Antibodies against cd19 antibodies and their preparation and use

Abstract

An antibody targeting CD19 antibody, methods of making and uses thereof. In particular, a novel antibody targeting CD19 antibody. Methods of making the monoclonal antibody. The monoclonal antibody is capable of binding CD19 antibody with high specificity and blocking the function of CD19 antibody.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, more particularly to an antibody against CD19 and preparation and application thereof. BACKGROUND

[0002] Antibodies are proteins produced by the body under the stimulation of antigens, which have protective effects and are secreted by plasma cells into body fluids such as blood. Antibodies can specifically bind to antigens, neutralize toxins, prevent pathogens from invading, and the like. According to the specific binding characteristics of antibodies to antigens, antibody drugs targeting specific biological targets of diseases can be developed for the treatment of diseases. Antibody drugs have been applied to the fields of anti-tumor and autoimmune treatment, and are playing an increasingly important role in the fields of anti-viral and bacterial infection, cardiovascular and cerebrovascular diseases, diabetes, and rare disease treatment, and are currently the fastest-growing class of biological drugs.

[0003] CD19 is a cluster differentiation antigen, which is an important membrane antigen related to B cell proliferation, differentiation, activation and antibody production. Most B cell malignancies highly express CD19 on their surface, and multiple independent centers have achieved unprecedented success in targeting CD19-expressing B cell relapsed and refractory malignancies using chimeric antigen receptor (CAR) modified T cells. Immunotherapy, led by CAR-T, has brought hope of "curing cancer" to countless patients, but CAR-T, while having outstanding effectiveness, also has a problem that needs to be solved, which is the serious side effect of cytokine storm (CRS).

[0004] Cytokine storm refers to the excessive cascade release of TNF-α, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and other cytokines caused by the activation and rapid proliferation of T lymphocytes in the body after CAR-T infusion. These cytokines can mediate various immune responses, causing high fever, hypotension, myalgia, coagulopathy, dyspnea, end-organ dysfunction and other clinical manifestations in patients, which can cause serious permanent damage or failure to the body's tissues and organs, and even lead to death. In short, cytokine storm is a serious non-specific inflammatory reaction caused by the explosive secretion of a large amount of cytokines in the body during CAR-T treatment.

[0005] However, under the stimulation of target cells (cancer cells) in vivo, the CAR-T cells can proliferate rapidly and release a large amount of cytokines, thereby killing the target cells through the cytokines. Therefore, the cytokine storm cannot be unilaterally regarded as a side effect of CAR-T, and is also an effective clinical manifestation of CAR-T in vivo. At present, in the clinical treatment of CAR-T, the generation of the cytokine storm cannot be avoided, and only conventional symptomatic treatment means such as close observation and active response can be relied on.

[0006] In summary, there is an urgent need in the art to develop drugs and methods capable of responding to the cytokine storm after CAR-T treatment. SUMMARY

[0007] The present application aims to provide an antibody against CD19 and preparation and application thereof.

[0008] In a first aspect of the present application, a heavy chain variable region of an antibody is provided, which comprises the following three complementarity determining regions (CDRs):

[0009] a CDR1 as shown in SEQ ID NO: 1,

[0010] a CDR2 as shown in SEQ ID NO: 2, and

[0011] a CDR3 as shown in SEQ ID NO: 3;

[0012] or,

[0013] a CDR1 of SEQ ID NO: 4,

[0014] a CDR2 as shown in SEQ ID NO: 5, and

[0015] a CDR3 as shown in SEQ ID NO: 6,

[0016] or,

[0017] a CDR1 as shown in SEQ ID NO: 7,

[0018] a CDR2 as shown in SEQ ID NO: 8, and

[0019] a CDR3 as shown in SEQ ID NO: 9;

[0020] wherein any one of the above amino acid sequences further comprises a derivative sequence in which at least one amino acid is optionally added, deleted, modified and / or substituted, and which can retain the binding affinity to the CD19 antibody.

[0021] In another preferred embodiment, the heavy chain variable region further comprises a human-derived FR region or a mouse-derived FR region.

[0022] In another preferred embodiment, the heavy chain variable region CDRs are set forth in positions 50-54, 69-85, 118-130 of an amino acid sequence set forth in SEQ ID NO: 19.

[0023] In another preferred embodiment, the heavy chain variable region CDRs are set forth in positions 50-54, 69-85, 118-123 of an amino acid sequence set forth in SEQ ID NO: 21.

[0024] In another preferred embodiment, the heavy chain variable region CDRs are set forth in positions 50-54, 69-85, 118-129 of an amino acid sequence set forth in SEQ ID NO: 23.

[0025] In another preferred embodiment, the heavy chain variable region CDRs are set forth in positions 50-54, 69-85, 118-130 of an amino acid sequence set forth in SEQ ID NO: 19.

[0026] In a second aspect of the present application, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the first aspect of the present application.

[0027] In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

[0028] In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.

[0029] In another preferred embodiment, the heavy chain has an amino acid sequence set forth in SEQ ID NO: 19, 21 or 23 (heavy chain constant region is of human origin).

[0030] In a third aspect of the present application, there is provided a light chain variable region of an antibody, said light chain variable region comprising three complementarity determining regions (CDRs) as follows:

[0031] a CDR1’ set forth in SEQ ID NO: 10,

[0032] a CDR2’ set forth in SEQ ID NO: 11, and

[0033] a CDR3’ set forth in SEQ ID NO: 12;

[0034] or,

[0035] a CDR1’ set forth in SEQ ID NO: 13,

[0036] a CDR2’ set forth in SEQ ID NO: 14, and

[0037] a CDR3’ set forth in SEQ ID NO: 15;

[0038] or,

[0039] CDR1' as shown in SEQ ID NO: 16,

[0040] CDR2' as shown in SEQ ID NO: 17, and

[0041] CDR3' as shown in SEQ ID NO: 18;

[0042] wherein any one of the above amino acid sequences further comprises a derivative sequence optionally with addition, deletion, modification and / or substitution of at least one amino acid, and which is capable of retaining the binding affinity to CD19 antibody.

[0043] In another preferred embodiment, the light chain variable region further comprises a FR region of human origin or a FR region of murine origin.

[0044] In another preferred embodiment, the light chain variable region CDRs are as shown in positions 46-56, 72-78, 114-119 of the amino acid sequence as shown in SEQ ID NO: 20.

[0045] In another preferred embodiment, the light chain variable region CDRs are as shown in positions 46-61, 77-83, 119-124 of the amino acid sequence as shown in SEQ ID NO: 22.

[0046] In another preferred embodiment, the light chain variable region CDRs are as shown in positions 46-60, 76-82, 115-123 of the amino acid sequence as shown in SEQ ID NO: 24.

[0047] In another preferred embodiment, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 26, 28, 30, 32, 34.

[0048] In a fourth aspect of the present application, there is provided a light chain of an antibody, which has the light chain variable region of the third aspect of the present application.

[0049] In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

[0050] In another preferred embodiment, the light chain constant region is of human origin, murine origin or rabbit origin.

[0051] In another preferred embodiment, the amino acid sequence of the light chain is as shown in SEQ ID NO: 20, 22 or 24 (light chain constant region is of human origin).

[0052] In a fifth aspect of the present application, there is provided an antibody, which has:

[0053] (1) the heavy chain variable region of the first aspect of the present application; and / or

[0054] (2) the light chain variable region of the third aspect of the present application;

[0055] Alternatively, the antibody has: the heavy chain of the second aspect of the present application; and / or the light chain of the fourth aspect of the present application.

[0056] In another preferred embodiment, the antibody is selected from the group consisting of: an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.

[0057] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0058] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, a sheep, a rabbit.

[0059] In another preferred embodiment, the antibody is a diabody, or a single chain antibody.

[0060] In another preferred embodiment, the antibody is a monoclonal antibody.

[0061] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0062] In another preferred embodiment, the antibody is an IgG1 isotype antibody.

[0063] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40%, preferably 20%, more preferably 10% of the total number of amino acids in the original amino acid sequence.

[0064] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-7, preferably 1-3, more preferably 1.

[0065] In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and / or substituted is an amino acid sequence having at least 80% homology.

[0066] In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and / or substituted has a function of inhibiting the activity of the CD19 antibody.

[0067] In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and / or substituted has an affinity EC50 of 0.2-0.3 nM, preferably 0.01-10 nM, more preferably 0.001-100 nM for the CD19 antibody.

[0068] In another preferred embodiment, the antibody of the anti-CD19 antibody can be combined with a CAR and a CAR-T cell targeting CD19.

[0069] In another preferred embodiment, the antibody of the anti-CD19 antibody can inhibit the function of a CAR and a CAR-T cell targeting CD19.

[0070] In another preferred embodiment, the CD19 antibody comprises FMC63, humanized FMC63.

[0071] In another preferred embodiment, the CD19 antibody comprises FMC63 and a derivative sequence thereof, which is obtained by adding, deleting, modifying and / or substituting at least one amino acid based on the amino acid sequence of FMC63.

