Anti-PD-L1 antibodies and methods for their production and use

Abstract

To provide anti-PD-L1 monoclonal antibodies that are used to upregulate the function of T-cells and enhance cell-mediated immune responses in the treatment of cancer and T-cell dysfunctional disorders.SOLUTION: The present invention provides an isolated mAb or its antigen-binding fragment having binding specificity to human PD-L1, wherein the mAb or its antigen-binding fragment comprises a light chain variable region comprising a specific amino acid sequence and a heavy chain variable region comprising a specific amino acid sequence.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62,524,553, filed June 25, 2017, which is expressly incorporated by reference in its entirety. [Technical field]

[0002] The disclosure herein relates generally to the technical field of antibodies, and more specifically to the production and use of anti-PD-L1 antibodies. [Background technology]

[0003] Cancer is a major health problem worldwide. In the United States alone, there will be an estimated 1,685,210 new cancer diagnoses and 595,690 deaths in 2016 (http: / / www.cancer.gov). Therefore, medicines that can reduce the severity or mortality from cancer are desirable.

[0004] In the immune system, resting T cells can be activated to respond to antigens through a primary signal delivered via the T cell receptor (TCR) by foreign antigenic peptides presented by antigen-presenting cells (APCs). In addition to this primary signal, there are secondary positive and negative co-stimulatory signals that further influence the T cell response. A secondary positive signal is required to fully activate T cells (Lafferty et al., Ausl. J. Exp. Biol. Med. Sci. 53:27-42 (1975)). A negative secondary signal can result in T cell suppression and tolerance.

[0005] Programmed Death 1 (PD-1) is a member of the CD28 family of receptors and is expressed on T cells and other cell types. PD-1 is one of the routes used to transmit negative secondary signals to T cells. PD-L1 is the cell surface ligand glycoprotein for PD-1, and binding to PD-1 has been shown to downregulate T cell activation (Freeman et al. J. Exp. Med. 192: 1027-34 (2000)).

[0006] Cancerous tumors may employ various mechanisms to avoid detection and / or destruction by the host immune system. One method utilized by various tumors is to suppress immune T cell responses by expressing PD-L1 on the surface of tumor cells. When PD-L1 binds to its receptor PD-1 on the surface of T cells, a negative costimulatory signal is sent to the T cells, suppressing them. In this way, tumor cells may evade the T cell-mediated response of the host immune system.

[0007] PD-L1, also known as B7-H1 or CD274, is a 40 kDa type 1 transmembrane protein that has been shown to be expressed in several human cancers and is associated with increased tumor aggressiveness and increased risk of death (Thomson et al. PNAS 101: 17174-9 (2004)). PD-L1 expression in cancer is thought to suppress the immune response against tumors through suppression of T cells (Dong et al. Nat. Med. 8: 793-800 (2002)).

[0008] Drugs that can bind to PD-L1 and block negative costimulatory signals from suppressing T cell responses have been shown to increase host immune responses against cancerous tumors, but the responses beneficial to cancer patients vary (see Reiss et al. Immunotherapy. 6:459-75 (2014)). The optimal PD-L1 binding site and associated affinity for developing the most effective tumor cell-specific anti-PD-L1 antibodies for cancer treatment remain unclear. Summary of the Invention

[0009] The disclosure herein provides, inter alia, anti-PD-L1 monoclonal antibodies, antigen-binding portions thereof, therapeutic compositions thereof, and / or nucleic acids encoding same, and their uses for upregulating T cell function and enhancing cellular immune responses in the treatment of cancer and other T cell dysfunction.

[0010] In one aspect, the application provides an isolated monoclonal antibody (mAb) or antigen-binding fragment thereof that has binding specificity for human PD-L1.

[0011] In one embodiment, an isolated mAb or antigen-binding fragment can have an amino acid sequence having a percentage homology to 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, or SEQ ID NO:80. The percentage homology can be 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or any range between 70-100%.

[0012] In one embodiment, the isolated mAb or antigen-binding fragment may have a binding affinity for PD-L1 with a Kd of less than or equal to 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM.

[0013] In one embodiment, the isolated mAb or antigen-binding fragment may exhibit one or more functional properties. Exemplary functional properties include high affinity binding to PD-L1, inhibition of binding of PD-1 to PD-L1, enhancement of T cell activation, ability to stimulate an antibody response, and / or ability to reverse the suppressive function of immune suppressive cells, such as T regulatory cells. In one embodiment, the isolated mAb or antigen-binding fragment may enhance T cell activation via T cell proliferation, IFN-γ and / or IL-2 secretion, or a combination thereof.

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

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

[0016] In one embodiment, the isolated mAb or antigen-binding fragment may have an IgG1 heavy chain comprising an amino acid sequence having a percentage homology to 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, or SEQ ID NO:79. In one embodiment, the percentage homology may be 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or any range between 70-100%.

[0017] In one embodiment, the isolated mAb or antigen-binding fragment may have a kappa light chain comprising an amino acid sequence having a percentage homology to 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, and SEQ ID NO:75. In one embodiment, the percentage homology may be 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or any range between 70-100%.

[0018] In one embodiment, the isolated mAb or antigen-binding fragment may have a variable light chain comprising an amino acid sequence having a percentage homology to 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, or SEQ ID NO:76. In one embodiment, the percentage homology may be 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or any range between 70-100%.

[0019] In one embodiment, the isolated mAb or antigen-binding fragment may have a variable heavy chain comprising an amino acid sequence having a percentage homology to 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, and SEQ ID NO:72, or SEQ ID NO:80. In one embodiment, the percentage homology may be 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or any range between 70-100%.

[0020] The application further provides isolated nucleic acids encoding the isolated mABs or antigen-binding fragments disclosed herein. In one embodiment, the nucleic acid encodes an amino acid sequence having a percentage homology to 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, or SEQ ID NO:79 of an IgG1 heavy chain. In one embodiment, the nucleic acid encodes an amino acid sequence having a percentage homology to 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, or SEQ ID NO:75 of a kappa light chain. In one embodiment, the nucleic acid encodes an amino acid sequence having a percentage homology to 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, or SEQ ID NO:76 of a variable light chain. In one embodiment, the nucleic acid encodes an amino acid sequence having a percentage homology to variable heavy chain 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, or SEQ ID NO:80. In one embodiment, the percentage homology can be 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or any range between 70-100%.

[0021] The present application further provides expression vectors comprising one or more of the nucleic acid sequences disclosed herein. In some embodiments, the nucleic acid sequence encodes a peptide or protein disclosed herein.

[0022] In one embodiment, the expression vector described herein is present and expressible in a cell. In one embodiment, the cell is a host cell. In one embodiment, the host cell can be a prokaryotic or eukaryotic cell.

[0023] In another aspect, the application provides a method for producing the disclosed antibodies or antigen-binding fragments. In one embodiment, the method comprises providing a host comprising an expression vector expressible in a host cell, the expression vector comprising a nucleic acid sequence disclosed herein to produce the antibody by expression of the nucleic acid sequence.

[0024] The application further provides an immunoconjugate comprising an anti-PD-L1 antibody or a fragment thereof. In one embodiment, the immunoconjugate comprises a drug unit or imaging agent linked to the anti-PDL1 mAb or antigen-binding fragment thereof via a linker.

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

[0026] In one embodiment, the Drug Unit in the immunoconjugate may be a chemotherapeutic agent, a growth inhibitor, or a Drug Unit selected from the group of a calicheamicin, an antimitotic agent, a toxin, a radioisotope, a therapeutic agent, or a combination thereof, hi one embodiment, the Drug Unit comprises a calicheamicin, an ozogamicin, a monomethyl auristatin E, an emtansine, a derivative or combination thereof.

