TW202024135A - Full human anti-cd30 single chain antibody and use thereof - Google Patents

Abstract

The present invention relates to novel antibodies and antibody fragments that specifically bind to CD30, especially fully human single-chain antibodies scFvs. The invention also relates to nucleic acids encoding these antibodies, vectors and host cells expressing said nucleic acids. In addition, the present invention also relates to a composition comprising the antibody of the present invention and its uses in therapy and diagnosis.

Description

Fully human anti-CD30 single chain antibody and its application

The present invention relates to novel antibodies and antibody fragments that specifically bind to CD30, especially single-chain antibodies (such as single-chain scFv). The present invention also relates to nucleic acids encoding these antibodies and antibody fragments, vectors and host cells expressing the nucleic acids. In addition, the present invention also relates to a composition comprising the antibody of the present invention, and its use in therapy and diagnosis.

CD30, also known as Ki-1 or TNFRSF8, is a member of the tumor necrosis factor (TNF) receptor superfamily. As a 105-120kD type I transmembrane glycoprotein receptor, CD30 has an intracellular domain, a transmembrane domain, and an extracellular domain, and there are multiple cysteine-rich regions in the extracellular domain. This extracellular domain can be fragmented to produce 88kD soluble form of CD30 (sCD30), which can be released into serum, and can be used in patients with autoimmune diseases such as rheumatoid arthritis, inflammatory states, and CD30-positive hematological malignancies Increased serum CD30 was detected in. Pierce JM et al., Expert Rev.Hematol.2017 Jan; 10(1):29-37.doi:10.1080/17474086.2017.1270202.Epub 2016 Dec 21.

In healthy individuals, CD30 expression is restricted to a small percentage of activated T and B lymphocytes. However, CD30 is widely expressed in classic Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). CD30 is also expressed to varying degrees in some other lymphoproliferative disorders, including, for example, T fine Cell lymphoma, such as non-specific peripheral T cell lymphoma (PTCL-NOS), immunoblastic T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL), extranodal NK/T cell lymphoma, Adult T-cell leukemia/lymphoma, cutaneous T-cell lymphoma (CTCL); various B-cell non-Hodgkin’s lymphomas, including diffuse large B-cell lymphoma (DLBCL), especially EBV-positive diffuse large B-cell lymphoma ; Granuloma fungoides.

CD30 has been proposed for diagnosis and prognosis, for example as a diagnostic biomarker for Hodgkin's lymphoma and anaplastic large cell lymphoma. Froese et al., J. Immunol. 139: 2081 (1987); Carde et al., Eur. J. Cancer 26: 474 (1990). Smith CA, etc., Cell 1993; 73: 1349-1360.

In addition, the overexpression of CD30 in some lymphoma subtypes and some non-lymphoid tumors has also made it an ideal therapeutic target for some diseases, such as Hodgkin’s lymphoma and anaplastic large cell lymphoma. The emergence of such new targeted therapeutic agents will likely increase the cure rate of traditional chemotherapy and radiotherapy, and reduce long-term toxicity. Wang et al., Advances in CD30- and PD-1-targeted therapies for classical Hodgkin lymphoma, Journal of Hematology & Oncology, 2018, 11:57. A variety of CD30 targeted therapies have been proposed, including, for example, antibody-drug conjugates (ADC ), bispecific antibodies and chimeric antigen receptor modified T cells (CAR-T cells).

In terms of CD30 antibody-drug conjugates (ADC), it has been reported that the chimeric CD30 monoclonal antibody is linked to the microtubule destroying drug-methyl auristatin E (MMAE, monomethyl auristatin E) via a protease cleavable linker. Conjugate Bentuximab (brentuximab vedotin). The compound has been proven to be well tolerated in phase I and II clinical trials in Hodgkin’s lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, and even CD30-expressing B-cell lymphoma. It is important to be able to achieve disease control, including disease control in patients with multiple relapses or refractory diseases disease. CA van der Weyden et al., Blood Cancer Journal (2017) 7, e603; doi: 10.1038/bcj.2017.85.

