Therapeutic antibodies against ror-1 protein and methods for use of same

[0001] Tyrosine kinases are important mediators of signaling cascades that play a key role in various biological processes, such as growth, differentiation, metabolism and apoptosis in response to external and internal stimuli. The results of the conducted studies indicate the role of tyrosine kinases in the pathophysiology of cancer. Schlessinger J. (2000) Cell, 103: 211-225; and Robinson et al. (2000) Oncogene, 19: 5548-5557. MacKeigan et al. Have applied a large-scale RNAi-based approach to the identification of kinases that can regulate the survival and apoptosis of human cancer cell lines (HeLa); it was found that RNAi for ROR 1 showed the strongest activity in inducing apoptosis from RNAi directed to each of the 73 different genes encoding the kinase. MacKeigan et al. (2005) Nat Cell Biol., 7: 591-600. researchers,

[0002] The receptor protein ROR 1, a receptor tyrosine kinase receptor similar to orphan receptor one, is a molecule expressed at high concentration during embryogenesis, which plays an important role in the development of the bone, lung and nervous system. Expression of the ROR 1 receptor protein in mammalian cells after delivery decreases substantially to virtually undetectable levels. The ROR 1 receptor protein is a membrane receptor with an intracellular kinase domain and the extracellular Frizzled domain with high cysteine ​​content, which is often found in the Wnt family of receptors. The ROR 1 receptor protein belongs to the ROR family, which has been preserved in evolutionary species of Caenorhavditis elegans, Drosophila, mice and humans. Wilson C, Goberdhan DC, Steller H. Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family of Trk-related receptor tyrosine kinases. Proc Natl Acad Sci USA. 1993; 90: 7109-7113; Oishi et al. (1997) J Biol Chem., 272: 1191611923; Masiakowski et al. (1992) J Biol Chem., 267: 26181-26190; Forrester et al. (2002) Cell Mol Life Sci., 59: 83-96; and Oishi et al. (1999) Genes Cells, 4: 41-56. It is not known what the actual functional role of the ROR 1 receptor protein plays during embryogenesis, although it is thought to be the Wnt protein receptor that regulates cellular polarity and cell-to-cell interactions. and Oishi et al. (1999) Genes Cells, 4: 41-56. It is not known what the actual functional role of the ROR 1 receptor protein plays during embryogenesis, although it is thought to be the Wnt protein receptor that regulates cellular polarity and cell-to-cell interactions. and Oishi et al. (1999) Genes Cells, 4: 41-56. It is not known what the actual functional role of the ROR 1 receptor protein plays during embryogenesis, although it is thought to be the Wnt protein receptor that regulates cellular polarity and cell-to-cell interactions.

[0003] Although it is primarily an embryonic protein, ROR 1 is only expressed in some tumor cells, including chronic lymphocytic leukemia (CLL), small lymphoma, B-cell lymphoma, Burkitt's lymphoma and other cancers ( e.g. breast cancer), but not in healthy cells or tissues in adults.

A recent study showed that the ROR 1 receptor protein, at the mRNA level and protein level, is expressed in large amounts in B cells of chronic lymphocytic leukemia, but not in healthy B cells.

In addition, the ROR 1 receptor protein has been found to be a Wnt5a receptor that can induce NF-κΒ activation when co-expressed with the ROR 1 receptor protein in HEK293 cells, and may prolong the survival of chronic lymphocytic leukemia cells in vitro. This indicates that the ROR 1 receptor protein is a receptor for cell survival of chronic lymphocytic leukemia for Wnt5a. Another study showed that the ROR 1 receptor protein was also expressed in acute lymphocytic leukemia (ALL). Shabani et al. (2007) Tumour Biol. 28: 318-326; and Baskar et al. (2008) Clin Cancer Res., 14: 396-404. ROR 1 receptor protein has been expressed in a variety of hematological and solid tumors.

[0004] It is necessary to develop methods that allow therapeutic control of the expression of the ROR 1 protein. Polyclonal antibodies against the ROR 1 peptide are commercially available. The inventors have developed a monoclonal antibody against the ROR 1 receptor protein called 4A5 that reacts with the native ROR 1 receptor protein and allows the detection of ROR 1 receptor protein on the cell surface for flow cytometry analysis.

[0005] This antibody is discussed in Fukuda, T. et al., PNAS (2008) 105 (8): 3047-3052.

[0006] However, there are no strong therapeutic antibodies available with the demonstrated ability to inhibit tumor cell proliferation induced by the ROR 1 receptor protein to a significant therapeutically extent to slow down or prevent growth and metastasis.

SUMMARY OF THE INVENTION [0007] The present invention provides antibodies and a combination of antibodies that inhibit in vivo and in vitro ROR 1 receptor protein proliferation in cancer patients, including lymphomas, chronic lymphocytic leukemia, small lymphocytes, and cell lymphoma. B marginal zone, Burkitt's lymphoma, cancer, renal cell carcinoma, colon cancer, breast cancer, squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer, but not in the blood or spleen lymphocytes of non-leukemia patients and healthy adults.

[0008] Antibodies of the present invention are also useful in the differentiation between tumor cells expressing the ROR 1 receptor protein (ROR 1) and healthy cells. For example, an immunological assay that detects a ROR 1 receptor protein in a sample taken from a subject after combining the sample with a receptor-specific ROR 1 specific antibody of the present invention and detection of immunoreactivity between the antibody and the ROR 1 receptor protein in the sample is provided.

[0009] According to another aspect of the present invention, the ROR 1 tumor is diagnosed in a subject by detecting the presence or amount of the ROR 1 receptor protein in a sample.

[0010] The present invention relates to compositions containing purified, isolated monoclonal antibodies and combinations thereof that bind specifically to the ROR 1 receptor protein.

BRIEF DESCRIPTION OF THE DRAWINGS [0011]

Figure 1 shows a series of graphs showing the effect of flow cytometry analysis of the expansion of CD5 + B220low B cell leukemia in ROR 1 Tg mice after an adoptive adoption of 1 χ 10⁷splenocytes from mice

ROR 1 xTCL1 Tg. The top panel shows expansion in the period from 2 to 4 weeks after the adoption transfer. The percentage of leukemia cells in a line graph comparing mCD5 cells (x-axis) with mB220 (y-axis) is indicated above the goal on CD5 lymphocytes⁺B220^low. The lower panel shows the relative expression of the ROR 1 receptor protein (x-axis) using the mouse mAb 4A5 antibody against the receptor protein

ROR 1.