[0027] In one embodiment, the drug unit is selected from a cytotoxic agent, an immunomodulatory agent, an imaging agent, or a combination thereof. In one embodiment, the cytotoxic agent is selected from a growth inhibitory agent selected from the group of tubulin binding agents, DNA intercalators, DNA alkylators, enzyme inhibitors, immunomodulators, antimetabolites, radioisotopes, or a combination thereof, or a chemotherapeutic agent. In one embodiment, the cytotoxic agent is selected from calicheamicin, ozogamicin, monomethyl auristatin E, emtansine, derivatives or combinations thereof. In one embodiment, the immunomodulatory agent activates or suppresses 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] The present application further provides a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises an isolated mAb or antigen-binding fragment disclosed herein and a pharma- ceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises an immunoconjugate disclosed herein and a pharma- ceutically acceptable carrier.

[0030] In one embodiment, the pharmaceutical composition may further comprise a radioisotope, a radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof. In one embodiment, the pharmaceutical composition may comprise a drug unit, an isolated mAb or an antigen-binding fragment that specifically binds to human PD-L1, and a pharma- ceutically acceptable carrier. Exemplary Drug Units useful in the present application include capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, mustine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, folinic acid, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, folic acid, and the like. including, but not limited to, lumestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandertanib, afatinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, everolimus, temsirolimus, dabrafenib, vemurafenib, trametinib, vintafolide, apatinib, crizotinib, periforsine, olaparib, bortezomib, tofacitinib, calicheamicin, monomethylauristatin E, etansine, ozogamicin, or derivatives or combinations thereof.

[0031] In another aspect, the application provides methods of treating cancer using the anti-PD-L1 antibodies and antigen-binding fragments disclosed herein. In one embodiment, the method comprises administering to a subject an effective amount of an isolated mAb or antigen-binding fragment disclosed herein. In some aspects, the cancer has cells that express PD-L1.

[0032] In one embodiment, the method comprises the step of directly injecting into the tumor site an effective amount of the monoclonal antibodies, antigen-binding fragments thereof, and immunoconjugates disclosed herein.

[0033] In one embodiment, the method comprises administering to a subject an effective amount of an isolated mAb or antigen-binding fragment disclosed herein and co-administering an effective amount of a therapeutic agent. In some embodiments, the therapeutic agent can be an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. Examples of therapeutic agents include, but are not limited to, capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, derivatives or combinations thereof.

[0034] The anti-PD-L1 antibodies or antigen-binding fragments disclosed herein can be used to treat a variety of cancers, including, but not limited to, breast cancer, colon 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.

[0035] In some embodiments, the subject to be treated is a human. In one embodiment, a solution comprising an effective concentration of an isolated mAb or antigen-binding fragment that specifically binds to human PD-L1 is provided, wherein the solution is the plasma of the subject. [Brief description of the drawings]

[0036] The foregoing and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, which illustrate only some embodiments prepared in accordance with the present disclosure and therefore should not be considered as limiting its scope, and the present disclosure may be explained with further specificity and detail through the use of the accompanying drawings.

[0037] [Figure 1]Figure 1 shows an example of a PD-L1 binding ELISA performed on supernatants from B cell culture plates. Shaded wells indicate wells identified as positive for anti-PD-L1 antibodies. [Diagram 2] Figure 2 shows an example of a BioLayer Interferometry assay of the ability of antibody supernatants to block the binding of PD-L1 to PD-1. The black trace uses a blocking antibody supernatant, and the grey trace uses a non-blocking antibody supernatant. [Diagram 3] Figure 3 shows a biolayer interference analysis of the ability of antibodies AB1-AB5 to block PD-L1 binding to PD-1. Starting with PD-L1 binding, followed by baseline and PD-L1 binding. The negative control is an anti-PD-L1 antibody that can bind to PD-L1 but does not block PD-L1 binding to PD-1. [Figure 4] Figure 4 shows an example of binding kinetics data for two anti-PD-L1 antibodies binding to PD-L1. The data shown show 5 minutes of association followed by 15 minutes of dissociation for various concentrations of PD-L1. [Diagram 5] FIG. 5 shows an example of an antibody that binds to the lung cancer cell line A441. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like components unless otherwise indicated by context. The illustrative embodiments described in the detailed description, drawings, and claims 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 will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0039] The disclosure herein provides, inter alia, isolated antibodies, methods for making such antibodies, bispecific or multispecific molecules, antibody-drug conjugates and / or immunoconjugates comprised of such antibodies or antigen-binding fragments, pharmaceutical compositions comprising the antibodies, bispecific or multispecific molecules, antibody-drug conjugates and / or immunoconjugates, methods for making the disclosed antibodies, antigen-binding fragments, and compositions, and methods of using the same for the treatment of cancer.

[0040] In one aspect, the disclosure herein provides isolated monoclonal antibodies that bind to human PD-L1 and antigen-binding fragments thereof. The antibodies and antigen-binding fragments thereof exhibit one or more desirable functional properties, such as high affinity binding to PD-L1, the ability to inhibit binding of PD-L1 to PD-1, the ability to enhance T cell activation, including proliferation, IFN-γ and / or IL-2 secretion, the ability to stimulate an antibody response, and / or the ability to reverse the suppressive function of immune suppressor cells, such as regulatory T cells. In one embodiment, the antibodies and antigen-binding fragments thereof may be derived from structural features, such as specific heavy and light chain amino acid sequences, and / or complementarity determining regions (CDRs) that are composed of specific amino acid sequences.

[0041] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal, polyclonal, chimeric, single chain, bispecific or bipotent, and humanized antibodies, and active fragments thereof. Examples of active fragments of molecules that bind known antigens include Fab, F(ab')2, scFv, and Fv fragments, including the products of a Fab immunoglobulin expression library, as well as epitope-binding fragments of any of the above antibodies and fragments. In some embodiments, antibodies may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a binding site that has immunological binding specificity for an antigen. Immunoglobulins are any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. In one embodiment, an antibody may be a whole antibody and any antigen-binding fragment derived from a whole antibody. A typical antibody typically refers to a heterotetrameric protein having two heavy (H) chains and two light (L) chains. Each heavy chain is composed of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain is composed of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions (CDRs) and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) is typically composed of three CDRs and four FRs arranged in the following order: From the amino terminus to the carboxy terminus are FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Within the variable regions of the light and heavy chains are binding domains that interact with an antigen.

[0042] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies; that is, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by a particular method. For example, the monoclonal antibodies to be used in accordance with the disclosure herein may be made by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

[0043] Monoclonal antibodies may also include "chimeric" antibodies (immunoglobulins) in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the desired biological activity is exhibited (US Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0044] Monoclonal antibodies can be produced using a variety of methods, including mouse hybridoma or phage display (for review see Siegel. Transfus. Clin. Biol. 9:15-22 (2002)), or by molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453-7 (2011)). In the present disclosure, antibodies were generated by immunizing rabbits with both 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 the immunized animals were cultured in vitro and screened for production of anti-PD-L1 antibodies. Antibody variable genes were isolated using recombinant DNA techniques, and the resulting antibodies were recombinantly expressed and further screened for desirable characteristics such as the ability to inhibit binding of PD-1 to PD-L1, the ability to bind to non-human primate PD-L1, the ability to enhance human T cell activation, etc. This general method of antibody discovery is similar to that described in Seeber et al. PLOS One. 9:e86184 (2014).

[0045] The term "antigen- or epitope-binding portion or fragment" refers to a fragment of an antibody capable of binding to an antigen, in this case PD-L1. These fragments may be capable of antigen-binding function of the intact antibody as well as additional functions. Examples of binding fragments include, but are not limited to, a single-chain Fv fragment (scFv), consisting of the VL and VH domains of a single arm of an antibody connected to a single polypeptide chain by a synthetic linker, or a Fab fragment, which is a monovalent fragment consisting of the VL, constant light (CL), VH, and constant heavy 1 (CH1) domains. Antibody fragments are even smaller sub-fragments and can consist of a single CDR domain, particularly a domain as small as the CDR3 region from either the VL and / or VH domain (see, e.g., Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are produced using conventional methods known to those of skill in the art. Antibody fragments can be screened for utility using the same techniques used for intact antibodies.