In terms of bispecific antibodies, bispecific antibodies targeting CD30 and CD16 and bispecific antibodies targeting CD30 and CD64 have been developed. The latest promising development is the bispecific antibody AFM13 that targets CD30 and CD16A. These immunotherapies use bispecific antibodies as a bridge to connect CD30-positive tumor cells with immune effector cells (such as NK cells/macrophages).

In CAR-T cell immunotherapy, Baylor researchers recently announced a phase I clinical study of CD30 CAR-T cells in 7 cases of HL and 2 cases of ALCL. The results showed that lasting complete remission was obtained in some patients (CR ,complete reaction).

Single-chain scFv antibody is a small molecule genetically engineered antibody. It uses genetic engineering methods at the DNA level to connect the heavy chain variable region (VH) and light chain variable region (VL) of a natural antibody (usually by a section). A small molecule recombinant antibody made up of artificially synthesized connecting peptides (or "linkers"). Compared with intact antibody molecules, scFv single-chain antibodies have the following advantages: they contain a complete antibody variable region and retain the antigen specificity and binding activity of the original antibody; they do not contain the Fc region of the antibody molecule, and therefore have weak immunogenicity. Human body is not easy to produce immune response; small molecular weight, strong penetrability, easy to penetrate into tissues, can enter the tissues that are not reachable by general intact antibodies when used for imaging diagnosis or treatment; no glycosylation modification is required to form functional Antibody molecules are conducive to mass production of prokaryotic expression systems; they are easy to operate and are suitable for use as genetic engineering components to prepare other antigen-specific binding molecules with new properties, such as full-length antibodies, scFv-Fc, etc.

In view of the value of CD30 as a target for disease diagnosis, prognosis and treatment, there is still a need for new CD30 specific binding molecules in the art. The present invention provides a fully human monomer that binds to CD30 with high target specificity and high affinity, especially to CD30 expressed on the surface of tumor cells, and has low side effects. Strand antibodies meet this demand. The fully human single-chain antibody of the present invention is not only suitable for use alone in the diagnosis or treatment of tumors and cancers, but also, more advantageously, is suitable for use as a genetic engineering component to prepare other diagnostic and therapeutic molecules with high CD30 targeting. , Such as various forms of antibodies, scFv-Fc, and antibody-based fusions and conjugates.

The invention provides a fully human anti-human CD30 antibody and its coding gene and application. By using transgenic mice and phage display technology, the present inventors screened out fully human antibodies against human CD30 and obtained the variable region gene sequences. On this basis, fully human single-chain scFv antibodies and scFv-hFc were constructed. antibody. The recombinant single-chain antibody molecule of the present invention not only binds to non-membrane-bound human CD30 with high affinity, but also binds to CD30 expressed on the cell surface with high affinity.

Therefore, the present invention provides antibodies that specifically bind to CD30, especially single-chain antibodies, and nucleic acid molecules encoding them, and their use in therapy and diagnosis.

In one aspect, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CD30 (preferably human CD30 protein). In a preferred embodiment, the antibody of the invention is a single chain antibody. In a preferred embodiment, the antibody of the invention is a single chain scFv antibody. In another preferred embodiment, the antibody of the invention is a scFv-Fc antibody. In some embodiments, the antibody of the present invention binds to human CD30 protein with a K _{D of} about 100 nM to 1 nM, wherein the K _D value is measured according to, for example, a biofilm layer optical interference technique (eg, Fortebio detection method). In some embodiments, the antibody of the present invention binds to the human CD30 protein expressed on the cell surface with an EC _{50 of} about 100 nM to 0.1 nM, wherein the EC ₅₀ value is measured according to, for example, flow cytometry (for example, FACS).

