Anti-ror1 antibodies and methods of making and using the same

Abstract

The present application provides anti-ROR1 monoclonal antibodies, antigen-binding portions thereof, therapeutic compositions thereof, and / or nucleic acids encoding the same, and their use in upregulating T cell function to enhance cell-mediated immune responses in the treatment of cancer and other T cell dysfunction.

Description

[0001] This application is a divisional application of Chinese invention patent application (application number: 2018800383325; invention title: anti-ROR1 antibody and its preparation and use method).

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62551035, filed August 28, 2017; U.S. Provisional Patent Application No. 62551032, filed August 28, 2017; U.S. Provisional Patent Application No. 62524554, filed June 25, 2017; U.S. Provisional Patent Application No. 62524557, filed June 25, 2017; U.S. Provisional Patent Application No. 62524558, filed June 25, 2017; U.S. Provisional Patent Application No. 62545603, filed August 15, 2017; U.S. Provisional Patent Application No. 62551032, filed August 28, 2017; and U.S. Provisional Patent Application No. 62551065, filed August 28, 2017, the entire disclosure of which is expressly incorporated herein by reference. Technical Field

[0004] This application generally relates to the field of antibody technology, and more specifically to the preparation and use of anti-ROR1 antibodies. Background Technology

[0005] Cancer is a major health problem worldwide. In the United States alone, an estimated 1,685,210 new cancer cases were diagnosed in 2016, with 595,690 deaths (http: / / www.cancer.gov). Therefore, any drug that can reduce the severity or mortality of cancer is ideal.

[0006] In the immune system, resting T cells are activated to respond to antigens via primary signals delivered by T cell receptors (TCRs) from exogenous antigenic peptides presented by antigen-presenting cells (APCs). In addition to these primary signals, secondary positive and negative co-stimulatory signals further influence T cell responses. Complete T cell activation requires a secondary positive signal (Lafferty et al., Ausl. J. Exp. Biol. Med. Sci. 5327-42 (1975)). Negative secondary signals can lead to T cell suppression and tolerance.

[0007] ROR1, also known as neurotrophic tyrosine kinase receptor-associated 1 (NTRKR1), is an enzyme encoded by the ROR1 gene in humans. (See Masikowski P and Carroll RD, J. Bio. Chem. 267(36):26181-90. 1992; Reddy UR, et al. Oncogene. 13(7):1555-9(1996)). ROR1 is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. ROR1 has recently been shown to be expressed on ovarian cancer stem cells, where it appears to play a functional role in promoting migration / invasion or spheroidization and tumor transplantation in immunodeficient mice in vitro. Treatment with a ROR1 (UC-961)-specific humanized mAb can inhibit the ability of ovarian cancer cells to migrate, form spheroids, or transplant into immunodeficient mice. Furthermore, this treatment inhibited the growth of tumor xenografts, and conversely, reduced the ability of tumor xenografts to be transplanted into immunodeficient mice, with a relative lack of cells characteristic of CSCs, suggesting that UC-961 treatment can impair CSC renewal. In summary, these studies indicate that ovarian CSCs express ROR1, which can serve as a target for anti-CSC therapy. (See Zhang S et al., PNAS. 111(48):17266-71(2014)).

[0008] ROR1 is expressed at low levels in many malignant tumors, even in normal adult tissues. Much like its physiological function, ROR1 in cancer can exhibit kinase activity-dependent or kinase activity-independent functions, possibly a result of tissue-specific expression of co-receptors or effector proteins. ROR1 gene knockout, EGFR signaling enhancement, and ROR1-mediated EMT gene upregulation inducing apoptosis support the view that ROR1 plays an important role in cancer progression. Further investigation is needed to elucidate the tumor-specific mechanisms of ROR1 overexpression and its contribution to cancer initiation and progression. Summary of the Invention

[0009] In particular, this disclosure provides anti-ROR1 monoclonal antibodies, their antigen-binding moieties, their therapeutic compositions, and / or nucleic acids encoding them.

[0010] In one embodiment, this disclosure provides one or more isolated monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind to human ROR1. In one embodiment, the isolated one or more mAbs or antigen-binding fragments comprise an antigenic peptide sequence having a sequence disclosed herein. In one embodiment, the isolated mAb or antigen-binding fragment is selected from the sequences disclosed herein.

[0011] In one embodiment, the isolated monoclonal antibody (mAb) or antigen-binding fragment has the same characteristics as SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:116, SEQ ID NO:120, SEQ ID NO:120, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:10 The amino acid sequences NO:124, SEQ ID NO:128, or SEQ ID NO:132 have a homology percentage. In one embodiment, the homology percentage is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0012] In one embodiment, the binding affinity Kd of the isolated mAb or antigen-binding fragment to ROR1 is no greater than 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. In one embodiment, ROR1 is human ROR1.

[0013] In one embodiment, the isolated mAb or antigen-binding fragment may exhibit one or more functional properties. Examples of functional properties include, but are not limited to, high affinity binding to ROR1, enhanced T cell activation, the ability to stimulate antibody responses, and / or the ability to reverse the suppressive function of immunosuppressive cells such as regulatory T cells. In one embodiment, enhanced T cell activation includes T cell proliferation, IFN-γ and / or IL-2 secretion, or combinations thereof. In one embodiment, immunosuppressive cells include regulatory cells.

[0014] In some embodiments, the isolated mAb or antigen-binding fragment may include a human framework region. In one embodiment, the isolated mAb or antigen-binding fragment may include a humanized antibody, a chimeric antibody, or a recombinant antibody.

[0015] In one embodiment, the isolated mAb or antigen-binding fragment is IgG. In one embodiment, the antigen-binding fragment may include Fv, Fab, F(ab')2, scFV, or scFV2 ​​fragments. In one embodiment, the isolated mAb may be a bispecific antibody, a trispecific antibody, or a multispecific antibody.

[0016] In one embodiment, this application provides isolated mAbs or antigen-binding fragments having binding specificity to ROR1 and IgG1 heavy chains. The IgG heavy chain comprises amino acid sequences having a homology percentage with SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:99, SEQ ID NO:107, SEQ ID NO:115, SEQ ID NO:123, or SEQ ID NO:131. This homology percentage is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0017] In one embodiment, this application provides a separated mAb or antigen-binding fragment having binding specificity to ROR1 and the κ light chain. In one embodiment, the κ light chain comprises an amino acid sequence having a homology percentage with SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:51, SEQ ID NO:59, SEQ ID NO:67, SEQ ID NO:75, SEQ ID NO:83, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:119, or SEQ ID NO:127. This homology percentage is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0018] In one embodiment, this application provides a separated mAb or antigen-binding fragment having binding specificity to ROR1 and a variable light chain. The variable light chain comprises an amino acid sequence having a percentage of homology with SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, or SEQ ID NO: 128. This percentage of homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0019] In one embodiment, this application provides a separated mAb or antigen-binding fragment having binding specificity to ROR1 and the variable heavy chain. The variable heavy chain comprises an amino acid sequence having a homology percentage with SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO:116, SEQ ID NO:124, or SEQ ID NO:132. The homology percentage is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0020] This application also provides isolated nucleic acids encoding at least a portion of the isolated mAb or antigen-binding fragment disclosed herein. In one embodiment, the isolated mAb or antigen-binding fragment has a percentage of homology with the IgG1 heavy chain SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:99, SEQ ID NO:107, SEQ ID NO:115, SEQ ID NO:123, or SEQ ID NO:131. In one embodiment, the isolated mAb or antigen-binding fragment has a percentage of homology with the κ light chain: SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:51, SEQ ID NO:59, SEQ ID NO:67, SEQ ID NO:75, SEQ ID NO:83, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:119, or SEQ ID NO:127. In one embodiment, the isolated mAb or antigen-binding fragment has a percentage of homology with the variable light chains: SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:84, SEQ ID NO:96, SEQ ID NO:104, SEQ ID NO:112, SEQ ID NO:120, or SEQ ID NO:128.In one embodiment, the isolated mAb or antigen-binding fragment has a homology percentage with the variable heavy chains: SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO:116, SEQ ID NO:124, or SEQ ID NO:132. This homology percentage is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0021] This application also provides an expression vector containing an isolated nucleic acid encoding an amino acid sequence having a percentage of homology with the amino acid sequences disclosed herein. The percentage of homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%. In one embodiment, the vector can be expressed in cells.

[0022] This application also provides a host cell comprising a nucleic acid encoding an amino acid sequence having a percentage of homology with the amino acid sequences disclosed herein. This percentage of homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%. In one embodiment, the host cell may be a prokaryotic cell or a eukaryotic cell.

[0023] On the other hand, this application provides a method for producing an antibody or antigen-binding fragment thereof that has binding specificity to human ROR1. In one embodiment, the method includes the steps of: providing a host cell containing an expression vector expressible in a host cell, the expression vector comprising a nucleic acid encoding at least a portion of an isolated mAb or antigen-binding fragment, or a peptide having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with said mAb or antigen-binding fragment; to produce an antibody by expressing said nucleic acid.

[0024] This application also provides immunoconjugates. In one embodiment, the immunoconjugate comprises a pharmaceutical unit or imaging agent linked to a separated mAb or antigen-binding fragment via a linker.

[0025] The connector can be cuttable or non-cuttable. In one embodiment, the connector is a chemical connector. In one embodiment, the connector comprises a covalent bond, such as an ester bond, ether bond, amine bond, amide bond, disulfide bond, imide bond, sulfone bond, phosphate bond, phosphorus ester bond, peptide bond, hydrazone bond, or a combination thereof. In one embodiment, the connector comprises a hydrophobic poly(ethylene glycol) connector. In one embodiment, the connector comprises a peptide bond.

[0026] In one embodiment, the pharmaceutical unit in the immunoconjugate includes a chemotherapeutic agent, a growth inhibitor, a calichiomycin-like pharmaceutical unit, an antimitotic agent, a toxin, a radioisotope, a therapeutic agent, or a combination thereof. In one embodiment, the pharmaceutical unit comprises calichiomycin, ozogamicin, monomethylaurestatin E, emtansine, or a combination thereof. In another embodiment, the pharmaceutical unit comprises calichiomycin, ozogamicin, monomethylaurestatin E, emtansine, or a combination thereof.

[0027] In one embodiment, the pharmaceutical unit is selected from cytotoxic agents, immunomodulators, imaging agents, or combinations thereof. In one embodiment, the cytotoxic agent is selected from growth inhibitors or chemotherapeutic agents derived from a class of microtubule-binding agents, DNA intercalating agents, DNA alkylating agents, enzyme inhibitors, immunomodulators, antimetabolites, radioisotopes, or combinations thereof. In one embodiment, the cytotoxic agent is selected from calichiomycin, ozomicin, monomethylaurestatin E, emtansine, derivatives thereof, or combinations thereof. In one embodiment, the immunomodulatory agent activates or inhibits immune cells, T cells, NK cells, B cells, macrophages, or dendritic cells.

[0028] In one embodiment, the imaging agent may be a radionuclide, a fluorescent agent, a quantum dot, or a combination thereof.