[0046] "Antigen or epitope-binding fragments" may be derived from the antibodies disclosed herein by a number of 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. Appropriate fractions containing Fab fragments can then be collected and concentrated, such as by membrane filtration. For further description of general techniques for isolation of active fragments of antibodies, 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.

[0047] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is capable of cross-linking antigen.

[0048] Fab fragments may contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0049] An "Fv" is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can also recognize and bind antigen, although with a lower affinity than the entire binding site.

[0050] The "light chains" of antibodies (immunoglobulins) from vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda (λ), based on the amino acid sequences of their constant domains.

[0051] Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1 and IgA-2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, delta, epsilon, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0052] "Humanized antibody" refers to a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one (or more) human immunoglobulins. Additionally, framework support residues may be altered to retain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (See, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio / Technology, 9:421 (1991)). In one embodiment, a "humanized antibody" can be obtained by genetic engineering approaches that allow the production of affinity-matured human-like polyclonal antibodies in large animals, such as rabbits (see, e.g., US Pat. No. 7,129,084).

[0053] As used herein, the terms "polypeptide," "peptide," and "protein" are interchangeable and defined to mean a biomolecule composed of amino acids linked by peptide bonds.

[0054] As used herein, the terms "a," "an," and "the" are defined to mean "one or more" and include the plural forms unless the context is inappropriate.

[0055] "Isolated" refers to a biological molecule that is free from at least some of the components with which it naturally occurs. "Isolated", when used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide is prepared by at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities.

[0056] By "recombinant" it is meant that the antibody is produced using recombinant nucleic acid techniques in an exogenous host cell.

[0057] The term "antigen" refers to an entity or fragment thereof that is capable of eliciting an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions thereof that are responsible for antigenic or antigenic determinants.

[0058] The term "immunogenic" as used herein also refers to a substance that induces or enhances the production of antibodies, T cells or other reactive immune cells against the immunogenic agent and contributes to a human or animal immune response. An immune response occurs when an individual produces sufficient antibodies, T cells and other reactive immune cells against an administered immunogenic composition disclosed herein to alleviate or ameliorate the disorder being treated.

[0059] "Specific binding" or "specifically binds" or "specific" for a particular antigen or epitope refers to binding that is distinct from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0060] Specific binding to a particular antigen or epitope is, for example, at least about 10 -4 M, at least about 10 -5 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, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, or greater, for an antigen or epitope, where KD refers to the off-rate of a particular antibody-antigen interaction. In some embodiments, an antibody that specifically binds to an antigen may have a KD for the antigen or epitope that is 20-, 50-, 100-, 500-, 1000-, 5000-, 10000-, or greater than that of a control molecule.

[0061] Specific binding to a particular antigen or epitope can also be exhibited by an antibody having a K A or K A for the antigen or epitope that is at least 20-, 50-, 100-, 500-, 1000-, 5000-, 10000-, or greater than that for a control epitope, where K A or K A refers to the association rate of a particular antibody-antigen interaction.

[0062] The "homology" between two sequences is determined by sequence identity. When the lengths of the two sequences compared to each other are different, sequence identity preferably relates to the percentage of nucleotide residues of the shorter sequence that are identical to the nucleotide residues of the longer sequence. Sequence identity can be conventionally determined using computer programs. Deviations that appear in the comparison of a given sequence with the above sequences disclosed herein can be caused by, for example, addition, deletion, substitution, insertion or recombination.

[0063] In another aspect, the present application provides pharmaceutical compositions. Formulation of pharmaceutical compositions according to the disclosure herein can be accomplished according to standard methodologies known to those skilled in the art. Antibodies according to the disclosure herein can be prepared in physiologically acceptable formulations and can include pharma- ceutically acceptable carriers, diluents and / or excipients using known techniques. For example, antibodies according to the disclosure herein and described herein, including antibodies, including functionally equivalent antibodies or functional portions thereof, particularly monoclonal antibodies, including functionally equivalent antibodies or functional portions thereof, are combined with pharma- ceutically acceptable carriers, diluents and / or excipients to form therapeutic compositions. Suitable pharmaceutical carriers, diluents and / or excipients are well known in the art and include, for example, phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like.

[0064] In a further aspect, the application provides a method of treating a subject using the mAbs, antigen-binding fragments thereof, and compositions disclosed herein. In one embodiment, the method is used to inhibit tumor cell growth. In some embodiments, the application provides a method of stimulating a protective autoimmune response, modifying an immune response, or stimulating an antigen-specific immune response using an antibody. In some embodiments, the method includes stimulating a protective autoimmune response, modifying an immune response, or stimulating an antigen-specific immune response using an antibody. In some embodiments, the method includes administering to a subject a composition disclosed herein to treat cancer.

[0065] The compositions disclosed herein may be administered to a subject in solid, liquid or aerosol form at an appropriate pharma- ceutically effective dose. Examples of solid compositions include pills, creams, and implantable dosage units. Pills may be administered orally. Therapeutic creams may be administered topically. Implantable dosage units may be administered locally, e.g., at the tumor site, or may be implanted, e.g., subcutaneously, for systematic release of the therapeutic composition. Examples of liquid compositions include formulations suitable for injection intramuscularly, subcutaneously, intravenously, intraarterially, and formulations for topical and intraocular administration. Examples of aerosol formulations include inhalation formulations for administration to the lungs.

[0066] The composition may be administered by standard routes of administration. For example, the composition may be administered by topical, oral, rectal, nasal, intradermal, intraperitoneal, or parenteral (e.g., intravenous, subcutaneous, or intramuscular) routes. In one embodiment, administration may be parenteral, e.g., 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 the group consisting of water, alcoholic / aqueous solutions, emulsions or suspensions including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0067] In one embodiment, the composition may be incorporated into a sustained release matrix, such as a biodegradable polymer, which is implanted near where delivery is desired, for example, at a tumor site. This method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and sustained administration for a predetermined period of time.

[0068] A sustained release matrix, as used herein, is a matrix made of materials, usually polymers, that are degradable by enzymatic or acid / base hydrolysis or dissolution. Once inserted into the body, the matrix is ​​acted upon by enzymes and body fluids. The sustained release matrix is ​​desirably selected from biocompatible materials such as liposomes, polylactides (polylactide acids), polyglycolides (polymers of glycolic acid), polylactide-co-glycolides (copolymers of lactic and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. A preferred biodegradable matrix is ​​a matrix of either polylactide, polyglycolide, or polylactide-co-glycolide (copolymers of lactic and glycolic acid).

[0069] Those skilled in the art will know that the dosage of the composition will depend on a variety of factors, such as, for example, the condition being treated, the particular composition used, the patient's weight, size, sex, general health, and other clinical factors such as body surface area, the particular compound or composition being administered, other drugs being administered concomitantly, and the route of administration.

[0070] Furthermore, the term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a human or animal, elicits a response sufficient to provide a therapeutic effect in that human or animal. The effective amount is readily determined by one of ordinary skill in the art following routine procedures.

[0071] Depending on the intended use, additional biologically active agents may be present in the pharmaceutical compositions disclosed herein. In one embodiment, the composition may be administered in combination with other compositions containing biologically active substances or compounds. Examples of biologically active substances or compounds include, but are not limited to, compounds against oxidative stress, anti-apoptotic compounds, metal chelators, inhibitors of DNA repair such as pirenzepine and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS), secretase activators, β- and γ-secretase inhibitors, tau protein, neurotransmitters, β-sheet breakers, anti-inflammatory molecules, "atypical anti-inflammatory drugs" such as clozapine, ziprasidone, risperidone, aripiprazole or olanzapine. "psychotropic drugs", or cholinesterase inhibitors (ChEIs) such as tacrine, rivastigmine, donepezil, and / or galantamine, and other medications, and nutritional supplements such as, for example, vitamin B12, cysteine, precursors of acetylcholine, lecithin, choline, ginkgo, acetyl-L-carnitine, idebenone, propentofylline, or xanthine derivatives, together with an antibody according to the disclosure herein, and optionally including a pharmacokinetically acceptable carrier and / or diluent and / or excipient and instructions for treating the disease.