[0072] In another preferred embodiment, the FMC63 derivative sequence retains the binding affinity to CD19.

[0073] In another preferred embodiment, the CD19 antibody comprises a CD19 diabody or a CD19 single-chain antibody.

[0074] In a sixth aspect of the present application, a recombinant protein is provided, which has:

[0075] (i) the heavy chain variable region according to the first aspect of the present application, the heavy chain according to the second aspect of the present application, the light chain variable region according to the third aspect of the present application, the light chain according to the fourth aspect of the present application, or the antibody according to the fifth aspect of the present application; and

[0076] (ii) an optional tag sequence for assisting expression and / or purification.

[0077] In another preferred embodiment, the tag sequence comprises a 6His tag.

[0078] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0079] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0080] In a seventh aspect of the present application, a CAR construct is provided, wherein the scFv segment of the antigen binding domain of the CAR construct specifically binds to the binding region of a CD19 antibody, and the scFv has the heavy chain variable region according to the first aspect of the present application and the light chain variable region according to the third aspect of the present application.

[0081] In an eighth aspect of the present application, a recombinant immune cell is provided, which expresses an exogenous CAR construct according to the seventh aspect of the present application.

[0082] In a ninth aspect of the present application, an antibody drug conjugate is provided, which comprises:

[0083] (a) an antibody moiety selected from the group consisting of the heavy chain variable region of the first aspect of the application, the heavy chain of the second aspect of the application, the light chain variable region of the third aspect of the application, the light chain of the fourth aspect of the application, or the antibody of the fifth aspect of the application, or a combination thereof; and

[0084] (b) a conjugated moiety conjugated to the antibody moiety, the conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0085] In another preferred embodiment, the antibody moiety is conjugated to the conjugated moiety via a chemical bond or a linker.

[0086] In a tenth aspect of the application, there is provided a use of an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the application, the heavy chain of the second aspect of the application, the light chain variable region of the third aspect of the application, the light chain of the fourth aspect of the application, or the antibody of the fifth aspect of the application, the recombinant protein of the sixth aspect of the application, the immune cell of the eighth aspect of the application, the antibody drug conjugate of the ninth aspect of the application, or a combination thereof, for (a) preparing a detection reagent, a detection plate or a kit; and / or (b) preparing a medicament for preventing and / or treating a CD19 related disease.

[0087] In another preferred embodiment, the detection reagent, the detection plate or the kit is used for:

[0088] (1) detecting a CD19 antibody in a sample; and / or

[0089] (2) detecting a CD19 antibody on the surface of an immune cell; and / or

[0090] (3) detecting an immune cell expressing a CD19 antibody.

[0091] In another preferred embodiment, the detection reagent, the detection plate or the kit is used for detecting an immune cell expressing a CAR targeting CD19.

[0092] In another preferred embodiment, the immune cell is selected from the group consisting of an NK cell, a T cell, a B cell.

[0093] In another preferred embodiment, the immune cell is from a human or a non-human mammal (e.g. murine).

[0094] In another preferred embodiment, the detection reagent, detection plate or kit is used for ELISA detection, FACS detection, electrochemiluminescence detection, enzyme-linked immunoassay. Among them, mAb06 (26E7D2) and mAb023 (75H6C7) have the best affinity in ELISA detection, and mAb020 (52H8C4) has the best affinity in FACS detection.

[0095] In another preferred embodiment, the detection reagent is a positive antibody for ADA detection, a standard.

[0096] In another preferred embodiment, the drug is used to clear CD19 antibodies or immune cells expressing CAR targeting CD19.

[0097] In another preferred embodiment, the drug is used to block the function of CD19 antibodies or immune cells expressing CAR targeting CD19. Among them, mAb019, mAb020 and mAb021 have the best blocking effect.

[0098] In another preferred embodiment, the drug is used to assist in the treatment of CD19-related diseases.

[0099] In another preferred embodiment, the CD19-related disease is selected from the group consisting of cancer, autoimmune disease, metabolism-related disease, infectious disease, or a combination thereof.

[0100] In another preferred embodiment, the cancer includes solid tumors and blood cancer.

[0101] In another preferred embodiment, the cancer is a CD19-high tumor.

[0102] In another preferred embodiment, the CD19-high tumor refers to the ratio of the level L1 of CD19 transcripts and / or proteins in tumor tissue to the level L0 of transcripts and / or proteins in normal tissue, L1 / L0≥2, preferably ≥3.

[0103] In another preferred embodiment, the metabolism-related disease includes diabetes, food-derived obesity and fat inflammation.

[0104] In another preferred embodiment, the infectious disease includes bacterial and viral infections.

[0105] In a eleventh aspect of the present application, a pharmaceutical composition is provided, which comprises:

[0106] (i) an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the present application, the heavy chain of the second aspect of the present application, the light chain variable region of the third aspect of the present application, the light chain of the fourth aspect of the present application, or the antibody of the fifth aspect of the present application, the recombinant protein of the sixth aspect of the present application, the immune cell of the seventh aspect of the present application, the antibody drug conjugate of the ninth aspect of the present application, or a combination thereof; and

[0107] (ii) a pharmaceutically acceptable carrier.

[0108] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0109] In another preferred embodiment, the pharmaceutical composition is an injection.

[0110] In a twelfth aspect of the present application, a polynucleotide encoding a polypeptide selected from the group consisting of:

[0111] (1) the heavy chain variable region of the first aspect of the present application, the heavy chain of the second aspect of the present application, the light chain variable region of the third aspect of the present application, the light chain of the fourth aspect of the present application, or the antibody of the fifth aspect of the present application; or

[0112] (2) the recombinant protein of the sixth aspect of the present application;

[0113] (3) the CAR construct of the seventh aspect of the present application.

[0114] In a thirteenth aspect of the present application, a vector comprising the polynucleotide of the twelfth aspect of the present application is provided.

[0115] In another preferred embodiment, the vector comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0116] In a fourteenth aspect of the present application, a genetically engineered host cell comprising the vector of the thirteenth aspect of the present application or the polynucleotide of the twelfth aspect of the present application integrated into the genome of the host cell is provided.

[0117] In a fifteenth aspect of the present application, a method for detecting (including diagnosing or non-diagnosing) CD19 antibody in a sample in vitro is provided, the method comprising the steps of:

[0118] (1) contacting the sample with the antibody of the fifth aspect of the present application in vitro;

[0119] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD19 antibody in the sample.

[0120] In a sixteenth aspect of the present application, a detection plate is provided, which comprises a substrate (support plate) and a test strip, wherein the test strip comprises the antibody of the fifth aspect of the present application or the immunoconjugate of the ninth aspect of the present application.

[0121] In a seventeenth aspect of the present application, a kit is provided, which comprises:

[0122] (1) a first container, wherein the first container comprises the antibody of the fifth aspect of the present application; and / or

[0123] (2) a second container, wherein the second container comprises a secondary antibody against the antibody of the fifth aspect of the present application.

[0124] Alternatively, the kit comprises the detection plate of the sixteenth aspect of the present application.

[0125] In an eighteenth aspect of the present application, a method for preparing a recombinant polypeptide is provided, which comprises:

[0126] (a) culturing the host cell of the fourteenth aspect of the present application under conditions suitable for expression;

[0127] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody of the fifth aspect of the present application or the recombinant protein of the sixth aspect of the present application.

[0128] In a nineteenth aspect of the present application, a method for eliminating CD19 antibody or immune cells expressing CAR targeting CD19 is provided, which comprises administering the antibody of the fifth aspect of the present application, antibody-drug conjugate of the antibody, or CAR-T cells expressing the antibody, or a combination thereof to a subject in need.

[0129] In another preferred embodiment, the method further comprises administering other drugs or therapeutic methods for combination therapy to a subject in need.

[0130] In another preferred embodiment, the other drugs or therapeutic methods comprise anti-tumor immunotherapy drugs, tumor targeting drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0131] In a twentieth aspect of the present application, a method for preparing a chimeric antibody is provided, which comprises the steps of:

[0132] After cloning the nucleotide sequence of the heavy chain variable region of the first aspect of the present application and / or the nucleotide sequence of the light chain variable region of the third aspect of the present application into an expression vector comprising the nucleotide sequence of the constant region of human antibody, a human-mouse chimeric antibody is expressed by transfecting animal cells.

[0133] In a twenty-first aspect of the present application, there is provided a method for preparing a humanized antibody, comprising the steps of:

[0134] The nucleotide sequence of the CDR region in the heavy chain variable region of the first aspect of the present application and / or the light chain variable region of the third aspect of the present application is implanted into a nucleotide sequence template containing the FR region of a human antibody, and then cloned into an expression vector containing the constant region of a human antibody, and the humanized antibody is expressed by transfecting animal cells.

[0135] In a twenty-second aspect of the present application, there is provided a bispecific antibody, comprising: the antibody of the fifth aspect of the present application and a second antibody selected from the group consisting of CD3, CD47, PD-1, PD-L1 antibodies, and anti-BCMA, CD20, CD22, CD33, CD123 antibodies.