[0029] This application also provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises the isolated mAb or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises the immunoconjugate disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical unit may further comprise a chemotherapeutic agent, a growth inhibitor, a calichiomycin-like pharmaceutical unit, an antimitotic agent, a toxin, a radioisotope, a therapeutic agent, or a combination thereof.

[0030] On the other hand, this application provides a method for treating a subject with cancer using the isolated mAb or antigen-binding fragment thereof disclosed herein. In one embodiment, the method includes the step of administering an effective amount of the isolated mAb or antigen-binding fragment disclosed herein to the subject.

[0031] In one embodiment, the method includes directly injecting an effective amount of a monoclonal antibody, its antigen-binding fragment, and the immunoconjugate disclosed herein into a tumor site.

[0032] The disclosed mAb, its antigen-binding fragment, or combination can be used to treat a variety of cancers. In some embodiments, the cancer has cells expressing ROR-1. Examples of cancers include, but are not limited to, breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer, gastric cancer, bladder cancer, cervical cancer, or brain cancer.

[0033] In one embodiment, the method further includes co-administering an effective amount of the therapeutic agent. Examples of therapeutic agents include chemotherapeutic agents, growth inhibitors, carliciin-like drug units, antimitotic agents, toxins, radioisotopes, antibodies, enzymes, or combinations thereof. In one embodiment, the therapeutic agent may be capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, folic acid, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testrolide, voroxycycline, formexane, fazodone, letrozole, erlotinib. Lafatinib, dasatinib, gefitinib, osimertinib, vandetanib, afatinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, nab-paclitaxel, everolimus, tesimolimus, dapafenib, vemurafenib, trametinib, folic acid-deacetylated vinca monohydrazide conjugate, apatinib, crizotinib, perifosine, olaparib, bortezomib, tofatinib, or their derivatives or combinations thereof.

[0034] The subject receiving treatment can be a human. In one embodiment, this application provides a solution containing an effective concentration of isolated mAb or antigen-binding fragments, wherein the solution is the subject's plasma.

[0035] Other embodiments will become apparent to those skilled in the art from the following detailed description, wherein the embodiments are described by showing the expected best mode. As will be appreciated, other and different embodiments are possible, and several details of the embodiments can be modified in a variety of obvious ways, all without departing from their spirit and scope. Therefore, the accompanying drawings and detailed description should be considered illustrative rather than limiting in nature. Attached Figure Description

[0036] The foregoing and other features of the invention will become more fully apparent from the accompanying drawings, the following description, and the appended specifications. It should be understood that these drawings depict only a few embodiments of the arrangements according to this disclosure and are therefore not intended to be limiting of its scope. The disclosure will be described with additional features and details using the drawings, in which:

[0037] Figure 1 A strategy for immunizing NZW rabbits with human or mouse ROR1 is provided;

[0038] Figure 2 An immunization schedule was provided;

[0039] Figure 3 Showing spleen and lymphoid tissue harvested from rabbits immunized with ROR1; showing the day of tissue harvest after final immunization.

[0040] Figure 4a and Figure 4b This is a summary of screening B cell cultures for ROR1-specific IgG and chimeric rabbit / human IgG; among which... Figure 4a The binding of rabbit IgG in B cell culture supernatant to various full-length forms of recombinant human or mouse ROR1, human ROR2, or truncated recombinant fragments of human ROR1 was analyzed. Figure 4b The binding of chimeric rabbit / human IgG to various full-length forms of recombinant human or mouse ROR1, ROR2, or truncated recombinant fragments of human ROR1 was analyzed.

[0041] Figure 5 The binding and dissociation rates of different ROR1-specific humanized rabbit antibodies were analyzed. The dissociation rate of full-length human IgG1 antibodies with humanized ROR1-specific variable region binding domains was analyzed using octet-based analysis.

[0042] Figure 6a , Figure 6b and Figure 6c This is a graph showing the results of rabbit serum analysis of human and mouse ROR1-specific IgG before and after immunization, according to one implementation scheme; it is also an ELISA analysis of the binding of rabbit serum IgG to human and mouse ROR1. Figure 6aThe binding of recombinant human ROR1 IgG to rabbit serum immunized with human and mouse cell-expressed ROR1 was analyzed by ELISA. Figure 6b The binding of recombinant mouse ROR1 IgG in rabbit serum immunized with human and mouse cell-expressed ROR1 was analyzed by ELISA. Figure 6c The control human IgG1 antibody that binds to recombinant human or mouse ROR1 was analyzed by ELISA. Detailed Implementation

[0043] In the following detailed description, reference is made to the accompanying drawings, which form a part of this document. In the drawings, similar symbols generally identify similar parts unless the context otherwise requires. The illustrative embodiments described in the specific embodiments, drawings, and schemes are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It is readily understood that all aspects of this disclosure, as generally described herein, and all aspects of this disclosure shown in the drawings, which can be arranged, replaced, combined, separated, and designed in various different configurations, are all expressly considered herein.

[0044] This disclosure provides, in particular, methods for preparing isolated antibodies and antigen-binding fragments specific to ROR1, bispecific or multispecific molecules, antibody-drug conjugates and / or immunoconjugates composed of such antibodies or antigen-binding fragments, pharmaceutical compositions containing such antibodies or antigen-binding fragments, bispecific or multispecific molecules, antibody-drug conjugates and / or immunoconjugates, and methods for treating cancer using the disclosed antibodies, antigen-binding fragments and compositions.

[0045] In one aspect, this application provides monoclonal antibodies that specifically bind to human or mouse ROR1. In one embodiment, the antibody exhibits one or more desired functional properties, such as high affinity binding to ROR1. In one embodiment, the antibody is derived from specific heavy and light chain amino acid sequences and / or structural features, such as rabbit / human chimeric antibodies composed of specific amino acid sequences.

[0046] Monoclonal antibodies can be produced using various methods, including mouse hybridoma or phage display (see Siegel. Transfus. Clin. Biol. 9:15-22 (2002) review) or molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453-7 (2011)). In one embodiment, antibodies are produced by immunizing rabbits with the extracellular domain (ECD) of human or mouse ROR1 or with HEK293 cells transiently transfected with mouse or human ROR1. Rabbits are known to produce antibodies with high affinity, diversity, and specificity (Weber et al., Exp. Mol. Med. 49:e305). B cells from immunized animals are cultured in vitro and screened for anti-ROR1 antibodies. Antibody variant genes are isolated using recombinant DNA technology and the resulting antibodies are recombinantly expressed. This general approach to antibody discovery is similar to that of Seeber et al., PLOS One. 9:e86184 (2014).

[0047] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with multi-epitope specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), provided they exhibit the desired biological activity. In some embodiments, antibodies can be monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, bispecific or bifunctional antibodies, simian antibodies, human antibodies, and humanized antibodies, as well as their active fragments. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, as well as products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the aforementioned antibodies and fragments. In some embodiments, antibodies can include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, i.e., molecules containing binding sites that specifically bind to antigens. Immunoglobulins can be any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules. In one embodiment, an antibody can be a complete antibody and any antigen-binding fragment derived from that complete antibody. A typical antibody is a heterotetrameric protein chain that typically comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The VH and VL regions can be further subdivided into a hypervariable complementarity-determining region (CDR) domain and a more conserved region called a framework region (FR). Each variable region (VH or VL) typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Binding regions that interact with the antigen exist within the variable regions of both the light and heavy chains.

[0048] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that individual antibodies comprising that group are identical except for the possibility of naturally occurring mutations present in small amounts.

[0049] Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized from hybridoma cultures, free from contamination by other immunoglobulins. The modifier “monoclonal” indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by a hybridoma method first described in Kohler & Milstein, Nature, 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567).

[0050] Monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular class or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another class or belonging to another antibody class or subclass, as well as fragments of those antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855).

[0051] Monoclonal antibodies can be produced using various methods, including mouse hybridoma or phage display (see Siegel. Transfus. Clin. Biol. 9:15-22 (2002) review) or molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453-7 (2011)). In this disclosure, antibodies are produced by immunizing rabbits with human PD-L1 protein and cells transiently expressing human PD-L1 on their cell surface. Rabbits are known to produce antibodies with high affinity, diversity, and specificity (Weber et al., Exp. Mol. Med. 49:e305). B cells from immunized animals are cultured in vitro and screened for anti-PD-L1 antibodies. Antibody variant genes are isolated using recombinant DNA technology, the resulting antibodies are recombinantly expressed, and further screened for desired characteristics, such as the ability to inhibit PD-L1 binding to PD-1, the ability to bind to non-human primate PD-L1, and the ability to enhance human T cell activation. The general approach to antibody discovery is similar to that of Seeber et al., PLOSOne.9:e86184 (2014).

[0052] The term "antigen or epitope binding portion or fragment" refers to an antibody fragment capable of binding an antigen (in this case, ROR1). These fragments can possess both the antigen-binding function and additional functions of a complete antibody. Examples of binding fragments include, but are not limited to, single-chain Fv fragments (scFv) or Fab fragments, wherein the single-chain Fv fragment (scFv) consists of the VL and VH domains of a single arm of an antibody in a single polypeptide chain linked by a synthetic linker, and the Fab fragment is a monovalent fragment consisting of the VL, constant light chain (CL), VH, and constant heavy chain 1 (CH1) domains. Antibody fragments can be even smaller subfractions and can consist of domains as small as a single CDR domain, particularly the CDR3 region from the VL and / or VH domains (see, for example, Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are generated using conventional methods known to those skilled in the art. The utility of antibody fragments can be screened using the same techniques as for complete antibodies.

[0053] The “antigen or epitope binding fragment” can be derived from the antibody disclosed herein by many techniques known in the art. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. The appropriate fraction containing the Fab fragment can then be collected and concentrated by membrane filtration or the like. For a further description of general techniques for isolating active antibody fragments, see, for example, Khaw, BA et al. J. nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

[0054] Antibody digestion with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking the antigen.

[0055] Fab fragments may contain a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that they have residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH in this paper refers to Fab', where the cysteine ​​residues of the constant domain have a free thiol group. F(ab')2 antibody fragments are initially generated as Fab' fragment pairs with a hinge cysteine ​​residue between them. Furthermore, chemical coupling of antibody fragments is also known.

[0056] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a tightly non-covalently bound heavy chain and a light chain variable domain. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In total, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind antigens, although with lower affinity than the entire binding site.

[0057] Based on the amino acid sequence of their constant structural domains, the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ).

[0058] Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; and IgA-1 and IgA-2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well-known.

[0059] "Humanized antibodies" refer to a class of engineered antibodies whose CDRs are derived from non-human donor immunoglobulins, and whose remaining immunoglobulin-derived portions are derived from one (or more) human immunoglobulins. Furthermore, the framework support residues can be modified to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (See, for example, Queen et al., Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson et al., Bio / Technology, 9: 421 (1991)).

[0060] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and are defined as referring to a biomolecule composed of amino acids linked together by peptide bonds.

[0061] As used herein, the terms “a” and “the” are defined to mean “one or more” and include plural forms, unless the context is inappropriate.