[0072] The pharmaceutical composition may further comprise a proteinaceous carrier, such as, for example, serum albumin or immunoglobulin, particularly of human origin. The proteinaceous pharmacoactive substance may be present in an amount of 1 ng to 10 mg per dose. In general, the dosing regime should be in the range of 0.1 μg to 10 mg, particularly in the range of 1.0 μg to 1.0 mg, more particularly in the range of 1.0 μg to 100 μg of the antibody disclosed herein, all individual numbers within these ranges being part of the disclosure. If the administration is by continuous infusion, a more suitable dose is in the range of 0.01 μg to 10 mg units per kilogram of body weight per hour, all individual numbers within these ranges being part of the disclosure herein.

[0073] The disclosure of the present specification may be more readily understood by reference to the following detailed description of certain embodiments contained herein. Although the disclosure of the present specification has been described with reference to certain details of certain embodiments thereof, such detailed description should not be considered as a limitation on the scope of the disclosure. All references cited or referred to in the disclosure of the present specification are specifically incorporated herein by reference in their entirety. EXAMPLES

[0074] Example 1: Generation of anti-PD-L1 antibodies Monoclonal antibodies against human PD-L1 were generated by immunizing New Zealand white rabbits. The first immunization was performed subcutaneously with 100μg of recombinant human PD-L1 extracellular domain mixed 1:1 v / v with complete Freund's adjuvant. Booster immunizations were performed at weeks 3, 6, 9, and 10 with 50μg of antigen in incomplete Freund's adjuvant. In addition to the antigen, 1x10 6 The animals were boosted with HEK-293 cells.

[0075] At weeks 9 and 12, the animals' sera were tested for anti-PD-L1 titers by ELISA. 96-well plates were coated overnight with goat anti-rabbit IgG antibody (Jackson ImmunoResearch) by passive adsorption at 4°C. The coated wells were washed and blocked with 1% milk for 1 h at room temperature, then incubated with human PD-L1 extracellular domain human Fc domain fusion protein for 1 h at room temperature. After washing, undiluted serum was added to the wells and serially diluted 1:10 across the plate for a total of 7 wells each. After incubation for 1 h at room temperature, the plates were washed and incubated with goat anti-human IgG Fc-specific horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch). The plates were washed and then incubated with Ultra TMB Substrate (Fisher Scientific) for 30 min at room temperature. The signals in the wells were detected by an absorbance plate reader at a wavelength of 450 nm to generate titer curves. Anti-PD-L1 titers are compared to ELISA performed on sera from the same animals obtained pre-immunization.

[0076] Rabbits with significantly higher anti-PD-L1 titers were harvested and selected for monoclonal antibody generation.

[0077] B cells from anti-PD-L1 positive rabbits were harvested from the spleen and lymph nodes 13 weeks after the first immunization. B cells were cultured in 96-well plates for 1 week to allow differentiation into plasma cells and secretion of antibodies. Supernatants from these plasma cell cultures were screened by ELISA as described above for the presence of PD-L1 specific antibodies as shown in Figure 1.

[0078] Anti-PD-L1 antibody-secreting B cells were isolated from positive wells using magnetic capture. Briefly, streptavidin magnetic beads (ThermoFisher Scientific) were coated with biotin-conjugated human PD-L1 extracellular domain protein. The coated beads were incubated with cells from anti-PD-L1 positive wells. The bead-cell complex was washed using a magnet to remove any non-specific cells, and the bead-cell complex was added directly to a tube containing the RT-PCR master mix.

[0079] The light and heavy chain variable 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 kappa sequences. Secondary PCR was performed separately for the light and heavy chains using nested primers containing restriction sites. The amplicons from the variable heavy chain PCR were cloned into an expression vector containing human IgG1. The light chain amplicons were cloned into an expression vector containing human IgK. The resulting clones were sequenced and analyzed.

[0080] The heavy and light chain expression plasmids generated from each well were transiently co-transfected to produce rabbit / human chimeric antibodies, and the resulting supernatant containing the recombinant antibodies was clarified by centrifugation.

[0081] Recombinant antibody supernatants were confirmed to contain anti-PD-L1 antibodies using biolayer interferometry on a ForteBio Octet Red 96 instrument. Antibody in the supernatant was captured using an anti-human Fc biosensor (Pall ForteBio). Binding to PD-L1 was monitored by real-time interferometry by placing the biosensor in a well containing recombinant human PD-L1 extracellular domain protein. Dissociation was measured after transferring the biosensor to a well containing 10X kinetics buffer (Pall ForteBio). Interferometric data were analyzed using software provided by the manufacturer.

[0082] The ability of antibodies to block biochemical binding between PD-1 and PD-L1 was measured by biolayer interference analysis on a ForteBio Octet Red 96 instrument. Biotin-conjugated human PD-L1 extracellular domain was immobilized using a streptavidin biosensor (Pall ForteBio). The biosensor was then placed into a well containing recombinant antibody supernatant. The biosensor was then transferred to a well containing human PD-1 extracellular domain protein. A positive binding signal for PD-1 binding indicates an antibody that is unable to block the binding of PD-L1 to PD-1, whereas a blocking antibody would not produce a positive binding signal when the biosensor was transferred to the PD-1 well. A positive binding signal occurs when a control antibody supernatant containing a known antibody that can bind to PD-L1 but cannot block subsequent PD-1 binding is transferred to the well containing PD-1. Data were monitored in real time using software provided by the manufacturer. An example of the overall data is shown in Figure 2. Anti-PD-L1 antibodies AB1-AB5 were tested in this assay and the results are shown in Figure 3.

[0083] Humanized versions of the anti-PD-L1 antibodies AB1-AB5 have been made, which are designated by the addition of "HU" after the original name. For example, AB1HU is a humanized version of antibody AB1.

[0084] Example 2: Binding Affinities of Anti-PD-L1 Antibodies The binding kinetics of selected anti-PD-L1 antibodies AB1-AB5 and AB1HU-AB5HU were measured by biolayer interference analysis on a ForteBio Octet Red 96 instrument. First, purified antibodies were generated by protein A chromatography using antibody supernatants from transiently transfected HEK-293. The assay was performed by immobilizing the purified antibodies onto an anti-human Fc biosensor. The binding and dissociation of PD-L1 to the biosensor was then observed at various concentrations of PD-L1. Specifically, eight anti-human Fc biosensors were placed in wells containing the same purified antibodies for 5 min. The biosensors were equilibrated in kinetics buffer (Pall ForteBio) for 1 min to establish a baseline. Binding of PD-L1 was observed by placing the biosensors in wells containing various concentrations of human PD-L1 extracellular domain for 5 min. The biosensors were transferred to kinetics buffer and the interference signal was monitored for 15 min, after which dissociation was measured. All steps of the assay were carried out at 30°C with shaking at 1000 RPM. On and off rates (k and k) and the equilibrium binding constant K were determined using the software provided by the manufacturer and fitted using a 1:1 binding global fit model including several tested concentrations. The results of the kinetic studies are shown in Table 1 and representative data are shown in Figure 4. [Table 1]

[0085] Example 3: Antibodies that bind to the cell surface expressing PD-L1 Human lung cancer cell line H441 was labeled with FVS520 viability dye (BD Biosciences, 1:2000 dilution) for 15 min at room temperature. Labeled cells were then diluted in FACS buffer (2% FBS in PBS) and added to a V-bottom 96-well plate (50,000 cells per well) and incubated with 0–100 nM anti-huPDL1 or negative control isotype-matched or positive control isotype-matched control antibodies for 30 min on ice. Cells were washed to remove excess primary antibody and then labeled with Alex Fluor 647-conjugated goat anti-human Fc secondary antibody (Jackson Immunoresearch, 1:1600 dilution). Cells were incubated on ice for 20 min and then washed with FACS buffer before being subjected to a FACSCalibur flow cytometer equipped with a Cytek AMS plate loader system. Figure 5 shows the median fluorescence intensity (MFI) of the AF647 channel after gating on live cells. Error bars represent SEM of duplicate samples.