In some embodiments, the antibody of the invention comprises the VH region sequence of any antibody shown in Table A or a variant thereof. In other embodiments, the antibody of the invention comprises the VL region sequence of any antibody shown in Table A or a variant thereof. In other embodiments, the antibody of the present invention comprises the VH and VL sequence pair of any antibody shown in Table A, or variants thereof. In some embodiments, the antibody of the present invention comprises the 6 CDR region sequences of any antibody shown in Table A, or a variant thereof, wherein the variant comprises at least one and no more than 10 in the 6 CDR regions, or No more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution), preferably heavy chain CDR3 remains unchanged. In one embodiment, the CDR sequence of the antibody is the CDR sequence shown in Table B.

In some embodiments, the antibodies of the invention are single chain scFv antibodies. Preferably, the scFv antibody contains a VH sequence, a VL sequence and a linker. Preferably, the scFv antibody contains from N-terminus to C-terminus: VL domain-linker-VH domain, or VH domain-linker-VL domain.

In some embodiments, the present invention also provides scFv-Fc antibodies formed by fusion of the single-chain scFv antibody of the present invention and a wild-type or altered Fc region. In some embodiments, the Fc region of the scFv-Fc antibody of the present invention is hypo- or afucosylated. In some embodiments, the scFv antibody is fused to the Fc region via the hinge region.

In another aspect, the present invention relates to fusions and conjugates constructed based on the antibodies of the present invention, especially single-chain antibodies.

In yet another aspect, the present invention relates to methods and compositions for treating CD30-related disorders, wherein an effective amount of the antibody or antigen-binding fragment thereof, or the fusion or conjugate of the present invention is administered to the subject. In some embodiments, a CD30-related disorder is a disorder mediated by or related to CD30 and/or CD30-expressing cells, such as a disease characterized by the growth of CD30-expressing neoplastic cells. In some preferred embodiments, the CD30-related disorder is a CD30-positive tumor, including Hodgkin’s lymphoma And non-Hodgkin’s lymphoma, preferably selected from Hodgkin’s lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma, adult T-cell lymphoma (ATL), angioimmunoblastic T Cellular lymphoma (AITL), the best anaplastic large cell lymphoma and classic Hodgkin’s lymphoma. In some embodiments, the antibody molecules of the invention are used in combination with other therapeutic agents. In yet another embodiment, the antibody molecule of the invention is conjugated to a therapeutic agent, for example to a cytotoxin or a radioisotope.

In another aspect, the present invention relates to a method and kit for detecting CD30 in a sample, wherein the method comprises: (a) contacting the sample with the antibody of the present invention or its antigen-binding fragment, fusion or conjugate; and (b) The formation of a complex between the antibody or its antigen-binding fragment or fusion or conjugate and the CD30 protein is detected. The detection can be in vitro or in vivo. In some embodiments, the sample is a tissue biopsy, serum, plasma, or whole blood from the patient. In some embodiments, the sample is from a patient with lymphoma.

Figure 1 schematically shows the expression vector selection strategy of the exemplary scFv-hFc recombinant single chain antibody of the present invention.

Figures 2A and 2B show the affinity of the exemplary scFv-hFc recombinant single-chain antibody of the present invention to Karpas299 cells determined by flow cytometry.

Figure 3 shows exemplary CDR sequences of the present invention.

Figure 4 shows the VH sequence of an exemplary antibody of the invention.

Figure 5 shows the VL sequence of an exemplary antibody of the invention.

Figure 6 shows an exemplary amino acid sequence of the human CD30 antigen full-length protein and its extracellular domain (ECD).

Figure 7 shows the amino acid sequence and nucleotide sequence of the linker, hinge region and Fc region used to construct the exemplary scFv-Fc construct of the present invention.

Figure 8 shows the amino acid sequences of the variable regions of the positive control antibodies V2AC10 and XL.

Detailed description of the invention

Unless clearly indicated to the contrary, the implementation of the present invention will adopt conventional chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology methods in the art. For descriptions of these methods, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated in July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub .Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I&II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998) Current Protocols in Immunology Q.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and journal monographs such as Advances in Immunology.

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purpose of the present invention, the following terms are defined below.

The term "about" when used in conjunction with a numerical value means to cover a numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

When the term "and/or" is used to connect two or more alternatives, it should be understood to mean any one of the alternatives or any two or more of the alternatives.