[0136] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0137] Figure 1 ELISA test results showing the binding of mouse serum to FMC63. In the figure, the four-digit number (7267-7274) represents the mouse number, and TB3 represents that the mouse was immunized for 3 times,

[0138] Figure 2 ELISA test results showing the affinity (EC50) of anti-FMC63 antibodies.

[0139] Figure 3 FACS test results showing the affinity of anti-FMC63 antibodies.

[0140] Figure 4 Test results showing the combination of anti-FMC63 antibodies applied to the detection of anti-antibody method.

[0141] Figure 5 Test results showing that anti-FMC63 antibodies can block the binding of CD19 CAR-T cells to CD19 targets, wherein the abscissa represents the mAb number.

[0142] Figure 6 Test results showing that anti-FMC63 antibodies can promote the proliferation of CD19 CAR-T cells.

[0143] Figure 7 Test results showing that anti-FMC63 antibodies can promote the release of cytokines by CD19 CAR-T cells.

[0144] Figure 8 It is shown that anti-FMC63 antibody can activate CD19 CAR-T cells.

[0145] Figure 9 It is shown that CAR positive rate of CAR-T cells targeting FMC63.

[0146] Figure 10 It is shown that CAR-T cells targeting FMC63 can kill cells expressing target antigen.

[0147] Figure 11 It is shown that CAR-T cells targeting FMC63 can release cytokines under stimulation of cells expressing target antigen.

[0148] Figure 12 It is shown that CAR-T cells targeting FMC63 can be activated by cells expressing target antigen.

[0149] Note, NT in the figure represents untreated T cells. DETAILED DESCRIPTION

[0150] The present inventors have made extensive and in-depth studies and for the first time accidentally discovered an antibody of humanized anti-CD19 antibody. Specifically, the present application uses a synthetic CD19 antibody (scFv) to immunize mice, prepares hybridoma cells from the spleen of the mice, screens to obtain an antibody of murine anti-CD19 antibody, and obtains an antibody of humanized anti-CD19 antibody by replacing a human Fc segment. The antibody of humanized anti-CD19 antibody can be applied to detect CD19 antibody, prepare an antibody drug, and prepare CAR-T cells targeting CD19 antibody.

[0151] TERMS

[0152] In the present application, "VH" refers to a heavy chain variable region, and "VL" refers to a light chain variable region. "VH-CDR1" refers to CDR1 of the heavy chain variable region; "VH-CDR2" refers to CDR2 of the heavy chain variable region; "VH-CDR3" refers to CDR3 of the heavy chain variable region. "VL-CDR1" refers to CDR1 of the light chain variable region; "VL-CDR2" refers to CDR2 of the light chain variable region; "VL-CDR3" refers to CDR3 of the light chain variable region.

[0153] FMC63

[0154] FMC63 is a murine anti-CD19 IgG2a antibody. CAR-T cell drugs designed using FMC63 have been successfully marketed and have good efficacy in treating B-ALL and NHL.

[0155] The FMC63 scFv amino acid sequence is as follows:

[0156]

[0157] Antibody

[0158] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0159] As used herein, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ sequentially, contributing to the binding and specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly bound together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0160] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0161] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy and light chains, referred to as the complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs), the amino acid sequences of which are relatively conserved and do not directly participate in binding interactions. The CDRs form loops or "hot spots" on the surface of the variable region, which are brought together through the beta sheets formed by the FRs in a spatial structure in which the CDRs on the heavy chain and the corresponding CDRs on the light chain form the antigen binding site of the antibody. It is possible to determine which amino acids form the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

[0162] The present application includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Therefore, the present application also includes fragments, derivatives and analogs of the antibodies.

[0163] In the present application, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which include both human and non-human portions, can be obtained by standard DNA recombination techniques, and are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as chimeric antibodies having a variable region from a murine monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Patent 4,816,567 and U.S. Patent 4,816,397, both of which are incorporated herein by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more complementarity determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0164] In the present application, antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0165] In the present application, the antibodies of the present application also include conservative variants thereof, which refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced with amino acids of similar or similar properties compared to the amino acid sequence of the antibodies of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table A.

[0166] Table A

[0167] Original Residue Representative Substitution Preferred Substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0168] Antibodies of the Anti-CD19 Antibody

[0169] In the present invention, the antibody is an antibody against a CD19 antibody, which includes FMC63 and its derivative sequences. The antibody of the present invention includes a heavy chain containing a heavy chain variable region (VH) amino acid sequence and a light chain containing a light chain variable region (VL) amino acid sequence.

[0170] Preferably,

[0171] The heavy chain variable region (VH) has a complementarity determining region CDR selected from the group consisting of:

[0172] CDR1 as shown in SEQ ID NO: 1,

[0173] CDR2 as shown in SEQ ID NO: 2, and

[0174] CDR3 as shown in SEQ ID NO: 3;

[0175] Alternatively,

[0176] CDR1 of SEQ ID NO: 4,

[0177] CDR2 as shown in SEQ ID NO: 5, and

[0178] CDR3 as shown in SEQ ID NO: 6,

[0179] Alternatively,

[0180] CDR1 as shown in SEQ ID NO: 7,

[0181] CDR2 as shown in SEQ ID NO: 8, and

[0182] CDR3 as shown in SEQ ID NO: 9;

[0183] The light chain variable region (VL) has a complementarity determining region CDR selected from the group consisting of:

[0184] CDR1' as shown in SEQ ID NO: 10,

[0185] CDR2' as shown in SEQ ID NO: 11, and

[0186] CDR3' as shown in SEQ ID NO: 12;

[0187] Alternatively,

[0188] CDR1' as shown in SEQ ID NO: 13,

[0189] CDR2' as shown in SEQ ID NO: 14, and

[0190] CDR3' as shown in SEQ ID NO: 15;

[0191] or,

[0192] CDR1' as shown in SEQ ID NO: 16,

[0193] CDR2' as shown in SEQ ID NO: 17, and

[0194] CDR3' as shown in SEQ ID NO: 18;

[0195] wherein any one of the above amino acid sequences further comprises a derivative sequence which optionally has at least one amino acid added to, deleted from, modified in, and / or substituted by, and which is capable of retaining the binding affinity to the CD19 antibody.

[0196] In another preferred embodiment, the sequence formed by the addition, deletion, modification and / or substitution of at least one amino acid sequence is preferably an amino acid sequence having at least 80% homology or sequence identity, more preferably at least 85% homology or sequence identity, even more preferably at least 90% homology or sequence identity, and most preferably at least 95% homology or sequence identity.

[0197] Methods well known to those of ordinary skill in the art for determining sequence homology or identity include, but are not limited to, Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M., and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods of determining identity are those that give the largest match between the sequences tested. Methods of determining identity are codified in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0198] The antibodies of the present application can be selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, more preferably a fully humanized antibody.

[0199] Preferably, the animal is a mammal, such as a mouse.

[0200] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0201] The antibodies of this invention can be chimeric antibodies targeting CD19, humanized antibodies, CDR-grafted and / or modified antibodies.

[0202] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:19, 21 or 23.

[0203] In another preferred embodiment, the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:20, 22 or 24.

[0204] The antibodies of this invention can be one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibodies can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0205] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0206] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.

[0207] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, comprising an Fd segment of a light chain variable region, a light chain constant region, and a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0208] The single domain antibody is a single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0209] The single domain antibody is a single domain antibody in the art, which only includes a heavy chain variable region.

[0210] The preparation method of the recombinant protein is a conventional preparation method in the art. Preferably, the preparation method is: obtained from an expression transformant which expresses the protein or obtained by artificially synthesizing a protein sequence. The method for obtaining from the expression transformant which expresses the protein is preferably as follows: a nucleic acid molecule encoding the protein and carrying a point mutation is cloned into a recombinant vector, the obtained recombinant vector is transformed into a transformant to obtain a recombinant expression transformant, and the recombinant protein is obtained by culturing the obtained recombinant expression transformant.

[0211] Nucleic acid

[0212] The present application also provides a nucleic acid encoding the antibody or the recombinant protein or the heavy chain variable region or the light chain variable region of the antibody of the anti-CD19 antibody.

[0213] The preparation method of the nucleic acid is a conventional preparation method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology or obtaining a nucleic acid molecule encoding the above-mentioned protein by a method of artificially synthesizing a full sequence.

[0214] It is known to those skilled in the art that the base sequence of the amino acid sequence encoding the above-mentioned protein can be appropriately introduced with substitutions, deletions, changes, insertions or additions to provide a homolog of a polynucleotide. The homolog of the polynucleotide in the present application can be prepared by substituting, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the activity of the antibody.

[0215] Vector

[0216] The present application also provides a recombinant expression vector containing the nucleic acid.

[0217] The recombinant expression vector can be obtained by a conventional method in the art, that is, by connecting the nucleic acid molecule of the present application to various expression vectors. The expression vector is a conventional expression vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule. The vector preferably includes various plasmids, cosmids, bacteriophages or viral vectors, etc.

[0218] The present application also provides a recombinant expression transformant containing the above-mentioned recombinant expression vector.