[0086] Although the disclosure herein has been particularly shown and described with reference to embodiments thereof, those skilled in the art will recognize that the foregoing and other changes in form and detail may be made therein without departing from the spirit and scope thereof. All references cited or referred to in the disclosure herein are hereby incorporated by reference in their entirety.

[0087] Sequence Listing SEQ ID NO:1 AB1 chimeric light chain full length nucleic acid sequence GACCCTGTGCTGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCCATCGCGTTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGTGTGCAGTGACAATGCACTTATTACTGTCAACAGGTTATAGTTGGGTAATGTTGATAATGTTTTCGGCGGAGGGACCGAGGTGGTGG TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCCGTCCACAAAGAGCTTCACAGGGGAGAGTGT கார்க்கை AB1 chimeric light chain variable light chain nucleic acid sequence GACCCTGTGCTGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCCCATCGCGTTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGTGTGCAGTGTGACAATGCCACTTATTACTGTCAACAGGTTATAGTTGGGTAATGTTGATAATGTTTTCGGCGGAGGGACCGAGGTGTGGTCAAA அக்க்கியுக்க்கு3 Full amino acid sequence of the AB1 chimeric light chain. The human kappa constant domain is underlined. DPVLTQTPASVEVAVGGTVTIKCQASQSISSHLNWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTETLTISSVQCDNAATYYCQQGYSWGNVDNVFGGGTEVVVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence number 4 AB1 chimeric light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. DPVLTQTPASVEVAVGGTVTIKC QASQSISSHLN WYQQKPGQPPKLLIY KASTLAS GVPSRFKGSGSGTTEFTLTISSVQCDNAATYYC QQGYSWGNVDNV FGGGTEVVVK sequence number 5 AB1 chimeric heavy chain full length nucleic acid sequence SEQ ID NO:6 AB1 chimeric heavy chain variable heavy chain nucleic acid sequence CAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGTAAAGCCTCTGGATTCTCCTTCAGTAGCGGGTACGACATGTGCTGGGTCCGCCGGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTGCTGCTGGTAGTGCTGGTATCACTT ACGACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTACAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGATCGGCGTTTTCGTTCGACTACGCCATGGACCTCTGGGGCCCAGGGACCCTCGTCACCGTCTCGAGC SEQ ID NO:7 Full length amino acid sequence of the AB1 chimeric heavy chain. The human gamma-1 constant domain is underlined. QEQLVESGGGLVQPEGSLTLTCKASGFSFSSGYDMCWVRRAPGKGLEWIACIAAGSAGITYDANWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARSAFSFDYAMDLWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:8 AB1 chimeric heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. QEQLVESGGGLVQPEGSLLTTCKASGFSFSS GYDMC WVRRAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISKTSSTTVTLQMTSLTAADTATYFCAR SAFSFDYAMDL WGPGTLVTVSS SEQ ID NO:9 AB1HU humanized light chain full length nucleic acid sequence GACATCCAGATGACCCAGTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGAAAGCCCTAAGCTCCTGATCTATAAGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGGTTATAGTTGGGTAATGTTGATAATGTTTTCGGCGGAGGACCAAGGTGGAGA TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCCGTCCACAAAGAGCTTCACAGGGGAGAGTGT sequence number 10 AB1HU humanized light chain variable light chain nucleic acid sequence GACATCCAGATGACCCAGTTCCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCCACTCTGGCATCTGGGGTCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGGTTATAGTTGGGTAATGTTGATAATGTTTTCGGCGGAGGACCAAGGTGGAGATCAAA sequence number 11 Full amino acid sequence of the AB1HU humanized light chain. The human kappa constant domain is underlined. DIQMTQSPSTLSASVGDRVTITCQASQSISSHLNWYQQKPKGAPKLLIYKASTLASGVPSRFSGSGSGTETLTISSLQPDDFATYYCQQGYSWGNVDNVFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence number 12 AB1HU humanized light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. DIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIK sequence number 13 AB1HU humanized heavy chain full length nucleic acid sequence SEQ ID NO:14 AB1HU humanized heavy chain variable heavy chain nucleic acid sequence GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCGGGTACGACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTGCTGCTGGTAGTGCTGGTATCACTTAC GACGCGAACTGGGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGATCGGCGTTTTCGTTCGACTACGCCATGGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC SEQ ID NO:15 AB1HU humanized heavy chain full length amino acid sequence. Human gamma 1 constant domain is underlined. EVQLLESGGGLVQPGGSLRLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:16 AB1HU humanized heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. EVQLLESGGGLVQPGGSLRLSCAASGFSSSS GYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDL WGQGTLVTVSS SEQ ID NO:17 AB2 chimeric light chain full length nucleic acid sequence GCCTATGATATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGCATTAGAGCTTCTTATCCTGGTTATCAGCAGAAACCAGGGCAGCGTCCCAGGCTCCTGATCTTTTCTGCATCCACTCTGGCATCTGGGGTCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTATTATTGTCAACAGGGTTATAAGTAAAAGTAATGTGGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGG TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCCGTCCACAAAGAGCTTCACAGGGGAGAGTGT sequence number 18 AB2 chimeric light chain variable light chain nucleic acid sequence GCCTATGATATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGCATTAGTAGCTTCTTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAGGCTCCTGATCTTTTCTGCATCCACTCTGGCA TCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCGATGCTGCCACTTATTATTGTCAACAGGGTTAAGTAAAAGTAATGTGGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA sequence number 19 Full amino acid sequence of the AB2 chimeric light chain. The human kappa constant domain is underlined. AYDMTQTPASVSAAVGGTVTINCQASESISSFLSWYQQKPGQRPRLLIFSASTLASGVPSRFKGSGSGTEFTLTISGVECADAATYYCQQGYSKSNVDNAFGGGTEVVVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence number 20 AB2 chimeric light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. AYDMTQTPASVSAAVGGTVTINC QASESISSFLS WYQQKPGQRPRLLIF SASTLAS GVPSRFKGSGSGTEFTLTISGVECADAATYYC QQGYSKSNVDNA FGGGTEVVVK sequence number 21 AB2 chimeric heavy chain full length nucleic acid sequence SEQ ID NO:22 AB2 chimeric heavy chain variable heavy chain nucleic acid sequence CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGTACAACTTCCGGAATCGACCTTAGTACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTAGTTATGTTGGTAACACAT ACTACGCGAGCTGGGCGAAAGGCCGATTCACCCTCTCCAAAACCTCGACCACGGTGGATCTGAGAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGGGATTTTATTAGTGGTTCCCACTTGTGGGGCCCGGGCACCCTGGTCACCGTCTCGAGC SEQ ID NO:23 Full amino acid sequence of the AB2 chimeric heavy chain. The human gamma-1 constant domain is underlined. QSLEESGGRLVTPGTPLTLTCTTSGIDLSTYDMIWVRQAPGKGLEYIGIISYVGNTYYASWAKGRFTLSKTSTTVDLRITSPTTEDTATYFCARDFISGSHLWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:24 AB2 chimeric heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. QSLEESGGRLVTPGTPLTLTCTTSGIDLS TYDMI WVRQAPGKGLEYIG IISYVGNTYYASWAKG RFTLSKTSTTVDLRITSPTTEDTATYFCAR DFISGSHL WGPGTLVTVSS SEQ ID NO:25 AB2HU humanized light chain full length nucleic acid sequence GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAATTGTCAGGCCAGTGAGCATTAGAGCTTCTTATCCTGGTTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTTTTCTGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATAAGTAAAAGTAATGTGGATAATGCTTTCGGGGGAGCCAAGGTGGAGA TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCCGTCCACAAAGAGCTTCACAGGGGAGAGTGT sequence number 26 AB2HU humanized light chain variable light chain nucleic acid sequence GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGTCAGGCCAGTGAGCATTAGAGCTTCTTATCCTGTATCAGCAGAAACCAGGAAAGCCCTAAGCTCCTGATCTTTTCTGCATCCACTCTGGCA TCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGTTATAGTAAAAGTAATGTGGATAATGCTTTCGGCGGAGGACCAAGGTGGAGATCAAA sequence number 27 Full amino acid sequence of the AB2HU humanized light chain. The human kappa constant domain is underlined. AYDMTQSPSSVSASVGDRVTINCQASESISSFLSWYQQKPGKAPKLLIFSASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSKSNVDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence no. 28 AB2HU humanized