[0062] "Isolated" refers to a biomolecule that does not contain at least some of its naturally occurring components. When used to describe the various polypeptides disclosed herein, "isolated" refers to a polypeptide that has been identified and isolated and / or recovered from the cells or cell cultures in which it is expressed. Typically, isolated polypeptides are prepared through at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities.

[0063] "Recombination" refers to the use of recombinant nucleic acid technology to produce antibodies in foreign host cells.

[0064] The term "antigen" refers to an entity or segment thereof that can induce an immune response in an organism, particularly animals, and more particularly mammals, including humans. This term includes immunogens and the regions responsible for antigenicity or antigenic determinants.

[0065] "Specific binding" or "specific for" refers to binding to a specific antigen or epitope that is significantly different from nonspecific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to a control molecule, typically a molecule with a similar structure that does not have binding activity. For instance, specific binding can be determined by competition with a target-like control molecule.

[0066] Specific binding to a particular antigen or epitope can be represented, for example, by the KD of the antibody against the antigen or epitope, said KD being at least about 10. -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M or higher, where KD refers to the dissociation rate of a specific antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a KD of 20, 50, 100, 500, 1000, 5,000, 10,000 or more times relative to the antigen or epitope compared to a control molecule.

[0067] The “homology” between two sequences is determined by sequence identity. If the two sequences being compared are of different lengths, sequence identity preferably involves the percentage of nucleotide residues in the shorter sequence that are identical to those in the longer sequence. Sequence identity can be routinely determined using computer programs. Deviations that occur in comparisons between a given sequence and the sequences described above in this disclosure can be caused by, for example, additions, deletions, substitutions, insertions, or recombinations.

[0068] This application also provides immunoconjugates comprising pharmaceutical units linked to antibody and antigen-binding fragments disclosed herein via adapters. The adapters may be cleavable or non-cleavable. In one embodiment, the adapter is a chemical adapter. In one embodiment, the adapter comprises a covalent bond, such as an ester bond, ether bond, amide bond, disulfide bond, imide bond, sulfone bond, phosphate bond, phosphorus ester bond, peptide bond, or a combination thereof. In one embodiment, the adapter comprises a hydrophobic poly(ethylene glycol) adapter. In one embodiment, the adapter comprises a peptide bond.

[0069] The drug unit may also be a chemotherapeutic agent, a growth inhibitor, a calichiomycin-like drug unit, an antimitotic agent, a toxin, a radioisotope, a therapeutic agent, or a combination thereof. In one embodiment, the therapeutic agent includes an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

[0070] In another aspect, this application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition may include the isolated antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition includes the immunoconjugate disclosed herein and a pharmaceutically acceptable carrier.

[0071] Antibodies and their fragments or immunoconjugates can be prepared in physiologically acceptable formulations using known techniques, and may contain pharmaceutically acceptable carriers, diluents, and / or excipients. For example, the antibodies disclosed herein may include any functionally equivalent antibody or its functional portion, and in particular, monoclonal antibodies comprising any functionally equivalent antibody or its functional portion may be combined with pharmaceutically acceptable carriers, diluents, and / or excipients to form therapeutic compositions.

[0072] Suitable drug carriers, diluents and / or excipients are well known in the art, including, for example, phosphate-buffered saline solutions, water, and emulsions such as oil / water emulsions.

[0073] The pharmaceutical composition may further comprise a protein carrier, such as serum albumin or immunoglobulin, particularly of human origin. In one embodiment, the protein pharmaceutically active substance may be present in an amount from 1 ng to 10 mg per dose.

[0074] In some embodiments, the administration regimen may be in the range of 0.1 μg to 10 mg of the antibody according to this disclosure, particularly in the range of 1.0 μg to 1.0 mg, and more particularly in the range of 1.0 μg to 100 μg, and the number of individuals falling within these ranges is also part of this disclosure. If administration is by continuous infusion, a more suitable dose may be in the range of 0.01 μg to 10 mg units / kg body weight / hour, and the number of individual values ​​falling within these ranges is also part of this disclosure.

[0075] "Pharmaceutical acceptable" means compounds, materials, compositions, and dosage forms that, to a reasonable extent medical judgment, are suitable for use in contact with human or animal tissues without excessive toxicity, irritation, or other problems or complications, in proportion to a reasonable benefit / risk ratio. Formulations of pharmaceutical compositions according to this disclosure can be obtained according to standard methods known to those skilled in the art.

[0076] Depending on the intended use, other bioactive agents may be present in the pharmaceutical compositions disclosed herein. In one embodiment, the compositions disclosed herein may be administered in combination with other compositions comprising at least one compound selected from the group consisting of therapeutic agents, including capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testosterone, voroxycycline, formetastatin, fazodazole, letrozole, erlotinib, lafafatinib, dasatinib, gefitinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, nab-paclitaxel, calichiomycin, antimitotic agents, monomethylolpropionate, emtansine, ozomicin, derivatives thereof, or combinations thereof.

[0077] On the other hand, this application provides a method for treating a subject using an anti-ROR1 antibody or other molecules containing an antigen-binding moiety of an anti-ROR1 antibody. In one embodiment, the method inhibits the growth of tumor cells. In some embodiments, the method uses the disclosed antibody or composition to stimulate a protective autoimmune response, a modified immune response, or an antigen-specific immune response.

[0078] In one embodiment, the method includes the step of administering an effective amount of the disclosed anti-ROR1 antibody or other molecule or composition to a subject in need of such treatment.

[0079] The composition can be administered to a subject in a suitable pharmaceutically effective dose in solid, liquid, or aerosol form. Examples of solid compositions include pills, creams, and implantable dosing units. Pills can be administered orally. Therapeutic creams can be applied topically.

[0080] Implantable dosing units can be administered, for example, locally at the tumor site, or implanted, for example, subcutaneously, to systemically release the therapeutic composition. Examples of liquid compositions include formulations suitable for intramuscular, subcutaneous, intravenous, and intra-arterial injection, as well as formulations for local and intraocular application. Examples of aerosol formulations include inhaled formulations for administration to the lungs.

[0081] The composition can be administered via standard routes of administration. Typically, the composition can be administered topically, orally, rectally, nasally, intradermally, intraperitoneally, or parenterally (e.g., intravenously, subcutaneously, or intramuscularly). Alternatively, the composition can be incorporated into a sustained-release matrix such as a biodegradable polymer, which can be implanted near the site of delivery, such as a tumor site. The method includes administering a single dose, administering repeated doses at predetermined time intervals, and administering continuously for a predetermined period.

[0082] In one implementation, administration may be parenteral, such as intravenous. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous solvents may be selected from water, alcohol / aqueous solutions, emulsions, or suspensions (including saline and buffer media). Parenteral carriers include sodium chloride solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's, or non-volatile oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements (e.g., those based on Ringer's glucose), etc.

[0083] It may also contain preservatives, such as antibacterial agents, antioxidants, chelating agents, inert gases, etc.

[0084] As is well known to those skilled in the art, the dosage of a composition will depend on various factors, such as the condition being treated, the specific composition used, and other clinical factors, such as the patient's weight, size, sex and general health status, body surface area, the specific compound or composition to be applied, other concurrently administered drugs, and the route of administration.

[0085] The term "therapeutic effective amount" refers to the amount of antibody that, when administered to a human or animal, elicits a response sufficient to produce a therapeutic effect in said human or animal. The effective amount is readily determined by those skilled in the art using conventional methods.

[0086] The disclosed mAbs, antigen-binding fragments, or compositions can be used to treat a variety of cancers. Cancers that express the ROR1 gene include breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer, gastric cancer, bladder cancer, cervical cancer, or brain cancer. In one embodiment, a therapeutically effective amount of a composition comprising an anti-ROR1 monoclonal antibody or its antigen-binding fragment or its immunoconjugate is administered to cure, prevent, improve, or delay the development or metastasis of cancer.

[0087] This disclosure can be more readily understood by referring to the detailed description of the specific embodiments included herein. Although the invention has been described in detail with reference to certain embodiments thereof, such details should not be construed as limiting the scope of the invention.

[0088] Example

[0089] Example 1: Generation of anti-ROR1 antibodies

[0090] Develop monoclonal antibodies against human ROR1 by immunizing New Zealand white rabbits. (For example...) Figure 1 As shown, animals were immunized subcutaneously with recombinant human or mouse ROR1 extracellular domain (ECD) or HEK293 cells, wherein the HEK293 cells were transiently transfected with mouse or human ROR1 at a 1:1 v / v ratio with complete or incomplete Freund's adjuvant (Group 1) or TiterMax Gold (Group 2), and alternately supplemented with 2% aluminum gel (Alum) plus CpG2007. Booster treatments were subsequently performed on days 7, 14, 21, 28, and 37. Figure 2 As shown.

[0091] At week 5, anti-ROR1 titers from animal serum were tested by ELISA. Serum obtained from each rabbit before immunization served as a negative control. After immunization, serum was collected again from each animal, and the presence of ROR1-specific IgG antibodies was compared with pre-immunization serum from the same animal. Figure 6a , Figure 6b and Figure 6c As shown, all animals immunized with human or mouse ROR1 produced detectable titers of human or mouse ROR1-specific IgG antibodies.

[0092] like Figure 3As shown, spleens and lymph nodes were harvested from two animals on days 4, 13, and 21 post-final immunization. ROR1-specific IgG+ B cells were sorted one per well into multiple 96-well tissue culture plates and cultured for 9 days to allow them to differentiate into plasma cells and secrete antibodies. The presence of ROR1-specific antibodies in the supernatant from these plasma cell cultures was screened by ELISA and flow cytometry (FACS) in a series of binding assays listed below:

[0093] Human ROR1 directly coated on a plate – ELISA for detecting ROR1-specific IgG

[0094] Mouse ROR1 directly coated on plates—ELISA for detecting ROR1-specific IgG

[0095] Human ROR2 directly coated on plates—ELISA for detecting ROR1-specific IgG

[0096] Biotinylated human ROR1 plate coated with avidin - ROR1-specific IgG detection ELISA

[0097] Biotinylated human ROR1 "Kringle domain" avidin-coated plates - ROR1-Kringle specific IgG ELISA detection

[0098] Biotinylated human ROR1 “Frizzled-Kringle domain” avidin-coated plates - ROR1-Frizzled-Kringle specific IgG ELISA

[0099] Biotinylated human ROR1 “Ig-Frizzled domain” with avidin-coated plates - ROR1-Ig-Frizzled specific IgG ELISA

[0100] Human ROR1 “Frizzled-Kringle domain” directly coated on plate - ROR1-Frizzled-Kringle specific IgG ELISA

[0101] Human ROR1 “Ig-Frizzled domain” directly coated on plate - ELISA for detecting ROR1-Ig-Frizzled specific IgG

[0102] Human ROR1-CHO cells - Detection of ROR1-specific IgG by FACS

[0103] On day 9 of B cell culture, the supernatant was separated from the B cells and stored in individual plates for later analysis. RNAlater tissue storage reagent was added to each well in the B cell culture plate to preserve RNA in the B cells for RT-PCR amplification of antibody variable regions.