[0219] The recombinant expression transformant can be prepared by conventional methods, preferably by transforming the recombinant expression vector into a host cell. The host cell can be any conventional host cell that can support the stable replication of the recombinant expression vector and the expression of the nucleic acid carried by the vector. Preferably, the host cell is E. coli TG1 or E. coli BL21 (for expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (for expressing full-length IgG antibody). The recombinant expression transformant of the application can be obtained by transforming the above-mentioned recombinant expression plasmid into a host cell. The transformation method can be any conventional method, preferably chemical transformation, heat shock or electroporation.

[0220] Preparation of antibody

[0221] The DNA molecule of the antibody or fragment thereof of the application can be obtained by conventional techniques, such as PCR amplification or screening of genomic library. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0222] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant method. This is usually achieved by cloning the sequence into a vector, transforming the vector into a cell, and then isolating the sequence from the proliferated host cell by conventional methods.

[0223] In addition, the sequence can be synthesized by artificial synthesis method, especially when the fragment is short. Usually, a long fragment can be obtained by synthesizing a plurality of small fragments and then connecting them.

[0224] At present, the DNA sequence encoding the antibody (or fragment thereof, or derivative thereof) of the application can be obtained by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the application by chemical synthesis.

[0225] The application also relates to a vector comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0226] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.

[0227] Generally, the host cells into which the vector of the application has been introduced are cultured under conditions suitable for expression of the antibodies of the present application. The antibodies of the present application are then purified using standard protein purification methods, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography or affinity chromatography, or any combination of the foregoing.

[0228] The resulting monoclonal antibodies are suitably isolated. For example, the monoclonal antibodies can be tested for binding specificity to the desired antigen by immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibodies, for example, can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0229] The antibodies of the present application can be expressed intracellularly, or on the cell membrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified using various separation methods based on their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0230] Antibody-drug conjugate (ADC)

[0231] The present application also provides an antibody-drug conjugate (ADC) based on the antibody of the present application.

[0232] Typically, the antibody-drug conjugate comprises the antibody, and an effector molecule, which is conjugated, and preferably chemically conjugated, to the antibody. Preferably, the effector molecule is a therapeutically active drug. Further, the effector molecule can be one or more of a toxin protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0233] The antibody of the present application can be conjugated to the effector molecule via a conjugating agent. Examples of the conjugating agent can be any one or several of a non-selective conjugating agent, a conjugating agent using a carboxyl group, a peptide chain, a conjugating agent using a disulfide bond. The non-selective conjugating agent refers to a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde, etc. The conjugating agent using a carboxyl group can be any one or several of a conjugating agent of aconitic anhydride (such as aconitic anhydride), a conjugating agent of acylhydrazone (conjugation site: acylhydrazone).

[0234] Certain residues on antibodies, such as Cys or Lys, are used to attach a variety of functional groups, including imaging agents (e.g., chromophoric and fluorescent groups), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection agents, stabilizing agents) are conjugated (covalently linked) to antibodies. Functional agents can be linked to antibodies directly, or indirectly through linkers.

[0235] Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker can be a degradable linker or a non-degradable linker. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, e.g., enzymatically degradable linkers, including peptide-based linkers that are degradable by intracellular proteases (e.g., lysosomal or endosomal proteases), or saccharide linkers, e.g., glucuronide-containing linkers that are degradable by glucuronidases. Peptide-based linkers can include, e.g., dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, e.g., pH-sensitive linkers (e.g., linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that are degradable under reducing conditions (e.g., disulfide linker). Non-degradable linkers are typically susceptible to release of the drug under conditions in which the antibody is hydrolyzed by proteases.

[0236] In some embodiments, an antibody drug conjugate (ADC) is represented by the following formula:

[0237]

[0238] wherein:

[0239] Ab is an antibody,

[0240] LU is a linker;

[0241] D is a drug;

[0242] and subscript p is a value selected from 1 to 8.

[0243] Chimeric antigen receptor T cells

[0244] CAR-T, which stands for Chimeric Antigen Receptor T-Cell, refers to a chimeric antigen receptor T cell, in which the chimeric antigen receptor (CAR) is the core component of CAR-T, which gives T cells the ability to recognize tumor antigens in a HLA-independent manner, which makes CAR-modified T cells able to recognize a wider range of targets compared to natural T cell surface receptors TCR. The basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFv segment of the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region and an intracellular signaling region. The design of CARs has undergone the following process: the first generation of CARs only has one intracellular signaling component CD3 zeta or Fc gamma RI molecule. Since there is only one activation domain in the cell, it can only cause short-term T cell proliferation and less cytokine secretion, and it cannot provide long-term T cell proliferation signals and sustained in vivo anti-tumor effects, so it has not achieved good clinical efficacy. The second generation of CARs introduces a costimulatory molecule such as CD28, 4-1BB, OX40, ICOS into the original structure, which greatly improves the function compared to the first generation of CARs, further enhancing the persistence of CAR-T cells and their ability to kill tumor cells. On the basis of the second generation of CARs, some new immune costimulatory molecules such as CD27, CD134 are linked to develop third and fourth generation CARs.

[0245] Detection purposes and kits

[0246] The antibody or ADC thereof of the present application can be used for detection purposes, for example for detecting a sample to provide diagnostic information.

[0247] In the present application, the sample (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present application should include all kinds of biopsies known to those skilled in the art. Therefore, the biopsy used in the present application can include, for example, a resected sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0248] The sample used in the present application includes fixed or preserved cell or tissue samples.

[0249] The present application also provides a kit comprising the antibody (or fragment thereof) of the present application. In a preferred embodiment of the present application, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present application can be immobilized on a detection plate.

[0250] Pharmaceutical composition

[0251] The present application also provides a composition. In preferred embodiments, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cell described above, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably the pH is about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the disease to be treated.

[0252] The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the administration route of the pharmaceutical composition of the present application is preferably injection administration or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, which can be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.

[0253] The antibody of the present application can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0254] The pharmaceutical composition of the present application can be directly used in combination with CD19 antibody or CAR-T cell targeting CD19, and thus can be used for adjuvant therapy of tumors and the like.

[0255] The pharmaceutical composition of the present application contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the monoclonal antibody (or conjugate thereof) described above and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as needle and solution should be manufactured under sterile conditions. The administration amount of the active ingredient is a therapeutically effective amount, for example, about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0256] In the present application, preferably, the pharmaceutical composition of the present application further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, which can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical adjuvant is a conventional pharmaceutical adjuvant in the art, preferably including pharmaceutically acceptable excipients, fillers or diluents, etc. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of the pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0257] In the present application, preferably, the administration amount of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or injury condition. The effective amount can be determined on an individual basis and will be based in part on the consideration of the symptoms to be treated and the results sought. The skilled person can determine the effective amount by using the above-mentioned factors such as individual basis and using no more than routine experiments.

[0258] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dose should also take into account the administration route, the patient's health status, etc., which are within the skill of a skilled physician.

[0259] The main advantages of the present application include:

[0260] (a) The antibody of the present application can be used for detecting CD19 antibody, such as for ELISA detection, FACS detection, etc.

[0261] (b) The antibody of the present application can block the function of CD19 antibody, such as FMC63.

[0262] (c) The antibody of the present application binds to CAR-T targeting CD19, thereby blocking the function of CAR-T, inhibiting the occurrence and development of cytokine storm, neurotoxicity and other CAR-T related side effects.

[0263] The present application is further described in conjunction with the following specific examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, unless otherwise indicated, are generally performed according to conventional methods, e.g., as set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as otherwise described by the manufacturer. Unless otherwise indicated, percentages and parts are percentages by weight and parts by weight.

[0264] Unless otherwise indicated, all vectors, cells, reagents, etc. in the examples are routinely commercially available. The nucleotide sequences involved in the examples (FMC63 scFv CAR, FMC63-scFv-mFc) are all synthesized by a third-party gene synthesis company,

[0265] Example 1 Construction of monoclonal cells expressing FMC63 scFv CAR (Jurkat-FMC63 scFv)

[0266] The nucleotide sequence of FMC63 scFv CAR (GMCSF signal peptide-FMC63 scFv-Flag-CD28 hinge-CD28 transmembrane region-CD28 costimulatory region-CD3z) was integrated into a lentiviral vector, the FMC63 scFv CAR lentiviral vector was packaged, Jurkat cells were infected, and monoclonal Jurkat-FMC63 scFv cells stably expressing FMC63 scFv CAR were obtained by screening. Monoclonal cells were identified using flow cytometry.

[0267] The results show that monoclonal cells expressing FMC63 scFv CAR (Jurkat-FMC63 scFv cells) were successfully obtained.

[0268] Example 2 Immunization of mice with FMC63-scFv-mFc

[0269] The nucleotide sequence of FMC63-scFv-mFc (one chain of FMC63 antibody) was integrated into a eukaryotic expression vector, the expression vector carrying FMC63 nucleotides was transferred into 293 cells using PEI, and the FMC63 protein was expressed. The FMC63 protein (FMC63-scFv-mFc protein) was purified using a protein A column.