light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. AYDMTQSPSSVSASVGDRVTINC QASESISSFLS WYQQKPGKAPKLLIF SASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYSKSNVDNA FGGGTKVEIK sequence number 29 AB2HU humanized heavy chain full length nucleic acid sequence SEQ ID NO:30 AB2HU humanized heavy chain variable heavy chain nucleic acid sequence CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCCAGCCTGGGGGCTCCCTGAGACTCTCCTGTACAACTTCTGGAATCGACCTTAGTACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTGGGAATCATTAGTTATGTTGGTAACACA TACTACGCGAGCTGGGCGAAAGGCCGATTCACCCTCTCCAAAACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTTTATTAGTGGTTCCCACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC SEQ ID NO:31 AB2HU humanized heavy chain full length amino acid sequence. Human gamma 1 constant domain is underlined. QVQLVESGGGLVQPGGSLRLSCTTSGIDLSTYDMIWVRQAPGKGLEWVGIISYVGNTYYASWAKGRFTLSKTKNTVYLQMNSLRAEDTAVYYCARDFISGSHLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:32 AB2HU humanized heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. QVQLVESGGGLVQPGGSLRLSCTTSGIDLS TYDMI WVRQAPGKGLEWVG IISYVGNTYYASWAKG RFTLSKTKNTVYLQMNSLRAEDTAVYYCAR DFISGSHL WGQGTLVTVSS SEQ ID NO:33 AB3 chimeric light chain full length nucleic acid sequence GCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTCCAGGTTTATAATGGCTACTGGTTATCCTGGTTATCAGCAGAAGCCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCACTCTGGCATCTGGGGTCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAACGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAGTTATACTAGTAGACTGAGAATTCTTTCGCGGAGGGACCGAGGTGGTGG TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCCGTCCACAAAGAGCTTCACAGGGGAGAGTGT sequence number 34 AB3 chimeric light chain variable light chain nucleic acid sequence GCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAGTGTTTATAATGGCTACTGGTTATCCTGGTATCAGCAGAAGCCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCACTCTG GCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAACGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAGTTATACTAGTAGTACTGAGAATTCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA SEQ ID NO:35 Full amino acid sequence of the AB3 chimeric light chain. The human kappa constant domain is underlined. AVLTQTPSPVSAAVGGTVSISCQSSQSVYNGYWLSWYQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTQFTLTINDVQCDDAATYYCLGSYTSSTENSFGGGTEVVVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:36 AB3 chimeric light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. AVLTQTPSPVSAAVGGTVSISC QSSQSVYNGYWLS WYQQKPGQPPKLLIY GASTLAS GVPSRFKGSGSGTQFTLTINDVQCDDAATYYC LGSYTSSTENS FGGGTEVVVK SEQ ID NO:37 AB3 chimeric heavy chain full length nucleic acid sequence SEQ ID NO:38 AB3 chimeric heavy chain variable heavy chain nucleic acid sequence CAGGAGCAGCTGGAGGAGTCCGGGGGAGGCCTGGTCACGCCTGGAGGAACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTGATACTAATGTTTATATATACT ACGCGAACTGGGCAAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAGATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATATGTGGGTAATAATGATGATTATATTAACTTGTGGGGCCCGGGCACCCTGGTCACCGTCTCGAGC SEQ ID NO: 39 Full amino acid sequence of the AB3 chimeric heavy chain. The human gamma-1 constant domain is underlined. QEQLEESGGGLVTPGGTLTLTCTVSGFSLSSYWMSWVRQAPGKGLEWIGVIDTNVYIYYANWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARYVGNNDDYINLWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 40 AB3 chimeric heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. QEQLEESGGGLVTPGGTLTLTCTVSGFSLS SYWMS WVRQAPGKGLEWIG VIDTNVYIYYANWAKG RFTISKTSTTVDLKITSPTTEDTATYFCAR YVGNNDDYINL WGPGTLVTVSS SEQ ID NO:41 AB3HU humanized light chain full length nucleic acid sequence GACATCCAGATGACCCAGTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTTTATAATGGCTACTGGTTATCCTGGTTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCTAGGCAGTTATACTAGTAGTACTGAGAACTCTTTCGGCGGAGCCAAGGTGGAG ATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCAGGGCCTGAGCCCGTCACAAAGAGCTTCACAGGGGAGAGTGT sequence number 42 AB3HU humanized light chain variable light chain nucleic acid sequence GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAATGGCTACTGGTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCTAAGCTCCTGATCTATGGTGCATCCACTC TGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCTAGGCAGTTATACTAGTAGTACTGAGAACTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA sequence number 43 Full amino acid sequence of the AB3HU humanized light chain. The human kappa constant domain is underlined. DIQMTQSPSTLSASVGDRVTITCQSSQSVYNGYWLSWYQQKPKGAPKLLIYGASTLASGVPSRFSGSGSGTETLTISSLQPDDFATYYCLGSYTSSTENSFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence number 44 AB3HU humanized light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. DIQMTQSPSTLSASVGDRVTITC QSSQSVYNGYWLS WYQQKPGKAPKLLIY GASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC LGSYTSSTENS FGGGTKVEIK sequence number 45 AB3HU humanized heavy chain full length nucleic acid sequence sequence number 46 AB3HU humanized heavy chain variable heavy chain nucleic acid sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGTAGCTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTGATACTAATGTTTATATATACTACG CGAACTGGGCAAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATATGTGGGTAATAATGATGATTATATTAACTTGTGGGGCCAGGGAACCCTGTCACCGTCTCGAGC sequence number 47 AB3HU humanized heavy chain full length amino acid sequence. Human gamma 1 constant domain is underlined. EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYWMSWVRQAPGKGLEWIGVIDTNVYIYYANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYVGNNDDYINLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 48 AB3HU humanized heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. EVQLVESGGGLVQPGGSLRLSCAASGFSLS SYWMS WVRQAPGKGLEWIG VIDTNVYIYYANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYCAR YVGNNDDYINL WGQGTLVTVSS sequence number 49 AB4 chimeric light chain full length nucleic acid sequence GCATTCGAATTGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAAAGCATTAGCAACTACTTATCCTGGTATCAGCAGATTCCAGGGCAGCCTCCCAACCTCCTGATCTTATTAGCATCCAATCTGGCATCTGGGGTCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAGCTATTATGGTGGTGGTAGGCCTATACTTTCGCGGAGGGACCGAGGTGGTGGTC AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCGTCACAAAGAGCTTCACAGGGGAGAGTGT sequence number 50 AB4 chimeric light chain variable light chain nucleic acid sequence GCATTCGAATTGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAAAGCATTAGCAACTACTTATCCTGGTATCAGCAGATTCCAGGGCAGCCTCCCAACCTCCTGATCTTATTAGCATCCAATCTGG CATCTGGGGTCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAGCTATTATGGTGGTGGTAGTGCCTATACTTTCGGCGAGGGACCGAGGTGGTGGTCAAA sequence number 51 Full amino acid sequence of the AB4 chimeric light chain. The human kappa constant domain is underlined. AFELTQTPASVSEPVGGTVTIKCQASESISNYLSWYQQIPGQPPNLLIYYASNLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSYYGGGSAYTFGGGTEVVVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence number 52 AB4 chimeric light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. AFELTQTPASVSEPVGGTVTIKC QASESISNYLS WYQQIPGQPPNLLIY YASNLAS GVPSRFKGSGSGTTEFTLTISDLECADAATYYC QSYYGGGSAYT FGGGTEVVVK sequence number 53 AB4 chimeric heavy chain full length nucleic acid sequence SEQ ID NO:54 AB4 chimeric heavy chain variable heavy chain nucleic acid sequence CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTGTCTGGAATCGACCTCAGTGTCATCAATATGGGCTGGGTCCGCCAGGCTCCAGGGGAGGGCTGGAATGGATCGGAACCATTACTTATGTTGGTAACACATATTACG CGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACTTATTTCTGTGCCAGAGAATCTGGTACTATTTATTACAGTTACTTTAACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCGAGC SEQ ID NO:55 Full amino acid sequence of the AB4 chimeric heavy chain. The human gamma-1 constant domain is underlined. QSVEESGGRLVTPGTPLTLTCTVSGIDLSVINMGWVRQAPGEGLEWIGTITYVGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARESGTIYYSYFNLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:56 AB4 chimeric heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. QSVEESGGRLVTPGTPLTLTCTVSGIDLS VINMG WVRQAPGEGLEWIG TITYVGNTYYASWAKG RFTISKTSTTVDLKITSPTTEDTATYFCAR