examples 1 & 2. SEQ ID NOs 25, 26, 27 and 28 respectively show the VH amino acids sequences for F7, F9 and E2. SEQ ID NOs 29-30, 31 and 32 show the VL amino acids sequences for F7, F9 and E2. Alternative forms of F7 and F9, D1 as well as E2 were also created. These alternative forms, in which certain frame residues were replaced with a germline residue were referred to herein by F7GL, F9GL and D1GL. SEQ ID NOs 41, 42, 43, 44 and 44 respectively show the VH amino acids sequences for F7GL, E9GL, D1GL, and E2GL. The VL amino acids sequences for F7GL, F9GL D1GL, and E2GL can be found in SEQ ID Nos 45, 46, 47, and 48, respectively.

“VH and VL sequences may be mixed and matched, as each antibody can bind CXCR4. This disclosure can be used to make other anti-CXCR4 binding molecule. These?mixed-and-matched’ CXCR4 binding molecules can be used to test for CXCR4. You can test antibodies using the binding assays mentioned above and in the Examples (e.g. flow cytometry using CEM cells). When VH and L chains are mixed and matched, it is preferable that a VH sequence of a particular VH/VL pair is replaced by a structurally identical VH sequence. A VL sequence from a specific VH/VL pairing should be replaced by a structurally identical VL sequence.

“Accordingly, this disclosure provides an isolate monoclonal antibody or antigen binding section of the antigen-binding portion.

“In another aspect, the disclosure includes antibodies that contain heavy chain and light-chain CDR1s, CDR2s, and CDR3s from F7, F9 and D1, E2, or combinations thereof. SEQ ID NOs 1-4 show the amino acid sequences for the VH CDR1s from F7, F9 and D1 respectively. SEQ ID NOs 5-8 show the amino acid sequences for VH CDR2s F7, F9 and D1 respectively. SEQ ID NOs 9-12 show the amino acid sequences for VH CDR3s F7, F9 and D1 respectively. SEQ ID NOs 13-16 show the amino acid sequences for the Vk CDR1s F7, F9 and D1 respectively. SEQ ID NOs 17-20 show the amino acid sequences for F7, F9 and D1 respectively. SEQ ID NOs 21-24 show the amino acid sequences for F7, F9 and D1 CDR3s. The Kabat system is used to delineate the CDR regions (Kabat E. A., and others. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. 91-3242).”

“Accordingly, in another embodiment, this disclosure provides an isolate monoclonal antibody or antigen binding section thereof consisting of:

“(a) A heavy chain variable region CDR1 containing an amino acid sequence chosen from the group consisting SEQ ID NOs 1-4;

“(b) A heavy chain variable region CDR2 containing an amino acid sequence chosen from the group consisting SEQ ID NOs 5-8;

“(c) A heavy chain variable region CDR3 containing an amino acid sequence chosen from the group consisting SEQ ID NOs 9-12;

“(d) A light-chain variable region CDR1 containing an amino acid sequence chosen from the group consisting SEQ ID NOs 13-16;

“(e), a light-chain variable region CDR2 containing an amino acid sequence chosen from the group consisting SEQ ID NOs 17-20; and

“(f) A light-chain variable region CDR3 containing an amino acid sequence chosen from the group consisting SEQ ID NOs 21-24;

“Wherein the antibody binds CXCR4, preferentially human CXCR4”

“In a preferred embodiment of the antibody, it comprises:

“(a) A heavy chain variable region CDR1 consisting of SEQ ID NO. 1;

“(b) A heavy chain variable region CDR2 containing SEQ ID NO. 5;

“(c) A heavy chain variable region CDR3 containing SEQ ID NO 9;

“(d), a light-chain variable region CDR1 consisting of SEQ ID NO 13;

“(e), a light-chain variable region CDR2 consisting of SEQ ID NO 17; and

“(f) A light chain variable region CDR3 containing SEQ ID NO 21

“In an alternative preferred embodiment, the antibody includes:

“(a) A heavy chain variable region CDR1 that includes SEQ ID NO. 2;

“(b) A heavy chain variable region CDR2 comprising the SEQ ID NO 6;

“(c) A heavy chain variable region CDR3 consisting of SEQ ID NO. 10;”

“(d) A light chain variable region CDR1 consisting of SEQ ID No: 14;

“(e), a light-chain variable region CDR2 consisting of SEQ ID NO. 18; and”

“(f) A light chain variable region CDR3 containing SEQ ID NO 22

“In an alternative preferred embodiment, the antibody includes:

“(a) A heavy chain variable region CDR1 comprising the SEQ ID NO. 3;

“(b) A heavy chain variable region CDR2 containing SEQ ID NO 7;

“(c) A heavy chain variable region CDR3 consisting of SEQ ID NO. 11;

“(d), a light-chain variable region CDR1 consisting of SEQ ID No: 15;

“(e), a light-chain variable region CDR2 consisting of SEQ ID NO. 19; and

“(f) A light chain variable region CDR3 containing SEQ ID NO 23

“In an alternative preferred embodiment, the antibody includes:

“(a) A heavy chain variable region CDR1 comprising the SEQ ID No: 4;

Summary for “Human monoclonal antibody that binds CXCR4 and its uses”