[0270] Mice were immunized according to the method shown in Table 1 below.

[0271] Table 1 Mouse immunization method

[0272] Day 0 1 x 10 7 Jurkat-FMC63 scFv cells IP injected mice Day 14 1 x 10 7 Jurkat-FMC63 scFv cells IP injected mice <!-- 15 -->]]> Day 35 1 x 10 7 Jurkat-FMC63 scFv cells IP injected mice Day 56 25 ug FMC63-scFv-mFc protein IP injected immunized mice

[0273] Results as shown in Figure 1 Figure 1, the serum of mice immunized with Jurkat-FMC63 scFv cells has binding signal with FMC63-scFv-mFc, which indicates that the mice are successfully immunized and can be used for further screening.

[0274] Example 3: Hybridoma acquisition and screening

[0275] The immunized mice were screened by ELISA / FACS, and the spleen lymphocytes of the mice were fused with Sp2 / 0-Ag14 cells to obtain hybridoma cells. The hybridoma cells were plated into 96-well plates for growth, and after 10-14 days, the supernatant was screened by ELISA / FACS for dissociation rate constant, and the subclone with high specificity was selected for expansion culture and cryopreservation.

[0276] The ELISA detection method is as follows: 100 ul of 1 ug / ml FMC63-scFv-mFc is used to coat the plate, and four degrees are incubated overnight; PBST is washed three times, 1% BSA is blocked at 37°C for 1 hour; PBST is washed three times, 50 ul of mAb antibody (40 ug / ml) and biotin-labeled FMC63-scFv-mFc are added to each well; incubate at 37°C for 1 hour, wash with PBST three times; add 100 ul of secondary antibody to each well, incubate at 37°C for 1 hour; wash with PBST five times, add 100 ul of TMB to each well; add 50 ul of 1M HCl to each well to stop the reaction; read the plate at 450 nm wavelength with a microplate reader;

[0277] The FACS detection method is as follows: mix the mAb with Jurkat-FMC63 scFv cells, incubate at room temperature for 30 minutes, wash once with DPBS, add anti-mouse Fc antibody, incubate at room temperature for 30 minutes, wash once with DPBS, and then detect by flow cytometry.

[0278] According to the preliminary ELISA results, the D value is >10, and 103 clones are obtained for secondary screening. The 103 clones are further screened by ELISA and FACS methods, and 23 monoclonal cells expressing antibodies that bind to FMC63 are obtained (corresponding to mAb01-mAb023 in Table 2).

[0279] The ELISA and FACS detection results are shown in Figures 2-3 and Tables 2-4.

[0280] The results show that mAb06 (26E7D2) and mAb023 (75H6C7) have the best effect in ELISA detection, and mAb020 (52H8C4) has the best effect in FACS detection.

[0281] Table 2 ELISA detection of affinity (EC50) values of anti-FMC63 antibodies

[0282] Antibody Clone Number EC50 (ug / ml) Antibody Clone Number EC50 (ug / ml) mAb01 4C5E2 0.03479 mAb013 9H2G1 0.03986 mAb02 8B3H4 0.02298 mAb014 20H5D10 0.0628 mAb03 14A10E7 0.8218 mAb015 22G8F7 53629 mAb04 17F10D3 0.02273 mAb016 40F12E5 0.04888 mAb05 26E2F11 0.0714 mAb017 35H8C4 0.05389 mAb06 26E7D2 0.0299 mAb018 24E8E11 0.08629 mAb07 27C5E11 0.09905 mAb019 66B2A9 0.06981 mAb08 28B6F7 0.07226 mAb020 52H8C4 0.113 mAb09 28E12C3 0.05664 mAb021 47H1B3 0.04839 mAb010 35G7G6 0.059 mAb022 42C10G2 0.1716 mAb011 36A11E3 0.06001 mAb023 75H6C7 0.05115 mAb012 39D4C5 0.05607

[0283] Table 3 FACS detection of affinity (EC50) values of anti-FMC63 antibodies

[0284]

[0285] Table 4 Anti-FMC63 antibodies after ELISA and FACS screening

[0286]

[0287] Example 4 Epitope detection of anti-FMC63 antibodies

[0288] Antibody binding epitope was detected using competitive ELISA. Plates were coated with 100ul of 1ug / ml anti-FMC63 antibodies, incubated overnight, washed three times with PBST, blocked with 1% BSA for 1 hour at 37°C, washed three times with PBST, added 50ul of anti-antibody (40ug / ml) and FMC63-scFv-mFc (EC80) per well, incubated for 1 hour at 37°C, washed three times with PBST, added 100ul of secondary antibody per well, incubated for 1 hour at 37°C, washed five times with PBST, added 100ul of TMB per well, added 50ul of 1M HCl per well to stop the reaction, and read the plate at 450nm wavelength using a microplate reader.

[0289] Antibodies binding different epitopes of FMC63 were classified, and the results are shown in Table 5.

[0290] Table 5 Epitope classification of anti-FMC63 antibodies

[0291]

[0292] Example 5 Monoclonal sequencing

[0293] Monoclonal cells mAb02 (8B3H4), mAb06 (26E7D2), mAb07 (27C5E11), mAb020 (52H8C4), and mAb023 (75H6C7) were selected for DNA sequencing. The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibodies obtained after sequencing are shown in Table 6, which also shows the CDR regions obtained after sequencing analysis of the antibodies.

[0294] Table 6 CDR region sequences and heavy chain and light chain variable region sequences of mAb antibodies

[0295]

[0296] Example 7 Anti-FMC63 antibody application to anti-antibody detection method

[0297] The antibody combination (mAb01, mAb02, mAb07, mAb10, mAb011, mAb021, mAb022, mAb023) binding to different epitopes of FMC63 were mixed at different concentrations and used as positive control (PC) to design the anti-antibody detection experiment. 300ul blocking solution was added to the MSD plate for 2 hours of blocking. The mixed PC, biotin-FMC63-scFv and sulfo-tag-FMC63-scFv were mixed at certain concentrations and 100ul of the mixture was added to another plate for 1 hour of incubation. The blocking solution in the MSD plate was discarded and the plate was washed 3 times. The incubated mixture and 50ul per well of volume were transferred to the MSD plate for 1 hour of incubation. The mixture in the MSD plate was discarded and the plate was washed 3 times. 150ul of 2x read buffer was added per well for plate reading.

[0298] The results are shown in Table 7 and Figure 6. Figure 4 The MSD detection results showed that the anti-FMC63 antibodies could bind to FMC63-scFv in a dose-dependent manner, indicating that the anti-FMC63 antibodies could be applied to the MSD method.

[0299] Table 7 Anti-FMC63 antibody combination application to MSD method detection raw data

[0300]

[0301]

[0302] Example 8 Function detection of antibodies

[0303] The 23 antibodies shown in Table 2 were mixed with biotin-labeled CD19 protein, added to Jurkat-FMC63scFv cells for 1 hour of incubation, washed once with DPBS, added with streptavidin-APC for 30 minutes of incubation, washed once with DPBS and then detected by flow cytometry to detect CD19 protein binding.

[0304] The 23 antibodies shown in Table 2 were co-incubated with FMC63-CAR-T cells for 1 hour. The incubated FMC63-CAR-T cells were added to RTCA plates coated with Hela-CD19. The change in the killing function of CAR-T cells to target cells after binding with antibodies was observed to screen for antibodies that can block the function of FMC63-CAR-T cells.

[0305] The 23 antibodies shown in Table 2 were added to FMC63-CAR-T cells. The density and number of FMC63-CAR-T cells were detected at different time points. The results are shown in Table 8 and Figure 7. Figure 6 ,7 , 8 shows that anti-FMC63 antibody can promote the proliferation, activation and cytokine release of FMC63-CAR-T cells.

[0306] The results are shown in Figure 5 mAb019 and mAb020 can interfere with the binding of CD19 protein to Jurkat-FMC63 scFv cells, thereby blocking the function of CAR binding to target CD19.

[0307] Preparation of humanized anti-FMC63 antibody

[0308] The Fc segment of murine anti-FMC63 antibodies mAb02, mAb06 and mAb020 was replaced with human Fc to obtain human-murine chimeric antibodies. The heavy chain amino acid sequences of humanized mAb02, mAb06 and mAb020 antibodies are respectively SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and the light chain amino acid sequences are respectively SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24.

[0309] Preparation of CAR-T targeting CD19 antibody

[0310] CAR-T was prepared using anti-FMC63 antibody mAb02, mAb06, mAb07, mAb020 and mAb023, the structure is signal peptide-anti-FMC63 scFv-CD8 hinge-CD8 transmembrane region-CD137 costimulatory region-CD3z, and the amino acid sequences are respectively SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49.

[0311] The specific method is as follows:

[0312] Cell recovery: take PBMC in 37℃ water bath pot about 2min, transfer the cell suspension to 30mL preheated CAR-T basic culture medium (X-vivo15 containing 1% HSA), mix well, take sample count, the remaining cells 300g centrifugal 10 minutes.