ESGTIYYSYFNL WGQGTLVTVSS SEQ ID NO:57 AB4HU humanized light chain full length nucleic acid sequence GCATTCGAATTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGCCAGGCCAGTGAAAGCATTAGCAACTACTTATCCTGGTATCAGCAGATTCCAGGGAAAGTTCCTAAGCTCCTGATCTTATTAGCATCCAATCTGGCATCTGGGGTCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTTATTACTGTCAAAGCTATTATGGTGGTAGTGCCTAACTTTCGGCGGAGGGACCAAGGTGGAGAAT AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCGTCACAAAGAGCTTCACAGGGGAGAGTGT sequence number 58 AB4HU humanized light chain variable light chain nucleic acid sequence GCATTCGAATTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGCCAGGCCAGTGAAAGCATTAGCAACTACTTATCCTGGTATCAGCAGATTCCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCATCCAATCTG CATCTGGGGTCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTTATTACTGTCAAAGCTATTATGGTGGTGGTAGTGCCTATACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA sequence number 59 Full amino acid sequence of the AB4HU humanized light chain. The human kappa constant domain is underlined. AFELTQSPSSLSASVGDRVTIKCQASESISNYLSWYQQIPGKVKPLLIYYASNLASGVPSRFSGSGSGTDFLTISSLQPEDVATYYCQSYYGGGSAYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence number 60 AB4HU humanized light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. AFELTQSPSSLSASVGDRVTIKC QASESISNYLS WYQQIPGKVPKLLIY YASNLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QSYYGGGSAYT FGGGTKVEIK sequence number 61 AB4HU humanized heavy chain full length nucleic acid sequence sequence number 62 AB4HU humanized heavy chain variable heavy chain nucleic acid sequence GAGGTGCAGCTGGTGGAGTCTGGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCTGTACAGTGTCTGGAATCGACCTCAGTGTCATCAATATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAACCATTACTTATGTTGGTAACACATATTAC GCGAGCTGGGCGAAAGGCAGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTTAAATCACCAGTCCGACAACCGAGGACACGGCTGTATTACTGTGCGAGAGAATCTGGTACTATTATTACAGTTACTTTAACTTGTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGC sequence number 63 AB4HU humanized heavy chain full length amino acid sequence. Human gamma 1 constant domain is underlined. EVQLVESGGGLVQPGGSLRLSCTVSGIDLSVINMGWVRQAPGKGLEWIGTITYVGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTAVYYCARESGTIYYSYFNLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG sequence number 64 AB4HU humanized heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. EVQLVESGGGLVQPGGSLRLSCTVSGIDLS VINMG WVRQAPGKGLEWIG TITYVGNTYYASWAKG RFTISKTSTTVDLKITSPTTEDTAVYYCAR ESGTIYYSYFNL WGQGTLVTVSS sequence number 65 AB5 chimeric light chain full length nucleic acid sequence GCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCTGAAACCAGGGCAGTCTCCCAAGCTCCTGATCCATTCTGCATCCTCTCTGGGCATCTGGGGTCTCTTGCGGTTCAAAGGCAGTGGATCTGGGACACACTTCACTTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTATTATTGTCAACAGGTTATGGTAAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGG TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCCGTCCACAAAGAGCTTCACAGGGGAGAGTGT sequence no.66 AB5 chimeric light chain variable light chain nucleic acid sequence GCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCTGAAACCAGGGCAGTCTCCCAAGCTCCTGATCCATTCTGCATCCTCTCTGGGCATCTGGGGTCTCTTCGCGGTTCAAAGGCAGTGGATCTGGGACACACTTCACTTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTATTATTGTCAACAGGGTTATGGTAAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTCAAA sequence number 67 Full amino acid sequence of the AB5 chimeric light chain. The human kappa constant domain is underlined. AYDMTQTPASVEVAVGGTVTIKKQASEDIYSFLAWYQLKPGQSPKLLIHSASSLASGVSSRFKGSGSGTHFTLTISGVECADAATYYCQQGYGKNNVDNAFGGGTEVVVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence number 68 AB5 chimeric light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. AYDMTQTPASVEVAVGGTVTIKC QASEDIYSFLA WYQLKPGQSPKLLIH SASSLAS GVSSRFKGSGSGTHFTLTISGVECADAATYYC QQGYGKNNVDNA FGGGTEVVVK sequence number 69 AB5 chimeric heavy chain full length nucleic acid sequence SEQ ID NO:70 AB5 chimeric heavy chain variable heavy chain nucleic acid sequence CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCCGGAATCGACCTTAATACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAATCATTACTTATAGTGGTAGTAGAT ACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAGAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGGGATTATATGAGTGGTTCCCACTTGTGGGGCCCAGGCACCCTCGTCACCGTCTCGAGC SEQ ID NO:71 Full amino acid sequence of the AB5 chimeric heavy chain. The human gamma-1 constant domain is underlined. QSVEESGGRLVTPGTPLTLTCTVSGIDLNTYDMIWVRQAPGKGLEWIGIITYSGSRYYANWAKGRFTISKTSTTVDLRITSPTTEDTATYFCARDYMSGSHLWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:72 AB5 chimeric heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. QSVEESGGRLVTPGTPLTLTCTVSGIDLN TYDMI WVRQAPGKGLEWIG IITYSGSRYYANWAKG RFTISKTSTTVDLRITSPTTEDTATYFCAR DYMSGSHL WGPGTLVTVSS SEQ ID NO:73 AB5HU humanized light chain full length nucleic acid sequence GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGGCATCTGGGGTCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAAATAATGTTGATAATGCTTTCGGCGGAGGACCAAGGTGGAGA TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCCCCGTCCACAAAGAGCTTCACAGGGGAGAGTGT sequence no. 74 AB5HU humanized light chain variable light chain nucleic acid sequence GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGGCATCTGGGGTCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAAATAATGTTGATAATGCTTTCGGCGGAGGACCAAGGTGGAGATCAAA sequence no. 75 AB5HU humanized light chain full length amino acid sequence. Human kappa constant domain is underlined. AYDMTQSPSSVSASVGDRVTIKCQASEDIYSFLAWYQQKPGKAPKLLIHSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYGKNNVDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence no. 76 AB5HU humanized light chain variable light chain amino acid sequence. Complementarity determining regions are underlined. AYDMTQSPSSVSASVGDRVTIKC QASEDIYSFLA WYQQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNA FGGGTKVEIK sequence number77 AB5HU humanized heavy chain full length nucleic acid sequence SEQ ID NO:78 AB5HU humanized heavy chain variable heavy chain nucleic acid sequence CAGTCGGTGGAGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACCGCCTCTGGAATCGACCTTAATACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTTGGAATCATTACTTATAGTGGTAGTAGATACT ACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTATATGAGTGGTTCCCACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC SEQ ID NO:79 AB5HU humanized heavy chain full length amino acid sequence. Human gamma 1 constant domain is underlined. QSVEESGGGLVQPGGSLRLSCTASGIDLNTYDMIWVRQAPGKGLEWVGIITYSGSRYYANWAKGRFTISKDNTKNTVYLQMNSLRAEDTAVYYCARDYMSGSHLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:80 AB5HU humanized heavy chain variable heavy chain amino acid sequence. Complementarity determining regions are underlined. QSVEESGGGLVQPGGSLRLSCTASGIDLN TYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVSS SEQ ID NO:81 Human PD-L1 full length nucleic acid sequence TTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAACAAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGG CTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACA TGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGCACCACCACCAATTCCAAGAGAGAGGAAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCT GAATTGGTCATCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTTATTATGCCTGGTGTAGCACTGACATTCATCTTCCGTTTAAGAAAGGGGAGAATGATGGATGTAAAAAATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACG sequence number82 Human PD-L1 full-length amino acid sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELT CQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPLAHPPNERTHLVIlgAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET sequence number 83 Human PD-L1 extracellular domain nucleic acid sequence TTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAACAAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGA AAGTCAATGCCCCATACAACAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACT SEQ ID NO:84 Human PD-L1 extracellular domain amino acid sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT

Claims

1. 1. An isolated mAb, or antigen-binding fragment thereof, that has binding specificity for human PD-L1, The mAb or antigen-binding fragment thereof is an isolated mAb or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:76 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

80.

2. An isolated mAb or antigen-binding fragment thereof that has binding specificity for human PD-L1, the isolated mAb or antigen-binding fragment thereof comprises VL-CDR1: QASEDIYSFLA, VL-CDR2: SASSLAS, and VL-CDR3: QQGYGKNNVDNA, and VH-CDR1: TYDMI, VH-CDR2: IITYSGSRYYANWAKG, and VH-CDR3: DYMSGSHL; wherein said VL-CDR1, VL-CDR2 and VL-CDR3 represent the three complementarity determining regions of a light chain variable region, and said VH-CDR1, VH-CDR2 and VH-CDR3 represent the three complementarity determining regions of a heavy chain variable region.

3. 3. The isolated mAb or antigen-binding fragment of claim 1 or 2, having a binding affinity for PD-L1 with a Kd of 70 nM or less.

4. 3. The isolated mAb or antigen-binding fragment of claim 1 or 2, which exhibits one or more functional properties selected from the group consisting of high affinity binding to PD-L1, inhibition of binding of PD-L1 to PD-1, enhancing T cell activation, stimulating an antibody response, reversing the suppressive function of immune suppressor cells, or a combination thereof.

5. 5. The isolated mAb or antigen-binding fragment of claim 4, wherein the enhanced T cell activation comprises T cell proliferation, IFN-γ and / or IL-2 secretion, or a combination thereof.

6. 3. The isolated mAb or antigen-binding fragment of claim 1 or 2, wherein the isolated mAb comprises a humanized antibody, a chimeric antibody, or a recombinant antibody.

7. 3. The isolated mAb or antigen-binding fragment of claim 1 or 2, wherein the isolated mAb comprises an IgG.

8. 3. The isolated mAb or antigen-binding fragment thereof of claim 1 or 2, wherein the antigen-binding fragment comprises an Fv, Fab, F(ab')2, scFv or scFv2 fragment.

9. 3. The isolated mAb or antigen-binding fragment thereof of claim 1 or 2, wherein the mAb comprises a bispecific antibody, a trispecific antibody, or a multispecific antibody.

10. The IgG1 heavy chain for the isolated mAb or antigen-binding fragment thereof, 3. The isolated mAb or antigen-binding fragment thereof of claim 1 or 2, comprising the amino acid sequence set forth in SEQ ID NO:

79.

11. The kappa light chain for the isolated mAb or antigen-binding fragment thereof, 3. The isolated mAb or antigen-binding fragment thereof of claim 1 or 2, comprising the amino acid sequence set forth in SEQ ID NO:

75.

12. 3. An isolated nucleic acid encoding the isolated mAb or antigen-binding fragment of claim 1 or 2.

13. An expression vector comprising the isolated nucleic acid of claim 12.

14. 13. A host cell comprising the nucleic acid of claim 12, wherein the host cell is a prokaryotic or eukaryotic cell.

15. A method for producing an antibody, comprising culturing the host cell of claim 14 so that the antibody is produced.

16. 3. An immunoconjugate comprising the isolated mAb or antigen-binding fragment thereof of claim 1 or 2 and a drug unit, the Drug Unit is linked to the isolated mAb or antigen-binding fragment via a linker; The linker comprises a covalent bond selected from an ether bond, an amine bond, an amide bond, a disulfide bond, an imide bond, a sulfone bond, a phosphate bond, or a combination thereof; Immune complex.

17. 17. The immunoconjugate of claim 16, wherein the drug unit is a cytotoxic agent, an immunomodulatory agent, or a combination thereof.

18. 18. The immunoconjugate of claim 17, wherein the cytotoxic agent is selected from a growth inhibitory agent or a chemotherapeutic agent selected from the group consisting of tubulin binding agents, DNA intercalators, DNA alkylators, enzyme inhibitors, immunomodulators, antimetabolites, radioisotopes, or combinations thereof.

19. 18. The immunoconjugate of claim 17, wherein the cytotoxic agent is selected from calicheamicin, ozogamicin, monomethyl auristatin E, emtansine, derivatives or combinations thereof.

20. 18. The immunoconjugate of claim 17, wherein the immunomodulatory agent activates or suppresses immune cells, T cells, NK cells, B cells, macrophages, or dendritic cells.

21. 17. A pharmaceutical composition comprising the isolated mAb or antigen-binding fragment thereof of claim 1 or the immunoconjugate of claim 16, and a pharma- ceutically acceptable carrier.

22. 22. The pharmaceutical composition of claim 21, further comprising a chemotherapeutic agent, proliferation inhibitor, or drug unit selected from the group of a calicheamicin, an antimitotic agent, a toxin, a radioisotope, an anti-estrogen, 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.

23. 22. The pharmaceutical composition of claim 21, further comprising an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

24. A pharmaceutical composition for treating a subject having cancer, comprising:

3. A method for treating a patient having a disease comprising administering to said patient an effective amount of the isolated mAb or antigen-binding fragment thereof of claim 1 or 2, the cancer comprises cells that express PD-L1; Pharmaceutical compositions.

25. 25. The pharmaceutical composition of claim 24, wherein the cancer comprises breast cancer, colon 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.

26. The pharmaceutical composition of claim 25, wherein the treatment comprises co-administering an effective amount of a therapeutic agent, the therapeutic agent comprising an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

27. The therapeutic agent is selected from the group consisting of capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, mustine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, folinic acid, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testosterone, and the like.

27. The pharmaceutical composition of claim 26, comprising thiazide, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandertanib, afatinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, everolimus, temsirolimus, dabrafenib, vemurafenib, trametinib, vintafolide, apatinib, crizotinib, periforsine, olaparib, bortezomib, tofacitinib, or derivatives or combinations thereof.

28. 25. The pharmaceutical composition of claim 24, wherein the subject is a human.

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