As used herein, the term "comprising" or "including" means to include the element, integer or step, but does not exclude any other elements, integers or steps. In this document, when the term "comprises" or "includes" is used, unless otherwise specified, it also covers the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.

As used herein, the term "antigen-binding molecule" refers to a molecule containing an antigen-binding region or antigen-binding portion capable of binding to a target antigen, such as a protein or a polypeptide. In the present invention, an antigen-binding molecule that binds to CD30 is also referred to as a CD30-binding molecule. Antigen-binding molecules include, for example, antibodies and antigen-binding fragments thereof, single-chain scFv antibodies, various fusions and conjugates constructed based on scFv, such as scFv-Fc antibodies. As understood by those skilled in the art, the antigen-binding portion of an antibody usually contains from The amino acid residues of the "complementarity determining region" or "CDR". In some cases, depending on the context, "CD30 binding molecule" and "antibody of the invention" or "anti-CD30 antibody" can be used interchangeably.

As used herein, the term "antibody" refers to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region, which specifically recognizes and binds to an antigen. The term encompasses various antibody structures, including, but not limited to, monoclonal antibodies, single-chain antibodies or multi-chain antibodies, monospecific or multispecific antibodies (such as bispecific antibodies), fully human antibodies or chimeric antibodies, or human Sourced antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

Those skilled in the art understand that "whole antibody" (herein can be used interchangeably with "full-length antibody", "complete antibody" and "complete antibody") contains at least two heavy chains (H) and two light chains (L) . Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three structural domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of a domain CL. The variable region is a domain in the heavy or light chain of an antibody that participates in the binding of the antibody to its antigen. The constant region does not directly participate in the binding of antibodies to antigens, but exhibits a variety of effector functions. The light chain of an antibody can be classified into one of two types (called kappa ( κ ) and lambda ( λ )) based on the amino acid sequence of its constant domain. The heavy chain of an antibody can be divided into five main types depending on the amino acid sequence of the constant region of its heavy chain: IgA, IgD, IgE, IgG and IgM, and several of these types can be further divided into subclasses , Such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. The heavy chain molecules of IgG are γ chains, and the heavy chains of IgM, IgA, IgE and IgD are μ, α , ε and δ chains, respectively. See, for example, Fundamental Immunology, Ch. 7 (Paul, W. ed., second edition, Raven Press, NY (1989)) (which is incorporated herein by reference in its entirety for all purposes).

The term "antibody fragment" refers to a molecule that is not a complete antibody, which includes the part of the complete antibody that binds to the antigen to which the complete antibody binds. The antigen-binding fragment can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of the intact antibody. Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab') ₂ , Fab'-SH, Fv, single-chain Fv, double-chain antibody (diabody), three-chain antibody (triabody), four-chain antibody ( tetrabody), minibody (minibody); and multispecific antibodies formed from antibody fragments. The Fab fragment is a monovalent fragment composed of VL, VH, CL and CH1 domains. For example, a Fab fragment can be obtained by digesting a complete antibody with papain. The light chain (L chain) and heavy chain (H chain) of the Fab can be fused to construct a single polypeptide chain, that is, single chain Fab (scFab) (see, for example, US20070274985A1) by means of a linker. In addition, by pepsin digesting the complete antibody under the disulfide bond in the hinge region, F(ab') ₂ can be produced, which is a dimer of Fab' and a bivalent antibody fragment. F(ab') ₂ can be reduced by breaking the disulfide bond in the hinge region under neutral conditions, converting from F(ab') ₂ dimer to Fab' monomer. Fab' monomers are basically Fab fragments with hinge regions. The Fv fragment is composed of the VL and VH domains of one arm of the antibody. In addition, a recombination method can be used to link the genes independently encoding the two domains VL and VH of the Fv fragment together by the nucleic acid sequence encoding the linker peptide (linker) to form a single-chain Fv by recombinant expression. The VH region and the VL region are paired to provide an antigen binding site. A diabody is an antibody fragment with two antigen binding sites, and the fragment contains VL and VH connected by a short linker in the same polypeptide chain. In diabodies, because the linker is too short, the VH and VL domains on the same chain cannot be paired, and they are forced to pair with the complementary domains on the other chain and create two antigen binding sites. Diabodies can be bivalent or bispecific. For a more detailed description of diabodies, see, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., PNAS USA 90: 6444-6448 (1993) ). Tri-chain antibodies, tetra-chain antibodies and micro-antibodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003), and Shao Rongguang et al. (editor), Antibody Drug Research and Application, People's Medical Publishing House ( 2013). For a more detailed description of antibody fragments, see also: Fundamental Immunology, edited by WEPaul, Raven Press, NY (1993).