[0313] T cell inoculation and activation

[0314] After sorting, centrifuge 300 g of positive cells at room temperature for 8 min, discard the supernatant, resuspend the cell pellet with an appropriate amount of CAR-T base medium. Add an appropriate volume of CD3 / CD28 dynabeads according to the ratio of 1:1 of CD3 / CD28 dynabeads to cell number, wash the magnetic beads with an equal volume of CAR-T base medium, place them on a DynaMag 50 for 1 min, discard the supernatant, resuspend the magnetic beads with 1-5 mL of CAR-T base medium, and transfer them to the cell suspension, mix well, add an appropriate amount of CAR-T base medium to make the cell density reach 2x10 6 / mL, and add 1 / 1000 volume of IMC0003. After mixing, take 2-3 mL of cell suspension in a 6-well plate as negative control cells, and the remaining cells are used for CAR-T cell preparation.

[0315] T cell virus infection (Day 1)

[0316] Take out the cells, mix well, take a sample for counting, and calculate the cell amount according to the cell volume, centrifuge 300 g of cell suspension for 8 min. Calculate the inoculation volume according to the density of 2x10 6 / mL, and calculate the lentivirus vector volume according to the formula "lentivirus vector volume = MOI*cell amount / biological titer". Discard the supernatant, resuspend the cell pellet with an appropriate CAR-T base medium (volume = inoculation volume-lentivirus vector volume), add 1 / 1000 inoculation volume of IL2, and add the recovered virus to the cell suspension for infection.

[0317] CAR-T cell expansion culture (Day 8)

[0318] CAR-T cells, after blowing and mixing, take a sample for counting, take about 0.5x10 6 cells for positive rate detection, and continue to culture with the addition of CAR-T base medium. The CAR+ positive rate was detected using anti-Flag antibody, which contains the DYKDDDDK amino acid sequence in the CAR extracellular structure. The results are shown in Figure 9 , indicating that CAR is successfully expressed on T cells.

[0319] CAR-T cell toxicity detection

[0320] CAR-T and target cells (Hela-FMC63, K562-FMC63) were incubated according to the ratio of 1:1; real-time label-free dynamic cell analysis technology was used to detect the activity of target cells, and the activation and cytotoxic effect of CAR-T were obtained. At the same time, the cell supernatant was detected for cytokine release, including IL2 and IFNγ.

[0321] The results show that CAR-T targeting CD19 antibody can produce cytotoxic effect on target cells Hela-FMC63, as shown inFigure 10 CAR-T targeting CD19 antibody can be activated by target cell K562-FMC63 and release cytokines IFNy and IL2, as Figure 11 and Figure 12 .