[0104] We performed a molecular "rescue" of the antibody variable region after screening B cell culture wells for the desired antibodies using ELISA and FACS. The light and heavy chain variable region sequences were amplified by multiplex RT-PCR using degenerate primers designed to anneal to the leader sequences and constant regions of rabbit IgG and rabbit κ sequences. A second PCR was performed on the light and heavy chains, respectively, using nested primers containing restriction sites. Amplicons from the variable heavy chain PCR were cloned into an expression vector containing human IgG1. Light chain amplicons were cloned into an expression vector containing human IgK. The resulting clones were sequenced and analyzed.

[0105] Rabbit / human chimeric antibodies were generated by transient co-transfection of heavy and light chain expression plasmids produced from each well. Biolayer interferometry analysis on a Fortebio Octt Red 96 instrument confirmed the presence of anti-ROR1 antibody in the recombinant antibody supernatant. Antibodies in the supernatant were captured using an anti-human Fc biosensor (Pall Fortebio). Association with ROR1 was observed via real-time interferometry by placing the biosensor in wells containing the recombinant human ROR1 extracellular domain protein. Dissociation was measured after transferring the biosensor to wells containing 10X kinetic buffer (Pall Fortebio). Software provided by the manufacturer was used to analyze the interferometry data.

[0106] Figure 4a and 4b The primary BCC screening data and a summary of the corresponding screening data for 27 recombinant chimeric rabbit / human IgG antibodies are presented.

[0107] Figure 4a and 4b Of the 27 chimeric rabbit / human IgG antibodies listed above, 8 have humanized heavy chain and light chain variable regions. Eight of these humanized variants of the 27 antibodies showed binding kinetics similar to human ROR1 through octet locus analysis, summarized in... Figure 5 .

[0108] Solution 1 of the present invention provides an isolated mAb or its antigen-binding fragment having binding specificity to human ROR1, comprising the following components: SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:116, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:119 ... The amino acid sequences NO:120, SEQ ID NO:124, SEQ ID NO:128 or SEQ ID NO:132 have a homology percentage of not less than 90%.

[0109] The isolated mAb or antigen-binding fragment of Scheme 2 of the present invention, wherein the homology percentage is not less than 98%.

[0110] The isolated mAb or antigen-binding fragment of Scheme 3 of the present invention has a binding affinity for human ROR1 of not more than 70 nM (Kd).

[0111] The isolated mAb or antigen-binding fragment of Scheme 4 of the present invention exhibits one or more functional properties selected from the following: high affinity binding to human ROR1, inhibition of human ROR1 activity, induction of apoptosis, regulation of EGFR signaling pathway, upregulation of EMT gene, enhancement of T cell activation, stimulation of antibody response, reversal of the inhibitory function of immunosuppressive cells, or a combination thereof.

[0112] The isolated mAb or antigen-binding fragment of Scheme 5 of the present invention, wherein the enhanced T cell activation includes T cell proliferation, IFN-γ and / or IL-2 secretion or a combination thereof.

[0113] The isolated mAb or its antigen-binding fragment of Scheme 6 of the present invention, wherein the isolated mAb is a humanized antibody, a chimeric antibody or a recombinant antibody.

[0114] The isolated mAb or its antigen-binding fragment of Scheme 7 of the present invention, wherein the isolated mAb is IgG.

[0115] The isolated mAb or its antigen-binding fragment of embodiment 8 of the present invention, wherein the antigen-binding fragment comprises Fv, Fab, F(ab')2, scFV or scFV2 ​​fragments.

[0116] The isolated mAb or its antigen-binding fragment of Scheme 9 of the present invention, wherein the isolated mAb is a bispecific antibody, a trispecific antibody or a multispecific antibody.

[0117] Scheme 10 of the present invention provides an isolated mAb or its antigen-binding fragment having human ROR1 binding specificity, comprising an amino acid sequence having a homology percentage with SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:99, SEQ ID NO:107, SEQ ID NO:115, SEQ ID NO:123 or SEQ ID NO:131, wherein the homology percentage is not less than 90%.

[0118] Scheme 11 of the present invention provides a κ light chain of an isolated mAb or its antigen-binding fragment having human ROR1 binding specificity, comprising an amino acid sequence having a homology percentage with SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:51, SEQ ID NO:59, SEQ ID NO:67, SEQ ID NO:75, SEQ ID NO:83, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:119, or SEQ ID NO:127, wherein the homology percentage is not less than 90%.

[0119] Scheme 12 of the present invention is a variable light chain of an isolated mAb or its antigen-binding fragment having human ROR1 binding specificity, comprising an amino acid sequence having a homology percentage with SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120 or SEQ ID NO: 128, wherein the homology percentage is not less than 90%.

[0120] Scheme 13 of the present invention provides a variable heavy chain of an isolated mAb or its antigen-binding fragment having human ROR1 binding specificity, comprising an amino acid sequence having a homology percentage with SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO:116, SEQ ID NO:124, or SEQ ID NO:132, wherein the homology percentage is not less than 90%.

[0121] Scheme 14 of the present invention is an isolated nucleic acid encoding the isolated mAb or antigen-binding fragment according to Scheme 1, the IgG1 heavy chain according to Scheme 10, the κ light chain according to Scheme 11, the variable light chain according to Scheme 12, or the variable heavy chain according to Scheme 13.

[0122] The present invention provides an expression vector for isolated nucleic acids in embodiment 15, wherein the vector can be expressed in cells.

[0123] Scheme 16 of the present invention comprises a host cell of nucleic acid according to Scheme 14, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

[0124] Scheme 17 of the present invention, a method for producing antibodies, includes culturing host cells according to Scheme 16 to produce antibodies.

[0125] Scheme 18 of the present invention provides an immunoconjugate comprising an isolated mAb or antigen-binding fragment thereof as described in Scheme 1 and a pharmaceutical unit, wherein the pharmaceutical unit is connected to the isolated mAb or antigen-binding fragment via a linker, and wherein the linker comprises a covalent bond selected from ester bonds, ether bonds, amine bonds, amide bonds, disulfide bonds, imide bonds, sulfone bonds, phosphate bonds, phosphate ester bonds, peptide bonds, hydrazone bonds, or combinations thereof.

[0126] Scheme 19 of the present invention, according to the immunoconjugate of Scheme 18, wherein the pharmaceutical unit is selected from cytotoxic agents, immunomodulators, or combinations thereof.

[0127] Scheme 20 of the present invention, according to the immune conjugate of Scheme 18, wherein the cytotoxic agent is selected from growth inhibitors or chemotherapeutic agents, and the chemotherapeutic agent is selected from microtubule binding agents, DNA intercalating agents, DNA alkylating agents, enzyme inhibitors, immunomodulators, antimetabolites, radioisotopes or combinations thereof.

[0128] Scheme 21 of the present invention, according to the immunoconjugate of Scheme 20, wherein the cytotoxic agent is selected from calichimycin, ozomicin, monomethyl auristatin E, emtansine, derivatives thereof, or combinations thereof.

[0129] Scheme 22 of the present invention, according to the immune conjugate of Scheme 20, wherein the immunomodulator activates or inhibits immune cells, T cells, NK cells, B cells, macrophages or dendritic cells.

[0130] Scheme 23 of the present invention is a pharmaceutical composition comprising the isolated mAb or its antigen-binding fragment as described in Scheme 1 and a pharmaceutically acceptable carrier.

[0131] Scheme 24 of the present invention, the pharmaceutical composition according to Scheme 23, further comprises a chemotherapeutic agent, a growth inhibitor, a calichiomycin-like drug unit, an antimitotic agent, a toxin, a radioisotope, a therapeutic agent, an anti-estrogenic agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

[0132] Scheme 25 of the present invention is a pharmaceutical composition comprising the immunoconjugate according to Scheme 21 and a pharmaceutically acceptable carrier.

[0133] Scheme 26 of the present invention is a method for treating a subject suffering from cancer, the method comprising administering to the subject an effective amount of the isolated mAb or its antigen-binding fragment according to Scheme 1, wherein the cancer comprises cells expressing ROR1.

[0134] Scheme 27 of the present invention, according to the method described in Scheme 26, wherein the cancer includes breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer, gastric cancer, bladder cancer, cervical cancer, or brain cancer.

[0135] Scheme 28 of the present invention, according to the method of Scheme 26, further includes co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent includes an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

[0136] Scheme 29 of the present invention, according to the method of Scheme 28, wherein the therapeutic agent comprises capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, folic acid, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testrolide, voroxycycline, formestan, fazodazole, etc. Letrozole, Erlotinib, Lafatinib, Dasatinib, Gefitinib, Osimertinib, Vandetanib, Afatinib, Imatinib, Pazopanib, Lapatinib, Sunitinib, Nilotinib, Sorafenib, Nab-paclitaxel, Everolimus, Tesseirolimus, Dapafenib, Vemurafenib, Trametinib, Folic acid-deacetylated vinca monohydrazide conjugate, Apatinib, Crizotinib, Periforsine, Olaparib, Bortezomib, Tofatinib, and their derivatives or combinations thereof.

[0137] Scheme 30 of the present invention, according to the method of Scheme 26, wherein the subject is a human.

[0138] Scheme 31 of the present invention is a solution comprising an effective concentration of the isolated mAb or its antigen-binding fragment as described in Scheme 1, wherein the solution is the plasma of a subject.

[0139] While this disclosure has been specifically shown and described with reference to embodiments thereof, those skilled in the art will understand that foregoing and other changes in form and detail may be made without departing from the spirit and scope of the invention. All references cited or mentioned in this disclosure are incorporated herein by reference in their entirety.

[0140] sequence list

[0141] anti-ROR1 antibody sequence

[0142] SEQ ID NO:1

[0143] 226E12 chimeric light chain full-length nucleotide sequence

[0144] GCCTATGATATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGAATTTACAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTGCTAGTATGGTTGATGTTGAGAATATGTTCGGCGGAGGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0145] SEQ ID NO:2

[0146] 226E12 chimeric light chain variable light chain nucleotide sequence

[0147] GCCTATGATATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGAATTTACAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTGCTAGTATGGTTGATGTTGAGAATATGTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0148] SEQ ID NO:3

[0149] The full-length amino acid sequence of the 226E12 chimeric light chain. The human κ constant domain is underlined.

[0150] AYDMTQTPSSVSAAVGGTVTIKCQASQRIYSYLAWYQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQQGASMVDVENMFGGGTEVVVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0151] SEQ ID NO:4

[0152] The 226E12 chimeric light chain variable light chain amino acid sequence. Complementarity-determining regions are underlined.