Figure 2 shows a schematic analysis of the mAb antibody against the ROR 1 receptor protein after adoptive transfer and the introduction of ROR 1 XTCL1 leukemic splenocytes. ROR 1 Tg mice (4 mice per group) were dosed intravenously with 250 μg of 4A5 antibody, D10 antibody or mIgG control on day 0. On the next day intravenous transfer was carried out 1 χ 10⁷splenocytes from ROR 1 x TCL1 Tg mice.

All mice were then monitored weekly for the expansion of CD5 B leukemia cells⁺B220^low by flow cytometry from the 2nd week after the transfer.

Figure 3 shows a series of graphs illustrating results of flow cytometry analysis that show that antibodies against the ROR 1 receptor protein of the present invention inhibit the development of lymphocytic leukemia-like leukemia in ROR 1 Tg mice. Two weeks after the adoption transfer, PBMC analysis was carried out. The data showed that the D10 antibody against the ROR 1 receptor protein, but not the 4A5 antibody against the ROR 1 receptor protein, can significantly inhibit the expansion of CD5 B leukemia cells^dullB220⁺ and ROR 1^brightB220⁺.

Figure 4A is a series of graphs illustrating the results of an in vivo study of a mouse model of human breast cancer. Antibodies against the ROR 1 receptor protein inhibited the metastasis of breast cancer in mice deficient in rag - / - g - / -. 5E5 MDA-MB-231 breast cancer cells were transferred by intravenous injection into the rag - / - g - / - mice organism on day 1. Rag - / - g - / - deficient mice were also injected intravenously with the isotype control or antibody against the ROR receptor protein 1 (4A5, D10 and 4A5 plus D10) on days 1, 3, 7 and 14 at a dose of 100 mg per mouse. Figure 4A (center) also shows images obtained during IVIS imaging studies in vivo of the above-mentioned mice that were performed weekly. After 5 weeks, the mice were sacrificed and histological analysis was performed (Figure 4B).

Figure 5 shows a comparison of the nucleotide sequence of the heavy Ig (VH) chain of the 4A5 antibody with the immunoglobulin heavy chain nearest to the mouse and human germline (Ig).

Figure 6 shows a comparison of the sequence encoding the heavy chain Ig (VH) nucleotide of the G6 antibody with the immunoglobulin heavy chain nearest to the mouse and human germline (Ig).

Figure 7 shows a comparison of the sequence encoding the heavy chain Ig (VH) nucleotide of the G3 antibody with the immunoglobulin heavy chain nearest to the mouse and human germline (Ig).

Figure 8 shows a comparison of the nucleotide sequence of the heavy chain Ig (VH) antibody of the H10 antibody with the immunoglobulin heavy chain nearest to the mouse and human germline (Ig).

Figure 9 shows a comparison of the sequence encoding the heavy chain Ig (VH) nucleotide of antibody D10 to the immunoglobulin heavy chain of the closest murine and human germline (Ig).

Figure 10 is a diagram and a graph illustrating the highly conserved nature of human and mouse antibodies to the ROR 1 receptor protein.

Figure 11 shows a nucleotide comparison illustrating the structure of the domain and the homology sequence of the murine and human extracellular ROR 1 receptor protein.

Figure 12 is a graph illustrating the extracellular domain by which mAbs against the ROR 1 receptor protein bind to the ROR 1 receptor protein.

Figure 13 is a diagram illustrating chimeric ROR 1 proteins produced to determine the binding domain of each mAb antibody against the ROR 1 receptor protein.

Figure 14 is a diagram illustrating truncated ROR 1 proteins produced to determine subregions that bind each mAb antibody to the ROR 1 receptor protein.

Figure 15 is a diagram illustrating amino acids that have been converted to mouse amino acids to determine the residues necessary for binding mAbs to human ROR 1 receptor protein and western blot results showing that the glutamic acid residue 138 is needed to bind the D10 antibody to the human ROR receptor protein 1. Figure 16 is a graph showing the K D values ​​for antibody D10 (Figure 16a) and 4A5 (Figure 16b).

Figure 17 shows a series of graphs showing high activity of the antibody against the ROR 1 D10 receptor protein in in vivo assays.

Figure 18 shows a schematic analysis of the mAb antibody against the ROR 1X TCL1 receptor protein after adoption and introduction of ROR 1 ROR 1 X TCL1 leukemic splenocytes. ROR 1 Tg mice (5 mice per group) were injected intravenously with 250 μg of 4A5 antibody, D10 antibody or mIgG control on day 0. On the next day intravenous transfer was carried out 5 χ 10⁵splenocytes from ROR 1 X TCL1 Tg mice.

All mice were then monitored weekly for an increase in the number of CD5 B leukemia cells^dullB200⁺ by flow cytometry from the 2nd week after the transfer.

Figure 19 shows a series of graphs illustrating results of flow cytometry analysis that show that antibodies against the ROR 1 receptor protein inhibit the development of lymphocytic leukemia-like leukemia in ROR 1 Tg mice. Two weeks after the adoption transfer, PBMC analysis was carried out. The data showed that the D10 antibody against the ROR 1 receptor protein, but not the 4A5 antibody against the ROR 1 receptor protein, can significantly inhibit the expansion of CD5 B leukemia cells^dullB220⁺ and ROR 1^brightB220⁺. Figure 20 is a graph illustrating that the D10 antibody against the ROR 1 receptor protein inhibits the growth and expansion of ROR 1xTCL1 B-cell leukemia B cells in the blood of the animals receiving them up to two weeks after receiving the last infusion of the mAb antibody.

Figure 21 shows the rapid internalization of D10 antibody against the ROR 1 receptor protein in chronic lymphocytic leukemia cells.

Figure 22 shows a series of graphs illustrating results of flow cytometry analysis showing that antibodies D10 and 4A5 against the ROR 1 receptor protein are internalized in chronic lymphocytic leukemia cells. Chronic lymphocytic leukemia cells were incubated with the hROR Ab-Alex647 mouse antibody for 30 min at 4 ° C. The cells were then washed and left at 4 ° C or incubated for 4 hours. at 37 ° C, followed by flow cytometry. A background signal without staining is also shown.

Figure 23 is a graph illustrating the kinetics of the internalization of D10 and 4A5 antibodies against the ROR 1 receptor protein.