The term "monoclonal antibody" as used herein refers to an antibody derived from a substantially homogeneous antibody population, that is, except for possible variant antibodies that are usually present in very small amounts (for example, those containing natural mutations or in monoclonal antibody preparations) Except for the variant antibodies produced during the production process, each antibody constituting the population is the same and/or binds the same epitope. Monoclonal antibodies can be prepared by a variety of techniques including, but not limited to, fusion tumor methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci.

The terms "human antibody" or "fully human antibody" are used interchangeably herein and refer to antibodies that include variable regions in which both framework regions and CDR regions are derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acids that are not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or point-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDR-especially in CDR3 in. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences are derived from the germline of other mammalian species (eg, mice) and transplanted into human framework sequences.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, for example, (a) animals (such as mice) that use human immunoglobulin genes for transgenic or transchromosome Or antibodies isolated from fusion tumors prepared therefrom, (b) antibodies isolated from host cells transformed to express human antibodies such as transfectionomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries such as phage display libraries, and ( d) Antibodies prepared, expressed, produced or isolated by any other means including splicing of human immunoglobulin genes to other DNA sequences. These recombinant human antibodies have framework and CDR regions derived from variable regions of human germline immunoglobulin sequences. However, in certain embodiments, To perform in vitro mutagenesis of recombinant human antibodies (or somatic mutagenesis in vivo when using human Ig sequence transgenic animals), the amino acid sequences of the VH and VL regions of the recombinant antibodies thus obtained are derived from human germline VH It is in the germline repertoire of human antibodies that are related to and related to VL sequences, but do not naturally exist in the body.

The term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and the constant region sequence is derived from another species.

The term "humanized antibody" refers to an antibody in which CDR sequences derived from other mammalian species, such as the mouse germline, are linked to human framework sequences.

An "isolated" antibody is an antibody that has been separated from components in its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse Phase HPLC) confirmed. For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

An epitope is the area of the antigen that the antibody binds to. Epitopes can be formed by consecutive amino acids or by discontinuous amino acids juxtaposed by tertiary folding of the protein.

The term "CD30" includes human CD30 variants, isotypes, species homologs, and analogs that have at least one epitope identical to CD30 (eg, human CD30). Figure 6 shows an exemplary human CD30 sequence (SEQ ID NO: 162). The CD30 protein may also include fragments of CD30, such as extracellular domains and fragments of extracellular domains, such as fragments that maintain the ability to bind to any antibody of the present invention.

The term "specifically binds" means that the antibody selectively or preferentially binds to the antigen. If in bio-optical interferometry, the antibody is about 5x 10 ^-7 M or less, about 1x 10 ^-7 M or less, about 5x 10 ^-8 M or less, about 1x 10 ^-8 M or less, A K _{D of} about 5×10 ^-9 M or lower, which binds to human CD30, is an antibody that "specifically binds to human CD30".

"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between the members of a binding pair. The affinity of a molecule X to its partner Y can generally be represented by the equilibrium dissociation constant (K _D ), which is the ratio of the dissociation rate constant and the association rate constant (k _dis and k _{on, respectively} ). Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay herein.