[0153] AYDMTQTPSSVSAAVGGTVTIKC QASQRIYSYLA WYQQKPGQPPKLLIY RASTLAS GVPSRFKGSGSGTEYTLTISDLECADAATYY CQQGASMVDVENM FGGGTEVVVKSEQ ID NO:5

[0154] 226E12 chimeric heavy chain full-length nucleotide sequence

[0155]

[0156] SEQ ID NO:6

[0157] 226E12 chimeric heavy chain variable heavy chain nucleotide sequence

[0158] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGAATTCTCCCTCAGTAACTACTACATGAGCTGGGTCCGCCAGGCTCCAGGGGAGGGGCTGGAGTGGATCGGAGCCATTAATGCTGACAGTGATAATACATGGTACCCGAGCTGGGTGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAGATCACCAGTCCGACAATTGAGGACACGGCCACCTATTTCTGTGCCAGAAGTGTGAGTAATAATTTCGCCGAATATAACATCTGGGGCCCGGGCACCCTGGTCACCGTCTCGAGC

[0159] SEQ ID NO:7

[0160] The full-length amino acid sequence of the 226E12 chimeric heavy chain. The human γ-1 constant domain is underlined.

[0161] QSLEESGGRLVTPGTPLTLTCTASEFSLSNYYMSWVRQAPGEGLEWIGAINADSDNTWYPSWVKGRFTISKTSSTTVDLKITSPTIEDTATYFCARSVSNNFAEYNIWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0162] SEQ ID NO:8

[0163] The 226E12 chimeric heavy chain variable heavy chain amino acid sequence. Complementarity-determining regions are underlined.

[0164] QSLEESGGRLVTPGTPLTLTCTASEFSLS NYYMS WVRQAPGEGLEWIG AINADSDNTWYPSWVKG RFTISKTSSTTVDLKITSPTIEDTATYFCAR SVSNNFAEYNI WGPGTLVTVSS

[0165] SEQ ID NO:9

[0166] 323H7 chimeric light chain full-length nucleotide sequence

[0167] CAAGCCGTGGTGACCCAGACTCCATCGTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCGCCATCAGCGACCTGGAGTGTGACGATTCTGCCACTTACTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGCACCGAGGTGGAGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0168] SEQ ID NO:10

[0169] 323H7 chimeric light chain variable light chain nucleotide sequence

[0170] CAAGCCGTGGTGACCCAGACTCCATCGTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCGCCATCAGCGACCTGGAGTGTGACGATTCTGCCACTTACTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGCACCGAGGTGGAGGTCAAASEQ ID NO:11

[0171] The full-length amino acid sequence of the 323H7 chimeric light chain. The human κ constant domain is underlined.

[0172] QAVVTQTPSSVSAAVGGTVTISCQSSQSVYNNNDLAWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLAISDLECDDSATYYCAGGYDTDGLDTFAFGGGTEVEVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0173] SEQ ID NO:12

[0174] 323H7 chimeric light chain variable light chain amino acid sequence. Complementarity-determining regions are underlined.

[0175] QAVVTQTPSSVSAAVGGTVTISC QSSQSVYNNNDLA WYQQKPGQPPKLLIY YASTLAS GVSSRFKGSGSGTQFTLAISDLECDDSATYYCAG GYDTDGLDTFA FGGGTEVEVK

[0176] SEQ ID NO:13

[0177] 323H7 chimeric heavy chain full-length nucleotide sequence

[0178]

[0179] SEQ ID NO:14

[0180] 323H7 chimeric heavy chain variable heavy chain nucleotide sequence

[0181] CAGGAGCAGCTGAAGGAGTCCGGAGGAGGCCTGGTAACGCCTGGAGGAACCCTGACACTCACCTGCACAGCCTCTGGATTCACCATCAGTCGCTACCACATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGTCATATTTATGTTAATAATGATGACACAGACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAGATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATTGGATGTTGGTGGTGGTGGTGCTTATATTGGGGACATCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGC

[0182] SEQ ID NO:15

[0183] The full-length amino acid sequence of the 323H7 chimeric heavy chain. The human γ-1 constant domain is underlined.

[0184] QEQLKESGGGLVTPGGTLTLTCTASGFTISRYHMTWVRQAPGKGLEWIGHIYVNNDDTDYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARLDVGGGGAYIGDIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0185] SEQ ID NO:16

[0186] The amino acid sequence of the 323H7 chimeric heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0187] QEQLKESGGGLVTPGGTLTLTCTASGFTIS RYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASWAKG RFTISKTSTTVDLKITSPTTEDTATYFCAR LDVGGGGAYIGDI WGQGTLVTVSS

[0188] SEQ ID NO:17

[0189] 324C7 chimeric light chain full-length nucleotide sequence

[0190] GACATTGTGATGACCCAGACTCCAGCCTCTGTGGAGGTCGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAACATTGGTAGTGATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATACTACATCCAATCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGGTTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCAGTTACTGCTGTCAAGGCGGTTATTTTAGTGGTCGTAATATTTATGGGAATGCTTTCGGCGGAGGCACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0191] SEQ ID NO:18

[0192] 324C7 chimeric light chain variable light chain nucleotide sequence

[0193] GACATTGTGATGACCCAGACTCCAGCCTCTGTGGAGGTCGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAACATTGGTAGTGATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATACTACATCCAATCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGGTTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCAGTTACTGCTGTCAAGGCGGTTATTTTAGTGGTCGTAATATTTATGGGAATGCTTTCGGCGGAGGCACCGAGGTGGTGGTCAAA

[0194] SEQ ID NO:19

[0195] The full-length amino acid sequence of the 324C7 chimeric light chain. The human κ constant domain is underlined.

[0196] DIVMTQTPASVEVAVGGTVTIKCQASQNIGSDLAWYQQKPGQPPKLLIYTTSNLASGVPSRFKGSGSGTGFTLTISDLECADAASYCCQGGYFSGRNIYGNAFGGGTEVVVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0197] SEQ ID NO:20

[0198] The 324C7 chimeric light chain variable light chain amino acid sequence. Complementarity-determining regions are underlined.

[0199] DIVMTQTPASVEVAVGGTVTIKC QASQNIGSDLA WYQQKPGQPPKLLIY TTSNLAS GVPSRFKGSGSGTGFTLTISDLECADAASYCC QGGYFSGRNIYGNA FGGGTEVVVK

[0200] SEQ ID NO:21

[0201] 324C7 chimeric heavy chain full-length nucleotide sequence

[0202]

[0203] SEQ ID NO:22

[0204] 324C7 chimeric heavy chain variable heavy chain nucleotide sequence

[0205] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTGGCGCTGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATACATTGATAGTGGTGCTACCACATACTACGCGAGCTGGGCAAAAGGCCGATTCACCATCTCCAAAGCCTCGACCACGGTGGATCTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGGATACTACGGCATGGACCCCTGGGGCCAAGGCACCCTGGTCACCGTCTCGAGC

[0206] SEQ ID NO:23

[0207] The full-length amino acid sequence of the 324C7 chimeric heavy chain. The human γ-1 constant domain is underlined.

[0208] QSVEESGGRLVTPGTPLTLTCTVSGFSLSGAGVSWVRQAPGKGLEWIGYIDSGATTYYASWAKGRFTISKASTTVDLKIASPTTEDTATYFCARGYYGMDPWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0209] SEQ ID NO:24

[0210] The amino acid sequence of the 324C7 chimeric heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0211] QSVEESGGRLVTPGTPLTLTCTVSGFSLS GAGVS WVRQAPGKGLEWIG YIDSGATTYYASWAKG RFTISKASTTVDLKIASPTTEDTATYFCAR GYYGMDP WGQGTLVTVSS

[0212] SEQ ID NO:25

[0213] 323D10 chimeric light chain full-length nucleotide sequence

[0214] GCCATCGATTTGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAATCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACGAAACATCCAAACTGGCATCTGGGGTCCCACCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAAGTTATTATCGTATTAATAATATTGGTTACGATAATGCTTTCGGCGGAGGCACCGAGGTGGAGTTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0215] SEQ ID NO:26

[0216] 323D10 chimeric light chain variable light chain nucleotide sequence

[0217] GCCATCGATTTGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAATCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACGAAACATCCAAACTGGCATCTGGGGTCCCACCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAAGTTATTATCGTATTAATAATATTGGTTACGATAATGCTTTCGGCGGAGGCACCGAGGTGGAGTTCAAA

[0218] SEQ ID NO:27

[0219] The full-length amino acid sequence of the 323D10 chimeric light chain. The human κ constant domain is underlined.

[0220] AIDLTQTPASVEAAVGGTITINCQASESISSWLAWYQQKPGQRPKLLIYETSKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQSYYRINNIGYDNAFGGGTEVEFK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0221] SEQ ID NO:28

[0222] The 323D10 chimeric light chain variable light chain amino acid sequence. Complementarity-determining regions are underlined.

[0223] AIDLTQTPASVEAAVGGTITINC QASESISSWLA WYQQKPGQRPKLLIY ETSKLAS GVPPRFSGSGSGTQFTLTISGVQCDDAATYYC QSYYRINNIGYDNA FGGGTEVEFK

[0224] SEQ ID NO:29

[0225] 323D10 chimeric heavy chain full-length nucleotide sequence

[0226]

[0227] SEQ ID NO:30

[0228] 323D10 chimeric heavy chain variable heavy chain nucleotide sequence

[0229] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTAGGAATGCAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTAGCACTAGTGGTACCACATTCTACGCGAACTGGGTGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGACTATAACTACGCCATGGACATCTGGGGCCAAGGCACCCTGGTCACCGTCTCGAGCSEQ ID NO:31

[0230] The full-length amino acid sequence of the 323D10 chimeric heavy chain. The human γ-1 constant domain is underlined.

[0231] QSVEESGGRLVTPGTPLTLTCTVSGFSLSRNAMNWVRQAPGKGLEYIGYISTSGTTFYANWVKGRFTISKTSTTVDLKMTSLTTEDTATYFCARDYNYAMDIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0232] SEQ ID NO:32

[0233] The amino acid sequence of the 323D10 chimeric heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0234] QSVEESGGRLVTPGTPLTLTCTVSGFSLS RNAMN WVRQAPGKGLEYIG YISTSGTTFYANWVKG RFTISKTSTTVDLKMTSLTTEDTATYFCAR DYNYAMDI WGQGTLVTVSS

[0235] SEQ ID NO:33

[0236] 324E2 chimeric light chain full-length nucleotide sequence

[0237] GCTCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTAATAACAACGACTTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTACTGGGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCATTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTGGTAATATTTATGGTTTCGGCGGAGGCACCGAGGTGGAGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0238] SEQ ID NO:34

[0239] 324E2 chimeric light chain variable light chain nucleotide sequence

[0240] GCTCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTAATAACAACGACTTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTACTGGGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCATTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTGGTAATATTTATGGTTTCGGCGGAGGCACCGAGGTGGAGGTCAAA

[0241] SEQ ID NO:35

[0242] The full-length amino acid sequence of the 324E2 chimeric light chain. The human κ constant domain is underlined.

[0243] AQVLTQTPSSVSAAVGGTVTINCQSSQSVNNNDLAWFQQKPGQPPKRLIYWASKLASGVPSRFKGSGSGTQFILTISDLECDDAATYYCAGGYSGNIYGFGGGTEVEVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0244] SEQ ID NO:36

[0245] The 324E2 chimeric light chain variable light chain amino acid sequence. Complementarity-determining regions are underlined.