Figure 24 is a diagram illustrating amino acids that have been converted to mouse amino acids to determine the residues necessary for binding mAbs to human ROR 1 receptor protein and western blots showing that the isoleucine residue 111 is needed to bind the 4A5 antibody to the human ROR receptor protein 1 .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0012] The subject matter of the present disclosure is described in detail below. The subject matter of the present disclosure may have many different forms and is not limited to the embodiments described herein; these embodiments are provided to meet the relevant legal requirements. Having become familiar with the following descriptions and attached figures, one skilled in the art will recognize that many modifications and developments may be made to the subject of the present invention. It is to be remembered, therefore, that the subject matter of the present invention is not limited to specific disclosed embodiments and that the modifications and other embodiments are within the scope of the appended claims.

[0013] Antibodies of the present invention were produced monoclonal using the techniques described above. In short, naturally occurring antibodies are generally tetramers containing two light chains and two heavy chains. Under experimental conditions, the antibodies can be cleaved using the papain proteolytic enzyme, which causes the disruption of each of the heavy chains leading to the formation of three distinct units. Two units, which consist of a light chain and a heavy chain fragment of approximately the same mass as the weight of the light chain, are called Fab fragments (i.e., antigen-binding fragments). The third unit, consisting of two equal lengths of the heavy chain, is called the Fc fragment.

[0014] Since the Fab and F (ab ') 2 fragments are smaller than the intact antibody molecules, more antigen-binding domains are available than when using whole antibody molecules. It is known that the proteolytic cleavage of a typical IgG molecule with papain results in the production of two distinct antigen-binding fragments called Fab fragments that contain an intact light chain linked to the amino terminal portion of the adjacent heavy chain by disulfide bonding. The remaining part of the immunoglobulin molecule digested by papain is called the Fc fragment and consists of the carboxyl terminal portions of the intact antibody and connected by disulfide bonds. If the antibody is digested with pepsin, a fragment called F (ab ') 2 is produced that does not contain the Fc region,

Fab) and disulfide bonds between the remaining parts of the adjacent heavy chains (Handbook of Experimental Immunology. Vol 1:

Immunochemistry, Weir, DM, Editor, Blackwell Scientific Publications, Oxford (1986)).

[0015] As known to those skilled in the art, altered antibodies (e.g., chimeric, humanized, CDR-grafted, bifunctional, antibody polypeptide dimers (i.e., linkage of two elements of an antibody polypeptide chain, e.g. one arm of an antibody containing a heavy chain and a chain) lightweight, a Fab fragment containing the VL, VH, CL and CH antibody domains, or a Fv fragment containing the domain

VL and the VH domain), single chain antibodies (e.g., scFv fragment (i.e.

a single-chain Fv fragment) comprising a VL domain fused to the VH domain via a linker etc.) can also be generated using methods known in the art.

[0016] Monoclonal antibody (mAb) production technology can be used to generate mAbs against the ROR 1 receptor protein.

Hybridomas are made using splenic cells from mice immunized with ROR 1 antigens. Spleen cells from each immunized mouse are combined with mouse Sp 2/0 myeloma cells, e.g. using the polyethylene glycol fusion technique described in Galfre, G. and Milstein , C., Methods Enzymol., 73: 3-46 (1981). Growth of hybridomas, selection in HAT medium, cloning and screening of clones against antigens is carried out using standard methodologies (Galfre, G. and Milstein, C., Methods Enzymol., 73: 3-46 (1981)):

[0017] Clones selected in the HAT medium are injected into the mouse's body to produce large amounts of mAbs in the fluid ascites, as described in Galfre, G. and Milstein, C., Methods Enzymol., 73: 3-46 ( 1981), which can be purified using protein A column chromatography (BioRad, Hercules, Calif.). MAbs are selected based on their (a) specificity for the ROR 1 receptor protein, (b) high binding affinity, (c) isotype, and (d) stability.

[0018] Mammal antibodies may be screened or screened for specificity to the ROR 1 receptor protein using any of a number of standard techniques, including western blot analysis (Koren, E. et al., Biochim Biophys Acta 876: 91-100 (1986)) and the enzyme immunoassay (ELISA) (Koren, E. et al., Biochim Biophys. Acta 876: 91-100 (1986)).

[0019] Humanized forms of mouse antibodies can be made by combining the CDRs of non-human antibodies with human solids using recombinant DNA techniques (see, e.g., Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033, 1989 and WO 90/0786). Human antibodies can be obtained using phage display methods (see, e.g., Dower et al., WO 91/17271, McCafferty et al., WO 92/01047). In these methods phage libraries are produced, the elements of which cause the expression of various antibodies on their external surfaces. Antibodies are typically expressed as Fv or Fab fragments. Phage expressing antibodies with the required specificity are selected by affinity concentration.

[0020] Human antibodies can be selected by competitive binding experiments for the same epitope specificity as specific murine antibodies. Using these techniques, a humanized antibody against the ROR 1 receptor protein having the human IgG1 constant domain and the kappa human constant chain domain with the mouse heavy and light chain variable portions can be obtained. The humanized antibody has the specificity of binding of the mouse antibody to the ROR 1 receptor protein, specifically the mAb 4A5 antibody described in Examples 4 and 5.

[0021] It may be desirable to generate and use functional mAb antibody fragments for a particular application. Typical IgG immunoglobulin molecules have a well-known basic structure of a Y-shaped tetramer molecule with a molecular weight of about 150,000 to 200,000 daltons consisting of two identical light polypeptide chains (containing about 220 amino acids) and two identical heavy polypeptide chains (containing about 440 amino acids).

The heavy chains are connected to each other by at least one disulfide bond. Each light chain is connected to an adjacent heavy chain by disulfide bonding. The antigen-binding center or domain is located in each arm of the Y-shaped antibody molecule and is formed between the amino-terminal portions of each pair of light and heavy chains linked by disulfide bonds. The amino-terminal portions of the light and heavy chains consist of about the first 110 amino-terminal amino acids and are referred to as light chain and heavy chain variable parts. In addition, parts of the light and heavy chain variables include hypervariable portions that contain segments of the amino acid sequences, called complementarity determining regions (CDRs). The CDRs provide antibody specificity for one particular center on the antigen molecule referred to as an epitope. A typical IgG immunoglobulin molecule is therefore divalent, i.e. it can bind to two antigen molecules, because each antigen binding center can bind to a specific epitope of each antigen molecule. The carboxyl-terminal parts of the light and heavy chains are similar or identical to other antibody molecules and are referred to as solid parts. The amino acid sequence of the constant part of the heavy chain of a particular antibody determines the class of the antibody, e.g. IgG, IgD, IgE, IgA or IgM. Some antibody classes contain two or more identical antibodies associated with each other in multivalent antigen binding systems.