The "antibody that competes for binding" with a reference antibody that binds to an antigen such as CD30 refers to an antibody that blocks the binding of the reference antibody to the antigen (such as CD30) by 50% or more in a competition test, and vice versa, In the competition test, the reference antibody also blocks the binding of the antibody to the antigen (eg CD30) by 50% or more. An exemplary competitive test is described in: "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). The antibody that competes for binding may bind to the same epitope region as the reference antibody, such as the same epitope, adjacent epitope, or overlapping epitope.

The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a part of the constant region. The term includes native sequence Fc-regions and variant Fc-regions. In one embodiment, the human IgG heavy chain Fc-region extends from Cys226 of the heavy chain or from Pro230 to the carboxy terminus. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc-region or constant region is based on the EU numbering system, also known as the EU index, such as Kabat, EA, etc., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

The term "variant" in relation to antibodies refers herein to include at least one, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acids. Substitution, deletion and/or insertion to have amino acid changes in the target antibody region (e.g. heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region), wherein the variant substantially retains the biological properties of the antibody molecule before the change. In one aspect, the invention encompasses variants of any of the antibodies described herein. In one embodiment, the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the antibody before the change. In some embodiments, the change does not cause the antibody variant to lose its binding to the antigen, but can optionally confer properties such as increased antigen affinity and different effector functions. It is understood that the variable region of the heavy chain or the variable region of the light chain of an antibody, or each CDR region can be changed individually or in combination. In some embodiments, no more than 1, 2, 3, 4, or 5 amino acid changes in one or more or all three heavy chain CDRs. In some embodiments, no more than 1, 2, 3, 4, or 5 amino acid changes in one or more or all three light chain CDRs are made. In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes are made in all 6 CDRs. Preferably, the amino acid is changed to an amino acid substitution, preferably conservative substitution. In some embodiments, the antibody variant has at least 80%, 85%, 90% or 95% or 99% or higher amino acid identity with the parent antibody in the target antibody sequence region. For example, in one embodiment, the antibody of the present invention, compared with any antibody listed in Table A, has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In another embodiment, the antibody of the present invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94% in the light chain variable region compared with any antibody listed in Table A , 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In still another embodiment, the antibody of the present invention has at least 80%, 85%, 90%, 91%, 92% in the variable region of the heavy chain and the variable region of the light chain compared with any antibody listed in Table A. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.

In this context, "sequence identity" refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in the comparison window. The "percentage of sequence identity" can be calculated by the following method: the two best aligned sequences are compared in the comparison window to determine the presence of the same nucleic acid base (for example, A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) the number of positions to get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to generate the sequence identity percentage. The optimal alignment for determining the percent sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment within the full-length sequence being compared or within the target sequence region.

In the present invention, as far as the antibody sequence is concerned, the percent amino acid sequence identity is optimized by aligning the candidate antibody sequence with the reference antibody sequence, and in a preferred scheme, after performing the optimal alignment according to the Kabat numbering rules , To be determined. After the alignment, the target antibody region (e.g., the entire variable region of the heavy chain or light chain, or a part thereof, such as one or more CDR regions) is compared with the same region of the reference antibody. The percentage of sequence identity between the target antibody region and the reference antibody region is: the number of positions occupied by the same amino acid in both the target and reference antibody regions divided by the total number of aligned positions of the two regions (gaps are not counted) ) And multiply by 100 to get the percentage. In this article, if the target antibody region is not specified, it will be applicable to alignment over the full length of the reference antibody sequence. In some embodiments, in the case of antibodies, the sequence identity can be distributed across the entire heavy chain variable region and/or the entire light chain variable region, or the percent sequence identity can be limited to only the framework regions, and the corresponding CDR regions The sequence remains 100% the same.

A. CD30 binding molecules of the present invention I. Anti-CD30 antibody of the present invention

One aspect of the present invention provides antibodies that bind to CD30 (especially membrane-bound CD30) with high target specificity and high affinity, especially single-chain antibodies (e.g., single-chain scFv antibodies).