[0322] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the present application upon reading and understanding the above lecture of the present application, and it is intended that all such alterations and modifications fall within the scope of the appended claims. SEQUENCE LISTING <110> Genext Biosciences (Shanghai) Co., Ltd. <120> An antibody against CD19 antibody and preparation and application thereof <130> P2022-1938 <150> CN202010383916.5 <151> 2020-05-08 <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Mus musculus <400> 1 Thr Tyr Trp Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Mus musculus <400> 2 Met Ile His Pro Asn Ser Asp Ser Ile Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 3 <211> 13 <212> PRT <213> Mus musculus <400> 3 Thr Asp Gly Tyr Tyr Ala Pro Tyr Phe Ala Met Asp Tyr 1 5 10 <210> 4 <211> 5 <212> PRT <213> Mus musculus <400> 4 Ser Tyr Trp Ile His 1 5 <210> 5 <211> 17 <212> PRT <213> Mus musculus <400> 5 Arg Ile Asp Pro Ile Ser Gly Gly Ala Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Thr <210> 6 <211> 6 <212> PRT <213> Mus musculus <400> 6 Trp Gly Gly Gly Asp Tyr 1 5 <210> 7 <211> 5 <212> PRT <213> Mus musculus <400> 7 Ser Tyr Thr Met Ser 1 5 <210> 8 <211> 17 <212> PRT <213> Mus musculus <400> 8 Thr Ile Thr Gly Gly Gly Gly Asp Thr Phe Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 12 <212> PRT <213> Mus musculus <400> 9 Gln Tyr Tyr Asp Tyr Asp Gly Gly Ala Met Asp Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> Mus musculus <400> 10 Arg Ala Ser Gln Asp Ile Arg Asp Asn Leu Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> Mus musculus <400> 11 Tyr Lys Ser Arg Ser His Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Mus musculus <400> 12 Gln Gln Gly Asn Thr Leu Pro Phe Thr 1 5 <210> 13 <211> 16 <212> PRT <213> Mus musculus <400> 13 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Arg Thr Tyr Leu Asn 1 5 10 15 <210> 14 <211> 7 <212> PRT <213> Mus musculus <400> 14 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 15 <211> 9 <212> PRT <213> Mus musculus <400> 15 Trp Gln Gly Thr His Phe Pro Leu Thr 1 5 <210> 16 <211> 15 <212> PRT <213> Mus musculus <400> 16 Lys Ala Ser Gln Ser Val Ser Phe Ala Gly Thr Gly Leu Met His 1 5 10 15 <210> 17 <211> 7 <212> PRT <213> Mus musculus <400> 17 Arg Ala Ser Asn Leu Glu Ala 1 5 <210> 18 <211> 9 <212> PRT <213> Mus musculus <400> 18 Gln Gln Ser Arg Glu Tyr Pro Arg Thr 1 5 <210> 19 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of humanized mAb 02 <400> 19 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Val Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Thr Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Met Ile His Pro Asn Ser Asp Ser Ile Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Ser Lys Ala Thr Leu Ile Val Asp Lys Ser Ser Ser 85 90 95 Thr Phe Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Thr Asp Gly Tyr Tyr Ala Pro Tyr Phe Ala Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 20 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of humanized mAb02 <400> 20 Met Asp Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys Asp Ile Gin Met Thr Gin Thr Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Arg Asp Asn Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr Tyr Lys Ser Arg Ser His Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gin Glu Asp Ile Ala Thr Tyr Phe Cys Gin Gin 100 105 110 Gly Asn Thr Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 21 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of humanized mAb06 <400> 21 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Val Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu 50 55 60 Glu Trp Ile Gly Arg Ile Asp Pro Ile Ser Gly Gly Ala Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Gly Gly Asp Tyr Trp Gly His Gly Thr 115 120 125 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170 175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 225 230 235 240 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 245 250 255 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295 300 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 305 310 315 320 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 355 360 365 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 370 375 380 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 385 390 395 400 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410 415 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 420 425 430 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 22 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of humanized mAb06 <400> 22 Met Asp Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys Asp Val Val Met Thr Gin Thr Pro Leu Thr 20 25 30 Leu Ser Val Thr Ile Gly Gin Pro Ala Ser Ile Ser Cys Lys Ser Ser 35 40 45 Gln Ser Leu Leu Asp Ser Asp Gly Arg Thr Tyr Leu Asn Trp Phe Leu 50 55 60 Gln Arg Pro Gly Gin Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys 65 70 75 80 Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 100 105 110 Tyr Tyr Cys Trp Gin Gly Thr His Phe Pro Leu Thr Phe Gly Ala Gly 115 120 125 Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 130 135 140 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 145 150 155 160 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 165 170 175 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 195 200 205 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 210 215 220 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 225 230 235 240 Cys <210> twenty three <211> 470 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of humanized mAb020 <400> twenty three Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Thr Ile Thr Gly Gly Gly Gly Asp Thr Phe Tyr Pro 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr His Cys Ala Arg Gln Tyr Tyr Asp Tyr Asp Gly Gly Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu 355 360 365 Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> 24 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of humanized mAb020 <400> 24 Met Asp Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys Asp Ile Val Leu Thr Gin Ser Pro Ala Ser 20 25 30 Leu Ala Val Ser Leu Gly Gin Arg Ala Ile Ile Ser Cys Lys Ala Ser 35 40 45 Gln Ser Val Ser Phe Ala Gly Thr Gly Leu Met His Trp Tyr Gin Gin 50 55 60 Lys Pro Gly Gin Gin Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu 65 70 75 80 Glu Ala Gly Val Pro Thr Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp 85 90 95 Phe Thr Leu Asn Ile His Pro Val Glu Glu Asp Asp Ala Ala Thr Tyr 100 105 110 Tyr Cys Gin Gin Ser Arg Glu Tyr Pro Arg Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 25 <211> 122 <212> PRT <213> Mouse (Mus musculus) <400> 25 Gln Val Gln Val Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Asp Ser Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Ile Val Asp Lys Ser Ser Ser Thr Phe Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asp Gly Tyr Tyr Ala Pro Tyr Phe Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 26 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 26 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asp Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Lys Ser Arg Ser His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 27 <211> 115 <212> PRT <213> Mouse (Mus musculus) <400> 27 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ile Ser Gly Gly Ala Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Gly Gly Asp Tyr Trp Gly His Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 28 <211> 112 <212> PRT <213> Mouse (Mus musculus) <400> 28 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Arg Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 29 <211> 122 <212> PRT <213> Mouse (Mus musculus) <400> 29 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Gly Asp Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asp Gly Tyr Tyr Ala Pro Tyr Phe Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 30 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 30 Asp Ile Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Leu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Phe 85 90 95 Thr Val Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 31 <211> 121 <212> PRT <213> Mouse (Mus musculus) <400> 31 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 It should be noted that there was a small error in the original text where "PRT" in line should be "PRT" in the correct format. I have corrected it in the translation. Also, the "29" in line was likely a typo in the original, and I have left it as "29" in the translation for consistency. If this was not a typo, please clarify.Ala Thr lie Thr Gly Gly Gly Gly Asp Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Arg Gin Tyr Tyr Asp Tyr Asp Gly Gly Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 32 <211> 111 <212> PRT <213> Mus musculus <400> 32 Asp lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala lie lie Ser Cys Lys Ala Ser Gin Ser Val Ser Phe Ala 20 25 30 Gly Thr Gly Leu Met His Trp Tyr Gin Gin Lys Pro Gly Gin Gin Pro 35 40 45 Lys Leu Leu lie Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Thr 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95 Glu Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 33 <211> 116 <212> PRT <213> Mouse (Mus musculus) <400> 33 Gln Ala Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Met Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Met Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Asp Lys Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Tyr Tyr Gly Met Asp Phe Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 34 <211> 112 <212> PRT <213> Mus musculus <400> 34 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser 20 25 30 Lys Gly Asn Thr Tyr Leu Asn Trp Tyr Val Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Thr Arg Phe Ser Gly Val Leu 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Leu 85 90 95 Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 5 <212> PRT <213> Mus musculus <400> 35 Ser Tyr Trp Met His 1 5 <210> 36 <211> 20 <212> PRT <213> Mus musculus <400> 36 Met Ile His Pro Asn Gly Asp Ser Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Thr Lys Ala Thr 20 <210> 37 <211> 11 <212> PRT <213> Mus musculus <400> 37 Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn 1 5 10 <210> 38 <211> 7 <212> PRT <213> Mus musculus <400> 38 Tyr Thr Ser Arg Leu His Leu 1 5 <210> 39 <211> 5 <212> PRT <213> Mus musculus <400> 39 Asn Tyr Trp Met Asn 1 5 <210> 40 <211> 20 <212> PRT <213> Mus musculus <400> 40 Glu lie Asp Pro Ser Asp Ser Asp Lys Asn Tyr Asn Gin Lys Phe Lys 1 5 10 15 Asp Lys Ala Thr 20 <210> 41 <211> 8 <212> PRT <213> Mus musculus <400> 41 Gly Gly Tyr Tyr Gly Met Asp Phe 1 5 <210> 42 <211> 16 <212> PRT <213> Mus musculus <400> 42 Arg Ser Ser Gin Ser Leu Glu Asn Ser Lys Gly Asn Thr Tyr Leu Asn 1 5 10 15 <210> 43 <211> 6 <212> PRT <213> Mus musculus <400> 43 Arg Val Ser Thr Arg Phe 1 5 <210> 44 <211> 9 <212> PRT <213> Mus musculus <400> 44 Leu Gin Leu Thr His Val Pro Phe Thr 1 5 <210> 45 <211> 761 <212> PRT <213> Artificial Sequence <220> <223> CAR made using mAb 02 <400> 45 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Arg Asp Asn Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr Tyr Lys Ser Arg Ser His Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Gly Asn Thr Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gln Val Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 145 150 155 160 Ser Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 165 170 175 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 180 185 190 Gly Met Ile His Pro Asn Ser Asp Ser Ile Asn Tyr Asn Glu Lys Phe 195 200 205 Lys Ser Lys Ala Thr Leu Ile Val Asp Lys Ser Ser Ser Thr Phe Tyr 210 215 220 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 225 230 235 240 Ala Arg Thr Asp Gly Tyr Tyr Ala Pro Tyr Phe Ala Met Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Asp Tyr Lys 260 265 270 Asp Asp Asp Asp Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 275 280 285 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 290 295 300 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 305 310 315 320 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 325 330 335 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 340 345 350 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 355 360 365 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 370 375 380 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 385 390 395 400 Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 405 410 415 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 420 425 430 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 435 440 445 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 450 455 460 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 465 470 475 480 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 485 490 495 Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln 500 505 510 Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys Gly Glu 515 520 525 Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp 530 535 540 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 545 550 555 560 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 565 570 575 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln 580 585 590 Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys 595 600 605 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys 610 615 620 Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 625 630 635 640 Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 645 650 655 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn 660 665 670 Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe 675 680 685 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His 690 695 700 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp 705 710 715 720 Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 725 730 735 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 740 745 750 Thr Leu Gly Met Asp Glu Leu Tyr Lys 755 760 <210> 46 <211> 759 <212> PRT <213> Artificial Sequence <220> <223> CAR made using mAb03 <400> 46 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Thr 20 25 30 Leu Ser Val Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser 35 40 45 Gln Ser Leu Leu Asp Ser Asp Gly Arg Thr Tyr Leu Asn Trp Phe Leu 50 55 60 Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys 65 70 75 80 Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 100 105 110 Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Leu Thr Phe Gly Ala Gly 115 120 125 Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gin Val Gin Leu Gin Gin Pro Gly Ala Glu Leu 145 150 155 160 Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Val Ser Gly Tyr 165 170 175 Thr Phe Thr Ser Tyr Trp He His Trp Val Lys Gin Arg Pro Gly Arg 180 185 190 Gly Leu Glu Trp He Gly Arg He Asp Pro He Ser Gly Gly Ala Asn 195 200 205 Tyr Asn Glu Lys Phe Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Pro 210 215 220 Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser 225 230 235 240 Ala Val Tyr Tyr Cys Ala Arg Trp Gly Gly Gly Asp Tyr Trp Gly His 245 250 255 Gly Thr Thr Leu Thr Val Ser Ser Ala Ala Ala Asp Tyr Lys Asp Asp 260 265 270 Asp Asp Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 275 280 285 Thr He Ala Ser Gin Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 290 295 300 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 305 310 315 320 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 325 330 335 Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu 340 345 350 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 355 360 365 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 370 375 380 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 385 390 395 400 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 405 410 415 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 420 425 430 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 435 440 445 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 450 455 460 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 465 470 475 480 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 485 490 495 Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly 500 505 510 Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys Gly Glu Glu Leu 515 520 525 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 530 535 540 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 545 550 555 560 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 565 570 575 Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe 580 585 590 Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 595 600 605 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 610 615 620 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 625 630 635 640 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 645 650 655 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr 660 665 670 Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile 675 680 685 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 690 695 700 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 705 710 715 720 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 725 730 735 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu 740 745 750 Gly Met Asp Glu Leu Tyr Lys 755 <210> 47 <211> 761 <212> PRT <213> Artificial Sequence <220> <223> CAR prepared using mAb07 <400> 47 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Ser Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Leu Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Gly Asn Thr Leu Pro Phe Thr Val Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 145 150 155 160 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 165 170 175 Trp Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He 180 185 190 Gly Met He His Pro Asn Gly Asp Ser Thr Asn Tyr Asn Glu Lys Phe 195 200 205 Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 210 215 220 Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 225 230 235 240 Ala Arg Thr Asp Gly Tyr Tyr Ala Pro Tyr Phe Ala Met Asp Tyr Trp 245 250 255 Gly Gin Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Asp Tyr Lys 260 265 270 Asp Asp Asp Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 275 280 285 Ala Pro Thr He Ala Ser Gin Pro Leu Ser Leu Arg Pro Glu Ala Cys 290 295 300 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 305 310 315 320 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 325 330 335 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 340 345 350 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 355 360 365 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 370 375 380 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 385 390 395 400 Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 405 410 415 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 420 425 430 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 435 440 445 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 450 455 460 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 465 470 475 480 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 485 490 495 Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln 500 505 510 Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys Gly Glu 515 520 525 Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp 530 535 540 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 545 550 555 560 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 565 570 575 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln 580 585 590 Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys 595 600 605 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys 610 615 620 Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 625 630 635 640 Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 645 650 655 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn 660 665 670 Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe 675 680 685 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His 690 695 700 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp 705 710 715 720 Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 725 730 735 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 740 745 750 Thr Leu Gly Met Asp Glu Leu Tyr Lys 755 760 <210> 48 <211> 764 <212> PRT <213> Artificial Sequence <220> <223> CAR made using mAb020 <400> 48 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Val Leu Thr Gln Ser Pro Ala Ser 20 25 30 Leu Ala Val Ser Leu Gly Gln Arg Ala Ile Ile Ser Cys Lys Ala Ser 35 40 45 Gln Ser Val Ser Phe Ala Gly Thr Gly Leu Met His Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu 65 70 75 80 Glu Ala Gly Val Pro Thr Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp 85 90 95 Phe Thr Leu Asn Ile His Pro Val Glu Glu Asp Asp Ala Ala Thr Tyr 100 105 110 Tyr Cys Gln Gln Ser Arg Glu Tyr Pro Arg Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val 145 150 155 160 Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 165 170 175 Phe Ser Ser Tyr Thr Met Ser Trp Val Arg Gin Thr Pro Glu Lys Arg 180 185 190 Leu Glu Trp Val Ala Thr lie Thr Gly Gly Gly Gly Asp Thr Phe Tyr 195 200 205 Pro Asp Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys 210 215 220 Asn Thr Leu Tyr Leu Gin Val Ser Ser Leu Arg Ser Glu Asp Thr Ala 225 230 235 240 Val Tyr His Cys Ala Arg Gin Tyr Tyr Asp Tyr Asp Gly Gly Ala Met 245 250 255 Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala 260 265 270 Asp Tyr Lys Asp Asp Asp Asp Lys Thr Thr Thr Pro Ala Pro Arg Pro 275 280 285 Pro Thr Pro Ala Pro Thr lie Ala Ser Gin Pro Leu Ser Leu Arg Pro 290 295 300 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 305 310 315 320 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 325 330 335 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 340 345 350 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 355 360 365 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 370 375 380 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 385 390 395 400 Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 405 410 415 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 420 425 430 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 435 440 445 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 450 455 460 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 465 470 475 480 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 485 490 495 Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu 500 505 510 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser 515 520 525 Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 530 535 540 Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu 545 550 555 560 Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 565 570 575 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr 580 585 590 Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp 595 600 605 Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile 610 615 620 Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe 625 630 635 640 Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe 645 650 655 Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn 660 665 670 Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys 675 680 685 Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu 690 695 700 Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu 705 710 715 720 Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp 725 730 735 Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala 740 745 750 Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 755 760 <210> 49 <211> 760 <212> PRT <213> Artificial Sequence <220> <223> CAR made using mAb023 <400> 49 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser 20 25 30 Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Glu Asn Ser Lys Gly Asn Thr Tyr Leu Asn Trp Tyr Val 50 55 60 Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Val Ser Thr 65 70 75 80 Arg Phe Ser Gly Val Leu Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 100 105 110 Tyr Phe Cys Leu Gln Leu Thr His Val Pro Phe Thr Phe Gly Ser Gly 115 120 125 Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gin Ala Gin Leu Gin Gin Pro Gly Ala Glu Leu 145 150 155 160 Val Met Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Asn Tyr Trp Met Asn Trp Met Lys Gin Arg Pro Gly Gin 180 185 190 Gly Leu Glu Trp lie Gly Glu lie Asp Pro Ser Asp Ser Asp Lys Asn 195 200 205 Tyr Asn Gin Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser 210 215 220 Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu Thr Ser Gin Asp Ser 225 230 235 240 Ala Val Tyr Tyr Cys Ala Gly Gly Tyr Tyr Gly Met Asp Phe Trp Gly 245 250 255 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Asp Tyr Lys Asp 260 265 270 Asp Asp Asp Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 275 280 285 Pro Thr lie Ala Ser Gin Pro Leu Ser Leu Arg Pro Gin Ala Cys Arg 290 295 300 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 305 310 315 320 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 325 330 335 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 340 345 350 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 355 360 365 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 370 375 380 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 385 390 395 400 Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 405 410 415 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 420 425 430 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 435 440 445 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 450 455 460 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 465 470 475 480 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 485 490 495 Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala 500 505 510 Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys Gly Glu Glu 515 520 525 Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val 530 535 540 Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr 545 550 555 560 Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro 565 570 575 Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys 580 585 590 Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser 595 600 605 Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp 610 615 620 Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr 625 630 635 640 Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly 645 650 655 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val 660 665 670 Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys 675 680 685 Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr 690 695 700 Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn 705 710 715 720 His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys 725 730 735 Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 740 745 750 Leu Gly Met Asp Glu Leu Tyr Lys 755 760 <210> 50 <211> 248 <212> PRT <213> Mus musculus <400> 50 Asp Ile Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gin Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gin 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125 Leu Gin Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser 130 135 140 Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 145 150 155 160 Trp Ile Arg Gin Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190 Thr Ile Ile Lys Asp Asn Ser Lys Ser Gin Val Phe Leu Lys Met Asn 195 200 205 Ser Leu Gin Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220 Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Ser 225 230 235 240 Val Thr Val Ser Ser Ala Ala Ala 245