[0022] Fab fragments and F (ab ') 2 mAbs that bind to the ROR 1 receptor protein can be used instead of whole mAbs. Since Fab and F (ab ') 2 fragments are smaller than intact antibody molecules, more antigen-binding domains are available than when using whole antibody molecules. It is known that the proteolytic cleavage of a typical IgG molecule with papain results in the production of two distinct antigen-binding fragments called Fab fragments that contain an intact light chain linked to the amino terminal portion of the adjacent heavy chain by disulfide bonding. The remaining part of the immunoglobulin molecule digested by papain is called the Fc fragment and consists of the carboxyl terminal portions of the intact antibody and connected by disulfide bonds. If the antibody is digested with pepsin, a fragment called F (ab ') 2 is produced that does not contain an Fc region, but contains both antigen binding domains held together by disulfide bonds between adjacent light and heavy chains (as Fab fragments) and disulfide bonds between remaining parts of adjacent heavy chains (Handbook of Experimental Immunology. Vol 1:

Immunochemistry, Weir, DM, Editor, Blackwell Scientific Publications, Oxford (1986)).

[0023] With reference to specific antibodies, the term "specific binding" means the binding of an antibody to a predetermined antigen. Typically, the antibody has a binding affinity corresponding to a K D value of 10^-8 M or less and binds to a predetermined antigen with an affinity (expressed as K D) at least 10-fold less, and preferably at least 100-fold less than its binding affinity to a non-specific antigen (e.g., BSA, casein) other than the pre-antigen. specific antigen or closely related antigen. Alternatively, the antibody may have a binding affinity corresponding to a KA of about 10⁶ M^-1, about 10⁷ M^-1, about 10⁸ M^-1, 10⁹ M^-1 or higher and binds to a predetermined antigen with an affinity (expressed as KA value) at least 10-fold higher, and preferably at least 100-fold greater than its affinity for binding a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or closely related antigen.

[0024] Furthermore, the phrase "an antibody having ROR 1 receptor specific protein binding specificity" also refers to antibody fragments with at least 90% or 95% sequence identity to any of the SEQ ID NO sequence of the polypeptides:

2. 4, 6, 8, 12, 14, 16, 18 and 20, including variants modified by mutation to improve their utility (e.g., increase their ability to attack specific cell types, etc.). Such variants include variants in which one or more conservative substitutions have been made to the antibody heavy chain and / or antibody light chain.

[0025] Such variants include variants in which one or more substitutions have been made to the nucleotide sequence of the heavy chain and / or the antibody light chain nucleotide sequence. In some embodiments, the variant has a light chain and / or a heavy chain having a nucleotide sequence at least 80% or at least 90% or at least 95% identical to the nucleotide sequences of SEQ ID NO: 1, 3, 5, 7, 11, 13 , 15, 17 and 19.

[0026] The polynucleotide sequences encoding the structural features of the antibodies of the present invention include the sequences listed below. After each polynucleotide sequence, the amino acid sequence of the encoded polypeptide follows. The light chain sequences that "correspond to" the heavy chain sequences are sequences of the same antibody; i.e. the heavy chain sequences F2 correspond to the sequences of the light chain F2, the heavy chain sequences D10 correspond to the sequences of the light chain D10 etc.

SEQ ID NO: 1 - coding sequence for the variable portion of the mouse 4A5 heavy chain antibody against the ROR 1 receptor protein: GAAGTGAAACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTC

CTGTGCAGCCTCTGGATT

CACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGATTCCAGAGAAGAGGCTGGAGTGGG

TCGCATCCATTAGTCGTG gtggtaccacctactatccagacagtgtgaagggccgattcaccatctccagagataatgtc

AGGAACATCCTGTACCTG

CAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGGAAGATATGATTACGA

CGGGTACTATGCAATGGA

CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 2 - polypeptide sequence of the variable portion of the heavy chain of the murine 4A5 antibody against the ROR 1 receptor protein:

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQIPEKRLEWVASISRGGTTYYPDS

VKGRFTISRDNVRNILYL

QMSSLRSEDTAMYYCGRYDYDGYYAMDYWGQGTSVTVSS

SEQ ID NO: 3 - coding sequence of the light chain variable portion of the mouse 4A5 antibody against the ROR 1 receptor protein: GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTAT

CACTTGCAAGGCGAGTCC

GGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGA

TCTATCGTGCAAACAGAT

TGGTTGATGGGGTCCCATCAAGGTTCAGTGGCGGTGGATCTGGGCAAGATTATTCTCTCACC

ATCAACAGCCTGGAGTAT

GAAGATATGGGAATTTATTATTGTCTACAGTATGATGAATTTCCGTACACGTTCGGAGGGGG

GACCAAGCTGGAAATGAA

AC

SEQ ID NO: 4 - polypeptide sequence of the light chain variable portion of the mouse 4A5 antibody against the ROR 1 receptor protein: DIKMTQSPSSMYASLGERVTITCKASPDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRF SGGGSGQDYSLTINSLEY

EDMGIYYCLQYDEFPYTFGGGTKLEMK

SEQ ID NO: 5 - coding sequence for the variable part of the heavy chain of murine F2, F12 and G6 antibodies against the ROR 1 receptor protein: GAGGTCCAGCTACAGCAGTCTGGACCTGAGCTGGAGAAGCCTGGCGCTTCAGTGAAGATATC

CTGCAAGGCTTCTGGTTT

CGCATTCACTGGCTACAACATGAACTGGGTGAAACAGACCAATGGAAAGAGCCTTGAGTGGA

TTGGAAGTATTGATCCTT

ACTATGGTGGTTCTACCTACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAA

TCCTCCAGCACAGCCTAC

ATGCAACTCAAGAGCCTCACATCTGATGACTCTGCAGTCTATTACTGTGCAAGATCCCCGGG

GGGGGACTATGCTATGGA

CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 6 - polypeptide sequence of the variable part of the heavy chain of murine F2, F12 and G6 antibodies against the ROR 1 receptor protein:

EVQLQQSGPELEKPGASVKISCKASGFAFTGYNMNWVKQTNGKSLEWIGSIDPYYGGSTYNQ KFKDKATLTVDKSSSTAY

MQLKSLTSDDSAVYYCARSPGGDYAMDYWGQGTSVTVSS

SEQ ID NO: 7 - coding sequence for the light chain variable part of mouse F2, F12 and G6 antibodies against the ROR 1 receptor protein:

GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTGTAGGAGAGAGAGTCACTAT

CACTTGTAAGGCGAGTCA

GGGCATTAATAGCTATTCAGGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGA

TTTATCGTGGAAATAGAT

TGGTGGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACC

ATCAGCAGCCTGGAGTAT

GAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGG

GACCAAGCTGGAAATAAA

AC

SEQ ID NO: 8 - polypeptide sequence of the light chain variable part of murine F2, F12 and G6 antibodies against the ROR 1 receptor protein:

DIKMTQSPSSMYASVGERVTITCKASQGINSYSGWFQQKPGKSPKTLIYRGNRLVDGVPSRF

SGSGSGQDYSLTISSLEY

EDMGIYYCLQYDEFPYTFGGGTKLEIK

SEQ ID NO: 9 - coding sequence for a portion of the variable heavy chain of the murine G3 antibody against the ROR 1 receptor protein: CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGACTTCAGTGAAGCTGTC

CTGCAAGGCTTCTGGCTA

CAACTTCACCAACTACTGGATAAACTGGGTGAAGCTGAGGCCTGGACAAGGCCTTGAGTGGA

TTGGAGAAATTTATCCTG

GTAGTGGTAGTACTAATTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGCAGACACA

TCCTCCAGCACAGCCTAC

ATGCAACTCAGCAGCCTGGCATCTGAAGACTCTGCTCTCTATTACTGTGCAAGAGATGGTAA

CTACTATGCTATGGACTA.

CTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 10 - polypeptide sequence of the variable portion of the mouse G3 antibody heavy chain against the ROR 1 receptor protein:

QVQLQQPGAELVKPGTSVKLSCKASGYNFTNYWINWVKLRPGQGLEWIGEIYPGSGSTNYNE

KFKSKATLTADTSSSTAY

MQLSSLASEDSALYYCARDGNYYAMDYWGQGTSVTVSS

SEQ ID NO: 11 - coding sequence for the light chain variable portion of the murine G3 antibody against the ROR 1 receptor protein:

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCAT

CACTTGCAGGGCAAGTCA

GGACATTAACAATTATTTAAACTGGTATCAACAGAAACCAGATGGAACTGTTAAACTCCTGA

TCTACTACACATCAGCAT

TACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACC

ATTAGCAACCTGGAACAA

GAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCTCCGTACACGTTCGGAGG

GGGGACCAAGCTGGAAAT

AAAAG

SEQ ID NO: 12 - polypeptide sequence of the light chain variable portion of the murine G3 antibody against the ROR 1 receptor protein: DIQMTQTTSSLSASLGDRVTITCRASQDINNYLNWYQQKPDGTVKLLIYYTSALHSGVPSRF SGSGSGTDYSLTISNLEQ

EDIATYFCQQGNTLPPYTFGGGTKLEIK

SEQ ID NO: 13 - coding sequence for a portion of the variable heavy chain of the mouse D10 antibody against the ROR 1 receptor protein:

CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGACTCTGTCCATCAC

TTGCACTGTCTCTGGGTT

TTCATTAACCAGTTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGC

TGGGAGTAATATGGGCTG

GTGGATTCACAAATTATAATTCGGCTCTCAAGTCCAGACTGAGCATCAGCAAAGACAACTCC

AAGAGCCAAGTTCTCTTA

AAAATGACCAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGGAGAGGTAGTTC

CTATTCTATGGACTATTG

GGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 14 - polypeptide sequence of the variable part of the mouse D10 antibody heavy chain against the ROR 1 receptor protein: QVQLKESGPGLVAPSQTLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWGGFTNYNSA LKSRLSISKDNSKSQVLL

KMTSLQTDDTAMYYCARRGSSYSMDYWGQGTSVTVSS

SEQ ID NO: 15 - coding sequence of the light chain variable portion of the mouse D10 antibody against the ROR 1 receptor protein:

GAAATTGTGCTCTCTCAGTCTCCAGCCATCACAGCTGCATCTCTGGGCCAAAAGGTCACCAT

CACCTGCAGTGCCAGTTC

AAATGTAAGTTACATCCACTGGTACCAGCAGAGGTCAGGCACCTCCCCCAGACCATGGATTT

ATGAAATATCCAAACTGG

CTTCTGGAGTCCCAGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATC

AGCAGCATGGAGGCTGAA

GATGCTGCCATTTATTATTGTCAGCAGTGGAATTATCCTCTTATCACGTTCGGCTCGGGGAC

AAAGTTGGAAATACAA

SEQ ID NO: 16 - polypeptide sequence of the light chain variable part of the mouse D10 antibody against the ROR 1 receptor protein:

EIVLSQSPAITAASLGQKVTITCSASSNVS ¥ IHWYQQRSGTSPRPWIYEISKLASGVPVRFS

GSGSGTSYSLTISSMEAE

DAAIYYCQQWNYPLITFGSGTKLEIQ

SEQ ID NO: 17 - coding sequence for a portion of the variable heavy chain of mouse H10 and G11 antibodies against the ROR 1 receptor protein: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTC

CTGTGCAGCCTCTGGATT

CACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGG

TCGCTTCCATTAGTACTG

GTGCTAGCGCCTACTTTCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCC

AGGAACATCCTGTACCTG

CAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTATTGTGCAAGGATTACTACGTC

TACCTGGTACTTCGATGT

CTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO: 18 - polypeptide sequence of the variable part of the heavy chain of mouse H10 and G11 antibodies against the ROR 1 receptor protein:

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISTGASAYFPDS

VKGRFTISRDNARNILYL

QMSSLRSEDTAMYYCARITTSTWYFDVWGAGTTVTVSS

SEQ ID NO: 19 - coding sequence for the light chain variable portion of murine H10 and G11 antibodies against the ROR 1 receptor protein:

GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTAT

CACTTGCAAGGCGAGTCA

GGACATTAATAGTTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGA

TCTATCGTGCAAACAGAT

TGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACC

ATCAGCAGCCTGGAGTAT

GAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGG

GACCAAGCTGGAAATAAA

AC

SEQ ID NO: 20 - polypeptide sequence of the light chain variable part of murine H10 and G11 antibodies against the ROR 1 receptor protein:

DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRF

SGSGSGQDYSLTISSLEY

EDMGIYYCLQYDEFPYTFGGGTKLEIK [0027] In one aspect, antibodies having a heavy chain encoded by the polynucleotide sequence SEQ ID NO: 13 and a light chain encoded by the polynucleotide sequence SEQ ID NO: 15 are shown.