[0246] AQVLTQTPSSVSAAVGGTVTINC QSSQSVNNNDLA WFQQKPGQPPKRLIY WASKLAS GVPSRFKGSGSGTQFILTISDLECDDAATYYC AGGYSGNIYG FGGGTEVEVK

[0247] SEQ ID NO:37

[0248] 324E2 chimeric heavy chain full-length nucleotide sequence

[0249]

[0250] SEQ ID NO:38

[0251] 324E2 chimeric heavy chain variable heavy chain nucleotide sequence

[0252] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTAACAATGCAATAACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTAGTAGTAGTGGTACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCGGAGCATTTAGCGTCTGGGGCCCGGGCACCCTCGTCACCGTCTCGAGC

[0253] SEQ ID NO:39

[0254] The full-length amino acid sequence of the 324E2 chimeric heavy chain. The human γ-1 constant domain is underlined.

[0255] QSVEESGGRLVTPGTPLTLTCTVSGFSLSNNAITWVRQAPGKGLEYIGIISSSGTTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCAGAFSVWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0256] SEQ ID NO:40

[0257] The amino acid sequence of the 324E2 chimeric heavy chain variable heavy chain is shown. Complementarity-determining regions are underlined.

[0258] QSVEESGGRLVTPGTPLTLTCTVSGFSLS NNAIT WVRQAPGKGLEYIG IISSSGTTYYASWAKG RFTISKTSSTTVDLKMTSLTTEDTATYFCAG AFSV WGPGTLVTVSS

[0259] SEQ ID NO:41

[0260] 324C6 chimeric light chain full-length nucleotide sequence

[0261] GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTGATAGTTGGTTATCCTGGTATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACCAGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGCGCTTATGGTGTTAGTGGTACTAGTAGTTATTTATATACTTTCGGCGGAGGCACCGAGGTGGAGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0262] SEQ ID NO:42

[0263] 324C6 chimeric light chain variable light chain nucleotide sequence

[0264] GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTGATAGTTGGTTATCCTGGTATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACCAGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGCGCTTATGGTGTTAGTGGTACTAGTAGTTATTTATATACTTTCGGCGGAGGCACCGAGGTGGAGGTCAAASEQ ID NO:43

[0265] The full-length amino acid sequence of the 324C6 chimeric light chain. The human κ constant domain is indicated by underlining.

[0266] DVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPKLLIYQASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCAYGVSGTSSYLYTFGGGTEVEVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0267] SEQ ID NO:44

[0268] 324C6 chimeric light chain variable light chain amino acid sequence. Complementarity-determining regions are underlined.

[0269] DVVMTQTPASVEAAVGGTVTIKC QASQSIDSWLS WYQQKPGQPPKLLIY QASTLAS GVSSRFKGSGSGTEFTLTISDLECADAATYYC QCAYGVSGTSSYLYT FGGGTEVEVK

[0270] SEQ ID NO:45

[0271] 324C6 chimeric heavy chain full-length nucleotide sequence

[0272]

[0273] SEQ ID NO:46

[0274] 324C6 chimeric heavy chain variable heavy chain nucleotide sequence

[0275] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAGGTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGAACCATTTATACTAGTGGTAGTACATGGTACGCGAGCTGGACAAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACTAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATCCTATTATGGCGGTGATAAGACTGGTTTAGGCATCTGGGGCCCAGGCACCCTCGTCACCGTCTCGAGC

[0276] SEQ ID NO:47

[0277] The full-length amino acid sequence of the 324C6 chimeric heavy chain. The human γ-1 constant domain is underlined.

[0278] QSLEESGGRLVTPGTPLTLTCTASGFSLSRYYMTWVRQAPGKGLEWIGTIYTSGSTWYASWTKGRFTISKTSTTVDLKITSPTTEDTATYFCARSYYGGDKTGLGIWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0279] SEQ ID NO:48

[0280] The amino acid sequence of the 324C6 chimeric heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0281] QSLEESGGRLVTPGTPLTLTCTASGFSLS RYYMT WVRQAPGKGLEWIG TIYTSGSTWYASWTKG RFTISKTSTTVDLKITSPTTEDTATYFCAR SYYGGDKTGLGI WGPGTLVTVSS

[0282] SEQ ID NO:49

[0283] 338H4 chimeric light chain full-length nucleotide sequence

[0284] GACATTGTGATGACCCAGACTCCAGCCTCGGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATCTGGCATCTACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAAGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAAAGCAATTATAACGGTAATTATGGTTTCGGCGGAGGGACCGAGGTGGAGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0285] SEQ ID NO:50

[0286] 338H4 chimeric light chain variable light chain nucleotide sequence

[0287] GACATTGTGATGACCCAGACTCCAGCCTCGGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATCTGGCATCTACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAAGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAAAGCAATTATAACGGTAATTATGGTTTCGGCGGAGGGACCGAGGTGGAGGTCAAA

[0288] SEQ ID NO:51

[0289] The full-length amino acid sequence of the 338H4 chimeric light chain. The human κ constant domain is underlined.

[0290] DIVMTQTPASVSAAVGGTVTINCQASQNIYSYLSWYQQKPGQPPKRLIYLASTLASGVPSRFKSSGSGTEYTLTISDLECDDAATYYCQSNYNGNYGFGGGTEVEVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0291] SEQ ID NO:52

[0292] The amino acid sequence of the 338H4 chimeric light chain variable light chain. Complementarity-determining regions are underlined.

[0293] DIVMTQTPASVSAAVGGTVTINC QASQNIYSYLS WYQQKPGQPPKRLIY LASTLAS GVPSRFKSSGSGTEYTLTISDLECDDAATYYC QSNYNGNYG FGGGTEVEVK

[0294] SEQ ID NO:53

[0295] 338H4 chimeric heavy chain full-length nucleotide sequence

[0296]

[0297] SEQ ID NO:54

[0298] 338H4 chimeric heavy chain variable heavy chain nucleotide sequence

[0299] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTAGCTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGGCTGGAATGGATCGGAATCATTTATGCTAGTGGTAGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAATTTATGACGGCATGGACCTCTGGGGCCCAGGGACCCTCGTCACCGTCTCGAGC

[0300] SEQ ID NO:55

[0301] The full-length amino acid sequence of the 338H4 chimeric heavy chain. The human γ-1 constant domain is underlined.

[0302] QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGRGLEWIGIIYASGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARIYDGMDLWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0303] SEQ ID NO:56

[0304] The amino acid sequence of the 338H4 chimeric heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0305] QSVEESGGRLVTPGTPLTLTCTVSGFSLS SYAMS WVRQAPGRGLEWIG IIYASGSTYYASWAKG RFTISKTSTTVDLKITSPTTEDTATYFCAR IYDGMDL WGPGTLVTVSS

[0306] SEQ ID NO:57

[0307] 330F11 chimeric light chain full-length nucleotide sequence

[0308] GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAATAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTATTGTCAAAGCTATAATGGTGTTGGTAGGACTGCTTTCGGCGGAGGGACCGAGGTGGAGTTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0309] SEQ ID NO:58

[0310] 330F11 chimeric light chain variable light chain nucleotide sequence

[0311] GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAATAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTATTGTCAAAGCTATAATGGTGTTGGTAGGACTGCTTTCGGCGGAGGGACCGAGGTGGAGTTCAAA

[0312] SEQ ID NO:59

[0313] The full-length amino acid sequence of the 330F11 chimeric light chain. The human κ constant domain is underlined.

[0314] DVVMTQTPASVEAAVGGTVTIKCQASQSINNYLAWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTQFTLTISDLECADAATYYCQSYNGVGRTAFGGGTEVEFK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0315] SEQ ID NO:60

[0316] The amino acid sequence of the 330F11 chimeric light chain variable light chain. Complementarity-determining regions are underlined.

[0317] DVVMTQTPASVEAAVGGTVTIKC QASQSINNYLA WYQQKPGQPPKLLIY RASTLES GVPSRFKGSGSGTQFTLTISDLECADAATYYC QSYNGVGRTA FGGGTEVEFK

[0318] SEQ ID NO:61

[0319] 330F11 chimeric heavy chain full-length nucleotide sequence

[0320]

[0321] SEQ ID NO:62

[0322] 330F11 chimeric heavy chain variable heavy chain nucleotide sequence

[0323] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAATAACTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGGAGGGGCTGGAATGGATCGGAACCATTAGTAGTGGTGCGTATACATGGTTCGCCACCTGGGCGACAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAGCATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATATTCTTCTACTACTGATTGGACCTACTTTAACATCTGGGGCCCGGGCACCCTGGTCACCGTCTCGAGC

[0324] SEQ ID NO:63

[0325] The full-length amino acid sequence of the 330F11 chimeric heavy chain. The human γ-1 constant domain is underlined.

[0326] QSVEESGGRLVTPGTPLTLTCTVSGFSLNNYWMSWVRQAPGEGLEWIGTISSGAYTWFATWATGRFTISKTSTTVDLSITSPTTEDTATYFCARYSSTTDWTYFNIWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0327] SEQ ID NO:64

[0328] The amino acid sequence of the 330F11 chimeric heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0329] QSVEESGGRLVTPGTPLTLTCTVSGFSLN NYWMS WVRQAPGEGLEWIG TISSGAYTWFATWATG RFTISKTSTTVDLSITSPTTEDTATYFCAR YSSTTDWTYFNI WGPGTLVTVSS

[0330] SEQ ID NO:65

[0331] 226E12 Humanized Light Chain Full-Length Nucleotide Sequence

[0332] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGAATTTACAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATAGGGCATCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAACAGGGTGCTAGTATGGTTGATGTTGAGAATATGTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0333] SEQ ID NO:66

[0334] 226E12 humanized light chain variable light chain nucleotide sequence

[0335] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGAATTTACAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATAGGGCATCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAACAGGGTGCTAGTATGGTTGATGTTGAGAATATGTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0336] SEQ ID NO:67

[0337] The full-length amino acid sequence of the humanized light chain 226E12. The human κ constant domain is underlined.

[0338] DIQMTQSPSSLSASVGDRVTITCQASQRIYSYLAWYQQKPGKVPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGASMVDVENMFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0339] SEQ ID NO:68

[0340] The 226E12 humanized light chain variable light chain amino acid sequence. Complexity-defined regions are underlined.

[0341] DIQMTQSPSSLSASVGDRVTITC QASQRIYSYLA WYQQKPGKVPKLLIY RASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QQGASMVDVENM FGGGTKVEIK

[0342] SEQ ID NO:69

[0343] 226E12 Humanized Heavy Chain Full-Length Nucleotide Sequence

[0344]

[0345] SEQ ID NO:70

[0346] 226E12 humanized heavy chain variable heavy chain nucleotide sequence

[0347] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGTAACTACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGCCATTAATGCTGACAGTGATAATACATGGTACCCGAGCTGGGTGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAAGTGTGAGTAATAATTTCGCCGAATATAACATCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0348] SEQ ID NO:71

[0349] The full-length amino acid sequence of the humanized heavy chain 226E12. The human γ-1 constant domain is underlined.