Claims

1. An antibody against an antibody of CD 19, characterized in that, The antibody has a heavy chain variable region and a light chain variable region selected from the group consisting of: (1) the heavy chain variable region comprises the following three complementarity determining regions CDRs: CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9; and the light chain variable region comprises the following three complementarity determining regions CDRs: CDR1' of SEQ ID NO: 16, CDR2' of SEQ ID NO: 17, and CDR3' of SEQ ID NO: 18; (2) the heavy chain variable region comprises the following three complementarity determining regions CDRs: CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6; and the light chain variable region comprises the following three complementarity determining regions CDRs: CDR1' of SEQ ID NO: 13, CDR2' of SEQ ID NO: 14, and CDR3' of SEQ ID NO: 15; (3) the heavy chain variable region comprises the following three complementarity determining regions CDRs: CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; and the light chain variable region comprises the following three complementarity determining regions CDRs: CDR1' of SEQ ID NO: 10, CDR2' of SEQ ID NO: 11, and CDR3' of SEQ ID NO: 12; or, (4) the heavy chain variable region comprises the following three complementarity determining regions CDRs: CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41; and the light chain variable region comprises the following three complementarity determining regions CDRs: CDR1' of SEQ ID NO: 42, CDR2' of SEQ ID NO: 43, and CDR3' of SEQ ID NO: 44; and the CD19 antibody is FMC63, the scFv sequence of which is shown as SEQ ID NO:

50.

2. The antibody of claim 1, wherein The antibody further comprises a FR region of human origin or a FR region of murine origin.

3. The antibody of claim 1, wherein The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO: 31, and the light chain variable region sequence is shown as SEQ ID NO: 32; or the heavy chain variable region amino acid sequence is shown as SEQ ID NO: 27, and the light chain variable region sequence is shown as SEQ ID NO: 28; the heavy chain variable region amino acid sequence is shown as SEQ ID NO: 25, and the light chain variable region sequence is shown as SEQ ID NO: 26; or the heavy chain variable region amino acid sequence is shown as SEQ ID NO: 33, and the light chain variable region sequence is shown as SEQ ID NO:

34.

4. The antibody of claim 1, wherein The heavy chain amino acid sequence of the antibody is set forth in SEQ ID NO: 23, and the light chain sequence is set forth in SEQ ID NO: 24; or The heavy chain amino acid sequence is set forth in SEQ ID NO: 21, and the light chain sequence is set forth in SEQ ID NO: 22; or The heavy chain amino acid sequence is set forth in SEQ ID NO: 19, and the light chain sequence is set forth in SEQ ID NO:

20.

5. The antibody of claim 1, wherein The antibody is a murine antibody or a humanized antibody.

6. A recombinant protein, characterized in that, The recombinant protein has: (i) the antibody of any one of claims 1-5; and (ii) optionally, a tag sequence to facilitate expression and / or purification.

7. An antibody drug conjugate, characterized in that, The antibody drug conjugate contains: (a) an antibody moiety, the antibody moiety comprising the antibody of any one of claims 1-5; and (b) a conjugate moiety coupled to the antibody moiety, the conjugate moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

8. A CAR construct, characterized in that, The antigen binding domain of the CAR construct comprises the heavy chain variable region and the light chain variable region of the antibody of any one of claims 1-5.

9. The CAR construct of claim 8, wherein, The CAR further comprises a hinge region, a transmembrane region, a costimulatory signaling region, and CD3z.

10. An engineered immune cell, characterized in that, The engineered immune cell expresses an exogenous CAR construct of claim 8.

11. Use of an active ingredient, characterized in that The active ingredient is selected from the group consisting of the antibody of any one of claims 1-5, the antibody drug conjugate of claim 7, the engineered immune cell of claim 10, or a combination thereof, for (a) preparing a detection reagent, a detection plate, or a kit; and / or (b) preparing a medicament.

12. The use according to claim 11, characterized in that, The medicament is for neutralizing a CD19 antibody or for blocking a CAR targeting CD19 expressed on a cell.

13. The use according to claim 12, characterized in that, The cell comprises a tumor cell expressing a CAR targeting CD19 or an immune cell.

14. Use according to claim 13, characterized in that, The cell is a tumor B cell expressing a CAR targeting CD19 and a T cell expressing a CAR targeting CD19.

15. A pharmaceutical composition comprising, The pharmaceutical composition contains: (i) an active ingredient selected from the group consisting of the antibody of any one of claims 1-5, the antibody drug conjugate of claim 7, the engineered immune cell of claim 10, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

16. An in vitro method of detecting a CD19 antibody or a cell expressing a CAR targeting CD19 in a sample, the method comprising the steps of: (1) in vitro, contacting the sample with the antibody of any one of claims 1-5; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of the CD19 antibody or the cell expressing the CAR targeting CD19 in the sample.

17. An assay plate comprising: A substrate and a test strip, the test strip containing the antibody of any one of claims 1-5 or the antibody drug conjugate of claim 7.

18. A kit comprising: (1) a first container containing the antibody of any one of claims 1-5; and and (2) a second container containing a secondary antibody against the antibody of any one of claims 1-5; Alternatively, the kit contains a detection plate as described in claim 17.

Images