[0028] Also disclosed is an antibody comprising a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 1 and a light chain encoded by the polynucleotide sequence of SEQ ID NO: 3.

[0029] Also disclosed are antibodies having a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 5 and a light chain encoded by the polynucleotide sequence of SEQ ID NO: 7; or by the polynucleotide sequence SEQ ID NO: 9 and a light chain encoded by the polynucleotide sequence SEQ ID NO: 11; or by the polynucleotide sequence SEQ ID NO: 15 and the light chain encoded by the polynucleotide sequence SEQ ID NO: 17, [0030] In another aspect, antibodies are shown that comprise a heavy chain with the polypeptide sequence of SEQ ID NO: 14 and a light chain with the SEQ polypeptide sequence ID NO: 16.

[0031] Also disclosed are antibodies that comprise a heavy chain with the polypeptide sequence of SEQ ID NO: 2 and a light chain with the polypeptide sequence of SEQ ID NO: 4.

[0032] In one aspect of the disclosure, we provide isolated polynucleotides encoding an antibody that specifically binds the ROR 1 receptor protein, (a) consisting of a portion of the heavy chain encoded by polynucleotides with at least 90% sequence match to any of the sequences selected from the group containing sequences SEQ ID NO: 1, 5, 9, 13 or 17, (b) consisting of a corresponding part of a light chain encoded by polynucleotides with at least 90% sequence match to any one of the sequences selected from the group consisting of SEQ ID NO: 3, 7 , 11, 15 or 19, and (c) specifically binding the 3 'terminus or central portion of the Ig region of the extracellular domain of the human or murine ROR 1 receptor protein.

[0033] Also disclosed are antibodies that bind residues in the central Ig region of the extracellular domain of a human or murine ROR 1 receptor protein (amino acids 1-147 in a human molecule). In one aspect, the antibodies of the present invention bind to amino acids 70-130 of the human ROR 1 receptor protein. Examples of such antibodies include antibodies

4A5, G11, H10 and G3.

[0034] Alternatively or additionally, the residue corresponding to the residue of the human ROR 1 receptor protein in position 111 that is present in the extracellular domain is very important in terms of antibody binding capacity.

[0035] Also disclosed are antibodies that bind residues within the Ig 3 'region between the Ig domain and the CRD domain of a human or murine ROR 1 receptor protein (amino acids 1-165 in a human molecule). In one aspect, the antibodies of the present disclosure bind to amino acids 130-165 of the human ROR 1 receptor protein. Examples of such antibodies include antibodies D10, F2, F12 and G6.

[0036] Alternatively or additionally, the antibodies bind a glutamic acid residue corresponding to the residue found in the extracellular domain of the human ROR 1 receptor protein at position 138.

[0037] Alternatively or additionally, the residue corresponding to the residue of the human ROR 1 receptor protein present in the extracellular domain at position 138 is very important in terms of the binding capacity of the antibodies.

Alternatively or additionally, the encoded antibody has in vivo activity in reducing the burden of leukemia or lymphoma cells in the animal model approved in the field at a rate of 2-8 times or at least 2,

3, 4, 5, 6, 7 or 8 times greater than in the case of a human antibody against a wild-type ROR 1 receptor protein or a 4A5 monoclonal antibody (disclosed herein).

[0039] Alternatively or additionally, the encoded antibody has in vivo activity inhibited the expansion of CD5 B leukemia cells^dullB220⁺ and ROR 1^brightB220⁺.

Alternatively or additionally, the encoded antibody is internalized in leukemia or lymphoma cells at a rate of at least 2 times or at least 2, 3,

4, 5, 6, 7, 8, 9 or 10 greater compared to monoclonal antibody 4A5. Such antibodies are particularly useful as drug carriers for a selected cell.

[0041] An example of an antibody having all of the aforementioned functional features is an antibody D10 that has a heavy chain part encoded by the sequence of SEQ ID NO: 13 and a light chain part encoded by the sequence of SEQ ID NO: 15.

[0042] In another aspect, disclosed are polypeptides consisting of antibodies or containing antibodies that bind specifically to the ROR 1 receptor protein and (a) consist of a heavy chain portion at least 90% sequence aligned with any one of the SEQ sequences. ID. NO: 2, 6, 10, 14 or 18, (b) consisting of a corresponding part of the light chain of at least 90% sequence correspondence with any of the sequences of SEQ ID NO: 4, 8, 12, 16 or 20, and (c ) binding specifically to the 3 'terminus or central portion of the Ig region of the extracellular domain of the human or murine ROR receptor 1 receptor protein. In one aspect, the isolated polypeptide is an antibody. In another aspect, the polypeptide is a Fab or F (ab) '2 fragment.

[0043] In certain embodiments, the antibody of the present invention may comprise a detectable label. Such labels are known in the art. These include radioactive isotopes and fluorescent labels. The internalization of the connection indicating the passage through the transporters can therefore be detected by detecting the signal from the inside of the cell or any of the various reporters. The reporter may be a marker, e.g. a fluorophore, a chromophore, a radioisotope. Confocal imaging can also be used to detect tag internalization because it provides sufficient spatial resolution to distinguish between cell surface fluorescence and intracellular fluorescence; alternatively, confocal imaging can be used to track the movement of compounds over time. Another way to detect the internalization of the compound is to use a reporter that is a substrate of the enzyme expressed within the cell. After the internalization of the complex, the substrate is metabolized by the enzyme and generates an optical signal or causes a radioactive decay indicative of absorption. Light emission can be monitored by means of commercially available devices based on PMT technology or CCD based imaging systems. In addition, determination methods using the LCMS technique for the detection of transported compounds or electrophysiological signals indicative of transport activity are used. After the internalization of the complex, the substrate is metabolized by the enzyme and generates an optical signal or causes a radioactive decay indicative of absorption. Light emission can be monitored by means of commercially available devices based on PMT technology or CCD based imaging systems. In addition, determination methods using the LCMS technique for the detection of transported compounds or electrophysiological signals indicative of transport activity are used. After the internalization of the complex, the substrate is metabolized by the enzyme and generates an optical signal or causes a radioactive decay indicative of absorption. Light emission can be monitored by means of commercially available devices based on PMT technology or CCD based imaging systems. In addition, determination methods using the LCMS technique for the detection of transported compounds or electrophysiological signals indicative of transport activity are used.