[0350] EVQLLESGGGLVQPGGSLRLSCAASGFSLSNYYMSWVRQAPGKGLEWIGAINADSDNTWYPSWVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSVSNNFAEYNIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0351] SEQ ID NO:72

[0352] The 226E12 humanized heavy chain variable heavy chain amino acid sequence. Complementarity-determining regions are underlined.

[0353] EVQLLESGGGLVQPGGSLRLSCAASGFSLS NYYMS WVRQAPGKGLEWIG AINADSDNTWYPSWVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SVSNNFAEYNI WGQGTLVTVSS

[0354] SEQ ID NO:73

[0355] 323H7 Humanized Light Chain Full-Length Nucleotide Sequence

[0356] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0357] SEQ ID NO:74

[0358] 323H7 humanized light chain variable light chain nucleotide sequence

[0359] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAASEQ ID NO:75

[0360] The full-length amino acid sequence of the humanized light chain 323H7. The human κ constant domain is underlined.

[0361] DIQMTQSPSSLSASVGDRVTITCQSSQSVYNNNDLAWYQQKPGKVPKLLIYYASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYDTDGLDTFAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0362] SEQ ID NO:76

[0363] The 323H7 humanized light chain variable light chain amino acid sequence. Complementarity-determining regions are underlined.

[0364] DIQMTQSPSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYCAG GYDTDGLDTFA FGGGTKVEIK

[0365] SEQ ID NO:77

[0366] 323H7 Humanized Heavy Chain Full-Length Nucleotide Sequence

[0367]

[0368] SEQ ID NO:78

[0369] 323H7 humanized heavy chain variable heavy chain nucleotide sequence

[0370] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTCGCTACCACATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGACATATTTATGTTAATAATGATGACACAGACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCACCTATTTCTGTGCGAGATTGGATGTTGGTGGTGGTGGTGCTTATATTGGGGACATCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0371] SEQ ID NO:79

[0372] The full-length amino acid sequence of the humanized heavy chain 323H7. The human γ-1 constant domain is underlined.

[0373] EVQLLESGGGLVQPGGSLRLSCAASGFTISRYHMTWVRQAPGKGLEWIGHIYVNNDDTDYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTATYFCARLDVGGGGAYIGDIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0374] SEQ ID NO:80

[0375] The amino acid sequence of the 323H7 humanized heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0376] EVQLLESGGGLVQPGGSLRLSCAASGFTIS RYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDI WGQGTLVTVSS

[0377] SEQ ID NO:81

[0378] 324C7 Humanized Light Chain Full-Length Nucleotide Sequence

[0379] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCAGGCCAGTCAGAACATTGGTAGTGATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATACTACATCCAATCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAAGGCGGTTATTTTAGTGGTCGTAATATTTATGGGAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0380] SEQ ID NO:82

[0381] 324C7 humanized light chain variable light chain nucleotide sequence

[0382] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCAGGCCAGTCAGAACATTGGTAGTGATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATACTACATCCAATCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAAGGCGGTTATTTTAGTGGTCGTAATATTTATGGGAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0383] SEQ ID NO:83

[0384] The full-length amino acid sequence of the 324C7 humanized light chain. The human κ constant domain is underlined.

[0385] DIQMTQSPSSVSASVGDRVTITCQASQNIGSDLAWYQQKPGKAPKLLIYTTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGGYFSGRNIYGNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0386] SEQ ID NO:84

[0387] 324C7 humanized light chain variable light chain amino acid sequence. Complexity-defined regions are underlined.

[0388] DIQMTQSPSSVSASVGDRVTITC QASQNIGSDLA WYQQKPGKAPKLLIY TTSNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGGYFSGRNIYGNA FGGGTKVEIK

[0389] SEQ ID NO:85

[0390] 324C7 humanized heavy chain full-length nucleotide sequence - variant 1

[0391]

[0392] SEQ ID NO:86

[0393] 324C7 humanized heavy chain variable heavy chain nucleotide sequence - variant 1

[0394] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGTGGCGCTGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGGTACATTGATAGTGGTGCTACCACATACTACGCGAGCAGTGCAAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGATACTACGGCATGGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0395] SEQ ID NO:87

[0396] 324C7 humanized heavy chain full-length amino acid sequence - variant 1. The human γ-1 constant domain is underlined.

[0397] EVQLVESGGGLVQPGGSLRLSCAASGFSLSGAGVSWVRQAPGKGLEWIGYIDSGATTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYYGMDPWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0398] SEQ ID NO:88

[0399] 324C7 humanized heavy chain variable heavy chain amino acid sequence - variant 1. Complementarity-determining regions are underlined.

[0400] EVQLVESGGGLVQPGGSLRLSCAASGFSLS GAGVS WVRQAPGKGLEWIG YIDSGATTYYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GYYGMDP WGQGTLVTVSS

[0401] SEQ ID NO:89

[0402] 324C7 humanized heavy chain full-length nucleotide sequence - variant 2

[0403]

[0404] SEQ ID NO:90

[0405] 324C7 humanized heavy chain variable heavy chain nucleotide sequence - variant 2

[0406] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCTCCCTCAGTGGCGCTGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGGTACATTGATAGTGGTGCTACCACATACTACGCGAGCAGTGCAAAAGGCAGATTCACCATCTCCAAAGACAATGCCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGATACTACGGCATGGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0407] SEQ ID NO:91

[0408] 324C7 humanized heavy chain full-length amino acid sequence - variant 2. The human γ-1 constant domain is underlined.

[0409] EVQLVESGGGLVQPGGSLRLSCTASGFSLSGAGVSWVRQAPGKGLEWIGYIDSGATTYYASSAKGRFTISKDNAKNTVDLQMNSLRAEDTAVYYCARGYYGMDPWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0410] SEQ ID NO:92

[0411] 324C7 humanized heavy chain variable heavy chain amino acid sequence - variant 2. Complementarity-determining regions are underlined.

[0412] EVQLVESGGGLVQPGGSLRLSCTASGFSLS GAGVS WVRQAPGKGLEWIG YIDSGATTYYASSAKG RFTISKDNAKNTVDLQMNSLRAEDTAVYYCAR GYYGMDP WGQGTLVTVSS

[0413] SEQ ID NO:93

[0414] 323D10 Humanized Light Chain Full-Length Nucleotide Sequence

[0415] GCCATCGATTTGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGGCACAATCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAACATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAAGTTATTATCGTATTAATAATATTGGTTACGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0416] SEQ ID NO:94

[0417] 323D10 Humanized Light Chain Variable Light Chain Nucleotide Sequence

[0418] GCCATCGATTTGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGGCACAATCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAACATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAAGTTATTATCGTATTAATAATATTGGTTACGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0419] SEQ ID NO:95

[0420] The full-length amino acid sequence of the 323D10 humanized light chain. The human κ constant domain is underlined.

[0421] AIDLTQSPSTLSASVGGTITINCQASESISSWLAWYQQKPGKAPKLLIYETSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYYRINNIGYDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0422] SEQ ID NO:96

[0423] 323D10 humanized light chain variable light chain amino acid sequence. Complexity-defined regions are underlined.

[0424] AIDLTQSPSTLSASVGGTITINC QASESISSWLA WYQQKPGKAPKLLIY ETSKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSYYRINNIGYDNA FGGGTKVEIK

[0425] SEQ ID NO:97

[0426] 323D10 Humanized Heavy Chain Full-Length Nucleotide Sequence

[0427]

[0428] SEQ ID NO:98

[0429] 323D10 humanized heavy chain variable heavy chain nucleotide sequence

[0430] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACCGCCTCTGGATTCTCCCTCAGTAGGAATGCAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGATACATTAGCACTAGTGGTACCACATTCTACGCGAACAGCGTGAAAGGCAGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACTATAACTACGCCATGGACATCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0431] SEQ ID NO:99

[0432] The full-length amino acid sequence of the 323D10 humanized heavy chain. The human γ-1 constant domain is underlined.

[0433] EVQLVESGGGLVQPGGSLRLSCTASGFSLSRNAMNWVRQAPGKGLEYIGYISTSGTTFYANSVKGRFTISKDNTKNTVDLQMNSLRAEDTAVYYCARDYNYAMDIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0434] SEQ ID NO:100

[0435] 323D10 humanized heavy chain variable heavy chain amino acid sequence. Complementarity-determining regions are underlined.

[0436] EVQLVESGGGLVQPGGSLRLSCTASGFSLS RNAMN WVRQAPGKGLEYIG YISTSGTTFYANSVKG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR DYNYAMDI WGQGTLVTVSS

[0437] SEQ ID NO:101

[0438] 324E2 Humanized Light Chain Full-Length Nucleotide Sequence

[0439] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATTGGGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAACCTGCAGCCTGAAGATTTTGCAACTTATTACTGTGCAGGCGGTTATAGTGGTAATATTTATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0440] SEQ ID NO:102

[0441] 324E2 Humanized Light Chain Variable Light Chain Nucleotide Sequence

[0442] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATTGGGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAACCTGCAGCCTGAAGATTTTGCAACTTATTACTGTGCAGGCGGTTATAGTGGTAATATTTATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0443] SEQ ID NO:103

[0444] The full-length amino acid sequence of the humanized light chain 324E2. The human κ constant domain is underlined.

[0445] DIQMTQSPSSLSASVGDRVTITCQSSQSVNNNDLAWYQQKPGKAPKRLIYWASKLASGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCAGGYSGNIYGFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0446] SEQ ID NO:104

[0447] The 324E2 humanized light chain variable light chain amino acid sequence. Complexity-defined regions are underlined.

[0448] DIQMTQSPSSLSASVGDRVTITC QSSQSVNNNDLA WYQQKPGKAPKRLIY WASKLAS GVPSRFSGSGSGTEFTLTISNLQPEDFATYYC AGGYSGNIYG FGGGTKVEIK

[0449] SEQ ID NO:105

[0450] 324E2 Humanized Heavy Chain Full-Length Nucleotide Sequence

[0451]

[0452] SEQ ID NO:106

[0453] 324E2 humanized heavy chain variable heavy chain nucleotide sequence

[0454] CAGTCGGTGGAGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACCGCCTCTGGATTCTCCCTCAGTAACAATGCAATAACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAATCATTAGTAGTAGTGGTACCACATACTACGCGAGCTCCGCGAAAGGCAGATTCACCATCTCCAAAGACACCTCCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGGGAGCATTTAGCGTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0455] SEQ ID NO:107

[0456] The full-length amino acid sequence of the 324E2 humanized heavy chain. The human γ-1 constant domain is underlined.