[0044] In certain embodiments of therapeutic use, the selected antibody may be administered separately in combination with another antibody of the present invention or with one or more combination therapies for combination therapy to treat ROR 1 cancer. When at least one of the antibodies described in this document is administered as a therapeutic agent, it may exert a beneficial effect on an individual's organism through a variety of mechanisms. For example, in some embodiments, antibodies that bind specifically to the ROR 1 receptor protein are purified and administered to a patient to neutralize one or more forms of the ROR 1 receptor protein, to block one or more of the effects of the ROR 1 receptor protein or to block or inhibit the interaction of one or more forms of the ROR 1 receptor protein with another biomolecule; e.g. for the treatment of CLL or other ROR 1 tumors. In all of these treatments, a therapeutically effective dose of a pharmaceutical composition comprising antibodies and therapeutic agents is administered which a pharmacologist or clinician with average knowledge of in the field of human immunotherapy can determine.

[0045] In one embodiment, the present invention can be used in a method of treating cancer by administering to a human in need of such treatment a therapeutically effective dose of the antibody of the present invention.

[0046] In another embodiment, the present invention can be used in a method of treating cancer by administering to a human in need of such treatment a therapeutically effective dose of the antibody of the present invention.

[0047] Preferably, said methods allow to reduce the burden of leukemia or lymphoma cells (as shown in the animal model validated in the field and to an equivalent degree) at a rate of 2-8 times or at least 2, 3, 4, 5, 6, 7 or 8 times greater than in the case of a human antibody against the ROR 1 receptor protein of wild type or the 4A5 monoclonal antibody (disclosed herein).

[0048] Said methods also represent a therapeutic approach to inhibiting the expansion of CD5 B leukemia cells^dullB220⁺ and ROR 1^brightB220⁺.

[0049] As discussed herein, the antibodies of the present invention can be humanized antibodies and can be combined for therapeutic purposes with additional active or inert ingredients, e.g. in conventional pharmaceutically acceptable carriers or diluents, e.g. immunogenic adjuvants, and optionally with molecules having supporting or acting in combination therapy, such as anti-inflammatory and anti-fibrinolytic agents. Antibodies that are readily internalized in cells as demonstrated herein with respect to the D10 antibody are also useful as drug carriers for delivery to target cells (e.g., as shown in Figures 21-23).

[0050] When carrying out determinations, performing diagnostic tests and treatment, it is desirable to first prepare kits comprising a combination of antibodies described herein with other materials.

For example, for sandwich immunoassays, the kits may include an antibody that specifically binds the ROR 1 receptor protein optionally linked to a suitable carrier, a lyophilized preparation or enzyme labeled monoclonal antibody solution that binds to the same antigen together with a monoclonal or polyclonal antibody labeled with an enzyme. in the same manner, a standard solution of purified ROR 1 receptor protein, buffer solution, cleaning solution, pipettes, reaction vessel, etc. Additionally, kits may optionally contain markers and / or instructional materials with guidelines (i.e., protocols) for using the methods described herein in the determination environment. Instructional materials usually contain written or printed materials, but are not limited to them. Consideration is given to the use of a carrier on which such instructions can be recorded and forwarded to the end user. Such carriers include electronic storage media (eg magnetic disks, tapes, cassettes, processors) and optical media (e.g. CD ROMs). Such carriers may be references to websites containing such instructional materials.

[0051] In general, the method of diagnosing ROR 1 tumor by in vitro method involves combining the putative cancer cells taken from a human with an antibody of the present invention and detecting binding to the ROR 1 receptor protein expressed on these cells as compared to expression on post-embryonic human non-cancer cells. Such diagnostic methods involve providing a diagnostically effective amount of the antibodies of the present invention, which may be determined by a diagnostic or in vitro diagnostic expert of average knowledge in the field of human cancers.

[0052] The following examples are provided to illustrate the present invention, but they are not limiting in nature.

EXAMPLE 1: GENERATION OF MONOCLONAL ANTIBODIES AGAINST ROR 1 RECEPTOR PROTEIN In order to obtain mAbs generated from the hybridoma, mice were inoculated with DNA, protein and adenovirus constructs expressing the extracellular portion (AA 1-406) of the ROR 1 receptor protein that contains the Ig domains, CRD and

Kringle, as well as the adjacent regions of the connector (Figures 10-11). Because of the high degree of homology between mouse and human molecules, various cytokines and immune stimulating agents, such as complete Freund adjuvants, were injected to maximize the level of antibody production against the human ROR 1 receptor protein. The hybridoma produced from the mAb was obtained and screened for screening. the binding of the human and mouse ROR receptor 1 receptor protein. An example of an mAb antibody obtained from a hybridoma is D10 antibody.

EXAMPLE 2: MANUFACTURING OF ANTIBODY AGAINST PROTEIN

ROR RECEPTOR 1 BY USING FAG EXPRESSION METHODS [0054] A second set of antibodies was generated by using its own enriched library of phages (Alere, Inc. San Diego). Antibodies against the human ROR 1 receptor protein bind epitopes extending over the entire length of the extracellular domain of the ROR 1 receptor protein (Figure 12). An example of an antibody against the ROR 1 receptor protein obtained by phage display is antibody 4A5.

EXAMPLE 3: ANALYSIS OF IN VITRO ANTIBODIES AGAINST ROR 1 RECEPTOR PROTEIN [0055] Antibodies produced from hybridomas or phage display were screened for binding to the human and mouse ROR 1 receptor protein. D10 antibodies against the ROR 1 and 4A5 receptor protein were found to bind only human ROR 1 receptor protein and do not cross-react with the murine ROR 1 receptor protein.