[0457] QSVEESGGGLVQPGGSLRLSCTASGFSLSNNAITWVRQAPGKGLEYIGIISSSGTTYYASSAKGRFTISKDTSKNTVDLQMNSLRAEDTAVYYCAGAFSVWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0458] SEQ ID NO:108

[0459] The amino acid sequence of the 324E2 humanized heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0460] QSVEESGGGLVQPGGSLRLSCTASGFSLS NNAIT WVRQAPGKGLEYIG IISSSGTTYYASSAKG RFTISKDTSKNTVDLQMNSLRAEDTAVYYCAG AFSV WGQGTLVTVSS

[0461] SEQ ID NO:109

[0462] 324C6 Humanized Light Chain Full-Length Nucleotide Sequence

[0463] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTGATAGTTGGTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAATCTGCTTATGGTGTTAGTGGTACTAGTAGTTATTTATATACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0464] SEQ ID NO:110

[0465] 324C6 humanized light chain variable light chain nucleotide sequence

[0466] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTGATAGTTGGTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAATCTGCTTATGGTGTTAGTGGTACTAGTAGTTATTTATATACTTTCGGCGGAGGGACCAAGGTGGAGATCAAASEQ ID NO:111

[0467] The full-length amino acid sequence of the 324C6 humanized light chain. The human κ constant domain is underlined.

[0468] DIQMTQSPSTLSASVGDRVTITCQASQSIDSWLSWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSAYGVSGTSSYLYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0469] SEQ ID NO:112

[0470] 324C6 humanized light chain variable light chain amino acid sequence. Complexity-defined regions are underlined.

[0471] DIQMTQSPSTLSASVGDRVTITC QASQSIDSWLS WYQQKPGKAPKLLIY QASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSAYGVSGTSSYLYT FGGGTKVEIK

[0472] SEQ ID NO:113

[0473] 324C6 Humanized Heavy Chain Full-Length Nucleotide Sequence

[0474]

[0475] SEQ ID NO:114

[0476] 324C6 humanized heavy chain variable heavy chain nucleotide sequence

[0477] CAGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCTCCCTCAGTAGGTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAACCATTTATACTAGTGGTAGTACATGGTACGCGAGCTGGACAAAAGGCAGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATCCTATTATGGCGGTGATAAGACTGGTTTAGGCATCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0478] SEQ ID NO:115

[0479] The full-length amino acid sequence of the 324C6 humanized heavy chain. The human γ-1 constant domain is underlined.

[0480] QSLVESGGGLVQPGGSLRLSCTASGFSLSRYYMTWVRQAPGKGLEWIGTIYTSGSTWYASWTKGRFTISKDNTKNTVDLQMNSLRAEDTAVYYCARSYYGGDKTGLGIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0481] SEQ ID NO:116

[0482] The amino acid sequence of the 324C6 humanized heavy chain variable heavy chain. The complementarity-determining region is underlined.

[0483] QSLVESGGGLVQPGGSLRLSCTASGFSLS RYYMT WVRQAPGKGLEWIG TIYTSGSTWYASWTKG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR SYYGGDKTGLGI WGQGTLVTVSS

[0484] SEQ ID NO:117

[0485] 338H4 Humanized Light Chain Full-Length Nucleotide Sequence

[0486] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCGCCTGATCTATCTGGCATCTACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGCAATTATAACGGTAATTATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0487] SEQ ID NO:118

[0488] 338H4 Humanized Light Chain Variable Light Chain Nucleotide Sequence

[0489] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCGCCTGATCTATCTGGCATCTACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGCAATTATAACGGTAATTATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0490] SEQ ID NO:119

[0491] The full-length amino acid sequence of the humanized light chain 338H4. The human κ constant domain is underlined.

[0492] DIQMTQSPSSLSASVGDRVTINCQASQNIYSYLSWYQQKPGKVPKRLIYLASTLASGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQSNYNGNYGFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0493] SEQ ID NO:120

[0494] The 338H4 humanized light chain variable light chain amino acid sequence. Regions with defined complexity are underlined.

[0495] DIQMTQSPSSLSASVGDRVTINC QASQNIYSYLS WYQQKPGKVPKRLIY LASTLAS GVPSRFSGSGSGTDYTLTISSLQPEDVATYYC QSNYNGNYG FGGGTKVEIK

[0496] SEQ ID NO:121

[0497] 338H4 Humanized Heavy Chain Full-Length Nucleotide Sequence

[0498]

[0499] SEQ ID NO:122

[0500] 338H4 humanized heavy chain variable heavy chain nucleotide sequence

[0501] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCTCCCTCAGTAGCTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTGGATCGGAATCATTTATGCTAGTGGTAGCACATACTACGCGAGCTCGGCGAAAGGCAGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAATTTATGACGGCATGGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0502] SEQ ID NO:123

[0503] The full-length amino acid sequence of the 338H4 humanized heavy chain. The human γ-1 constant domain is underlined.

[0504] EVQLVESGGGLVQPGGSLRLSCTASGFSLSSYAMSWVRQAPGRGLEWIGIIYASGSTYYASSAKGRFTISKDNTKNTVDLQMNSLRAEDTAVYYCARIYDGMDLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0505] SEQ ID NO:124

[0506] The amino acid sequence of the 338H4 humanized heavy chain variable heavy chain. The complementarity-determining region is underlined.

[0507] EVQLVESGGGLVQPGGSLRLSCTASGFSLS SYAMS WVRQAPGRGLEWIG IIYASGSTYYASSAKG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR IYDGMDL WGQGTLVTVSS

[0508] SEQ ID NO:125

[0509] 330F11 Humanized Light Chain Full-Length Nucleotide Sequence

[0510] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAATAACTACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGGGCATCCACTCTGGAATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAAGCTATAATGGTGTTGGTAGGACTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0511] SEQ ID NO:126

[0512] 330F11 Humanized Light Chain Variable Light Chain Nucleotide Sequence

[0513] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAATAACTACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGGGCATCCACTCTGGAATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAAGCTATAATGGTGTTGGTAGGACTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0514] SEQ ID NO:127

[0515] The full-length amino acid sequence of the 330F11 humanized light chain. The human κ constant domain is indicated by underlining.

[0516] DIQMTQSPSTLSASVGDRVTITCQASQSINNYLAWYQQKPGKAPKLLIYRASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYNGVGRTAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0517] SEQ ID NO:128

[0518] 330F11 humanized light chain variable light chain amino acid sequence. Complexity-defined regions are underlined.

[0519] DIQMTQSPSTLSASVGDRVTITC QASQSINNYLA WYQQKPGKAPKLLIY RASTLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSYNGVGRTA FGGGTKVEIK

[0520] SEQ ID NO:129

[0521] 330F11 Humanized Heavy Chain Full-Length Nucleotide Sequence

[0522]

[0523] SEQ ID NO:130

[0524] 330F11 Humanized Heavy Chain Variable Heavy Chain Nucleotide Sequence

[0525] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAATAACTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAACCATTAGTAGTGGTGCGTATACATGGTTCGCCACCTGGGCGACAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATATTCTTCTACTACTGATTGGACCTACTTTAACATCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

[0526] SEQ ID NO:131

[0527] The full-length amino acid sequence of the 330F11 humanized heavy chain. The human γ-1 constant domain is underlined.

[0528] EVQLVESGGGLVQPGGSLRLSCAASGFSLNNYWMSWVRQAPGKGLEWIGTISSGAYTWFATWATGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSSTTDWTYFNIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0529] SEQ ID NO:132

[0530] The amino acid sequence of the 330F11 humanized heavy chain variable heavy chain. Complementarity-determining regions are underlined.

[0531] EVQLVESGGGLVQPGGSLRLSCAASGFSLN NYWMS WVRQAPGKGLEWIG TISSGAYTWFATWATG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YSSTTDWTYFNI WGQGTLVTVSS

Claims

1. An isolated mAb or antigen-binding fragment thereof having binding specificity to human ROR1, comprising a variable light chain amino acid sequence and a variable heavy chain amino acid sequence, wherein the variable light chain amino acid sequence comprises a complementarity-determining region (CDR) sequence as shown in QASESISSWLA, a CDR2 as shown in ETSKLAS, and a CDR3 as shown in QSYYRINNIGYDNA, and wherein the variable heavy chain amino acid sequence comprises a CDR sequence as shown in RNAMN, a CDR2 as shown in YISTSGTTFYANSVKG, and a CDR3 as shown in DYNYAMDI.

2. The isolated mAb or antigen-binding fragment according to claim 1, comprising the humanized light chain variable light chain amino acid sequence shown in SEQ ID NO:96 and the humanized heavy chain variable heavy chain amino acid sequence shown in SEQ ID NO:100, or comprising a light chain variable light chain amino acid sequence having at least 90% identity with SEQ ID NO:96 and a heavy chain variable heavy chain amino acid sequence having at least 90% identity with SEQ ID NO:

100.

3. The isolated mAb or antigen-binding fragment according to claim 2, comprising the humanized light chain full-length amino acid sequence shown in SEQ ID NO:95 and the humanized heavy chain full-length amino acid sequence shown in SEQ ID NO:99, or comprising a light chain full-length amino acid sequence having at least 90% identity with SEQ ID NO:95 and a heavy chain full-length amino acid sequence having at least 90% identity with SEQ ID NO:

99.

4. The isolated mAb or antigen-binding fragment according to claim 1 has a binding affinity for human ROR1 of not more than 70 nM (Kd).

5. The isolated mAb or its antigen-binding fragment according to claim 1, wherein the isolated mAb is a recombinant antibody.

6. The isolated mAb or its antigen-binding fragment of claim 5, wherein the recombinant antibody is a humanized antibody or a chimeric antibody.

7. The isolated mAb or its antigen-binding fragment according to claim 1, wherein the isolated mAb is IgG.

8. The isolated mAb or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment comprises Fv, Fab, F(ab')2, or scFV fragments.

9. The isolated mAb or its antigen-binding fragment according to claim 1, wherein the isolated mAb is a multispecific antibody.

10. The isolated mAb or its antigen-binding fragment according to claim 9, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

11. An isolated nucleic acid encoding an isolated mAb or antigen-binding fragment according to any one of claims 1-10.

12. An expression vector comprising the isolated nucleic acid according to claim 11, wherein the vector is expressible in cells.

13. A host cell comprising the nucleic acid according to claim 11, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

14. A method for producing antibodies, comprising culturing host cells according to claim 13 to produce antibodies.

15. A pharmaceutical composition comprising an isolated mAb or antigen-binding fragment thereof as described in any one of claims 1-10 and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, further comprising a therapeutic agent.

17. The pharmaceutical composition of claim 16, wherein the therapeutic agent is a chemotherapeutic agent, a radioisotope, an anti-estrogenic agent, or a combination thereof.

18. The pharmaceutical composition of claim 17, wherein the chemotherapeutic agent is a growth inhibitor.

19. The pharmaceutical composition according to claim 18, wherein the growth inhibitor is a cell cycle inhibitor.

20. The pharmaceutical composition of claim 19, wherein the cell cycle inhibitor is an antimitotic agent.

21. The pharmaceutical composition of claim 20, wherein the antimitotic agent is a calichiomycin drug unit, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

22. The pharmaceutical composition of claim 17, wherein the chemotherapeutic agent is a toxin.

23. The pharmaceutical composition of claim 17, wherein the chemotherapeutic agent is a kinase inhibitor.

24. The pharmaceutical composition of claim 23, wherein the kinase inhibitor is a receptor tyrosine kinase inhibitor.

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