EXAMPLE 4: DETERMINATION OF BINDING CENTERS OF ANTIBODIES AGAINST ROR 1 RECEPTOR PROTEIN Since mAbs against the ROR 1 receptor protein are species specific, a series of chimeric proteins have been made that were used to determine the binding center for each mAb against ROR 1 receptor protein ( Figure 13). For the purpose of the second level screening test, a number of deletion constructs were made to determine the actual extracellular domain of ROR 1 to which mAbs bind. After the binding domain was identified, truncated chimeric ROR 1 molecules were generated to identify specific sub regions that are recognized by mAb antibodies to the human ROR 1 receptor protein (Figure 14). The last step was to determine the actual amino acids attacked by these antibodies. For the purpose of this final screening test, mouse-transformed human amino acids in fragments of sub-domains were generated to determine the residues necessary for antibody mAb binding (Figure 15). Based on this screening paradigm, sub-domains of the mAbs were determined (Figure 15). The mAb D10 antibody against the human ROR 1 receptor protein was found to require a glutamic acid residue at position 138 to bind the Ig domain of the human ROR 1 receptor protein molecule. After replacing this amino acid with the lysine residue of the mouse D10 molecule molecule no longer binds the ROR 1 receptor protein. For the purpose of this final screening test, mouse-transformed human amino acids in fragments of sub-domains were generated to determine the residues necessary for antibody mAb binding (Figure 15). Based on this screening paradigm, sub-domains of the mAbs were determined (Figure 15). The mAb D10 antibody against the human ROR 1 receptor protein was found to require a glutamic acid residue at position 138 to bind the Ig domain of the human ROR 1 receptor protein molecule. After replacing this amino acid with the lysine residue of the mouse D10 molecule molecule no longer binds the ROR 1 receptor protein. For the purpose of this final screening test, mouse-transformed human amino acids in fragments of sub-domains were generated to determine the residues necessary for antibody mAb binding (Figure 15). Based on this screening paradigm, sub-domains of the mAbs were determined (Figure 15). The mAb D10 antibody against the human ROR 1 receptor protein was found to require a glutamic acid residue at position 138 to bind the Ig domain of the human ROR 1 receptor protein molecule. After replacing this amino acid with the lysine residue of the mouse D10 molecule molecule no longer binds the ROR 1 receptor protein. Based on this screening paradigm, sub-domains of the mAbs were determined (Figure 15). The mAb D10 antibody against the human ROR 1 receptor protein was found to require a glutamic acid residue at position 138 to bind the Ig domain of the human ROR 1 receptor protein molecule. After replacing this amino acid with the lysine residue of the mouse D10 molecule molecule no longer binds the ROR 1 receptor protein. Based on this screening paradigm, sub-domains of the mAbs were determined (Figure 15). The mAb D10 antibody against the human ROR 1 receptor protein was found to require a glutamic acid residue at position 138 to bind the Ig domain of the human ROR 1 receptor protein molecule. After replacing this amino acid with the lysine residue of the mouse D10 molecule molecule no longer binds the ROR 1 receptor protein.

[0057] In a similar manner, it was determined that the mAb 4A5 antibody to the human ROR 1 receptor protein requires an isoleucine residue at position 111 to bind the human ROR 1 molecule (Figure 24). After replacing this amino acid with the aspartic residue of the mouse molecule 4A5 molecule, it no longer binds the ROR 1 receptor protein. It was also determined that the G11, H10 and G3 antibodies against the ROR 1 receptor protein bind the same region as the 4A5 antibody.

[0058] Using the standard cross-blocking technique, the binding sites for the F2, F12 and G6 antibodies against the ROR 1 receptor protein were determined. Based on these experiments, it was determined that the F2, F12 and G6 antibodies blocked the D10 antibody against the ROR 1 receptor protein, indicating that they have a joint binding center.

EXAMPLE 5: DETERMINATION OF KD ANTIBODIES D10 AND 4A5 AGAINST ROLE BACK RECEPTOR PROTEIN [0059] The K D values ​​of antibodies against the ROR 1 receptor protein were determined using standard techniques. The KD of the D10 antibody was found to be 40 nM, and the 4A5 antibody was 4 nM (Figures 16A and B).

EXAMPLE 6: ANALYSIS OF IN VIVO ANTIBODIES AGAINST ROLE RECEPTOR PROTEIN [0060] Antibodies of D10 mAb were evaluated in several in vivo models. In a nicotine dependent mouse model of in vivo heterologous transplantation, two doses of the mAb mAb of 10 mg / kg per 4 CLL main cells in 76 mice were used. As shown in Figure 17, the mAb D10 antibody substantially eliminated CLL cells in a dose-dependent manner. In contrast, the mAb 4A5 antibody showed minimal activity during these studies, although the kDa value of this mAb antibody is 10-fold higher (4 compared to 40) than the mAb D10 antibodies.

[0061] In addition to this activity model, the mAb D10 antibody was also tested in a mouse immune normal transgenic model that spontaneously produces leukemic cells expressing the human ROR 1 receptor protein (Figures 18-20). Db and 4A5 mAbs were administered against the ROR 1 receptor protein and control IgG antibodies (10 mg / kg) before and after the adoptive transfer of ROR 1xTCL1 CLL B cells into the Balb C mouse organism. D10 antibody but no control IgG antibody or 4A5 antibody , inhibits the growth and expansion of B RART 1xTCL leukemia cells in the blood of the animals receiving them up to two weeks after receiving the last infusion of the mAb antibody.

[0062] It has been shown that in addition to the leukemia of the mAb antibody, the D10 antibody against the ROR 1 receptor protein is internalized in CLL cells and in B cell leukemia lines and lymphoma at a faster rate and to a greater extent than other MAb antibodies against the ROR 1 receptor protein, which bind other antigenic centers on the extracellular portion of the ROR 1 protein (Figures 21-23). Due to the lack of ROR 1 receptor protein in the postpartum tissues and its rapid rate of internalization, the mAb D10 antibody may be an excellent carrier drug protein; e.g. for use in the regulation of cytotoxicity using an antibody-drug conjugate (ADC). Based on the results of these preclinical studies, it was determined that the mAb D10 antibody can be effective in the treatment of leukemia, lymphoma and solid tumors in tumors,

[0063] Although, for greater clarity and easier comprehension, the above-mentioned subject of the invention is described in detail by means of illustrations and examples, it will be quite clear to those skilled in the art that certain changes and modifications may be made to the present invention, which, however, will not diverge from the scope of the attached claims.