The antibody of the present invention has one or more of the following characteristics:

(i) With high affinity, such as less than 100nM, such as less than 50nM, such as 1-30nM, preferably less than 10nM KD value, bind to human CD30 (such as the polypeptide of SEQ ID NO: 162);

(ii) With a high affinity, such as less than 100nM, such as less than 70nM, such as 0.1-30nM, preferably less than 20nM, more preferably less than 10 or 5nM, and most preferably less than 1nM EC50 value, and cell surface expressed human CD30 (such as SEQ ID NO: 162 polypeptide) binding;

(iii) The dissociation rate constant (K _dis ) binding to human CD30 (such as the polypeptide of SEQ ID NO: 162) is less than 100×10 ^-4 , preferably less than 60×10 ^-4 , for example, 30-10×10 ^-4 s ^-1 , preferably 5-1×10 ^-4 s ^-1 ;

(iv) It specifically binds to the epitope on CD30, especially on the ECD of the extracellular domain of CD30 (for example, it recognizes the same or similar epitope as any antibody listed in Table A);

(v) Showing the same or similar binding affinity and/or specificity as any antibody listed in Table A;

(vi) Inhibit (e.g., competitively inhibit) the binding of the antibody molecules described herein, such as any of the antibody molecules shown in Table A, to CD30;

(vii) It binds the same or overlapping epitope with any antibody shown in Table A;

(viii) Compete with any antibody shown in Table A to bind to CD30 and/or bind to the same epitope on CD30;

(ix) Having one or more biological properties of the antibody molecules described herein, such as any antibody molecule listed in Table A;

(x) Having one or more pharmacokinetic properties of the antibody molecules described herein, for example, any antibody shown in Table A;

(xi) Inhibit the growth of tumor cells expressing CD30;

(xii) Inducing the killing effect against CD30-expressing tumor cells in the presence of effector cells (for example, by antibody-dependent cytotoxicity (ADCC)).

In some embodiments, the anti-CD30 antibody molecule of the present invention binds to human CD30 (such as the polypeptide of SEQ ID NO: 162) with high affinity, for example, with the following dissociation equilibrium constant (K _D ), the K _{D being} less than about 100 nM, Less than or equal to about 80nM, 70nM, 60nM, or 50nM, more preferably less than or equal to about 40nM, 30nM, or 20nM, more preferably less than or equal to about 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM Or 1nM. In one embodiment, the KD value is determined by using a bio-optical interferometry method (eg, Fortebio affinity measurement method).

In some embodiments, the dissociation rate constant (K _dis ) of the anti-CD30 antibody molecule of the present invention binding to human CD30 (for example, the polypeptide of SEQ ID NO: 162) is less than 6×10 ^-3 s ^-1 , for example, 4-1×10 ^-3 s ^-1 , preferably less than 8×10 ^-4 s ^-1 , such as less than 5×10 ^-4 or 3×10 ^-4 s ^-1 , such as about 2×10 ^-4 s ^-1 . In some embodiments, the binding rate constant (K _on ) of the anti-CD30 antibody molecule binding to human CD30 (for example, the polypeptide of SEQ ID NO: 162) is greater than 1×10 ⁴ , 5×10 ⁴ , 1×10 ⁵ or 2× 10 ⁵ M ^-1 s ^-1 .

In some embodiments, the anti-CD30 antibody molecules of the present invention bind to cells expressing CD30 with high affinity, preferably lymphoma cells expressing human CD30 on the cell surface (such as Karpas299 cells), preferably by flow cytometry ( For example, FACS) determination, the EC50 value of the antibody binding to the cell is less than about 200nM, 150nM or 100nM, preferably less than or equal to about 80nM, 70nM, 60nM, or 50nM, more preferably less than or equal to about 40nM, 30nM or 20nM, better less than or etc It is about 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM or 2nM, preferably less than 1nM.

In one embodiment, the antibody molecule binds to human CD30 comprising the amino acid sequence SEQ ID NO:162. In some embodiments, the antibody molecule binds to an epitope on CD30, preferably on the extracellular domain of CD30.

In some embodiments, the antibody molecule is a full-length antibody. In other embodiments, the antibody molecule is an antibody fragment. For example, the antibody molecule of the present invention may comprise or may be Fab, scFab, Fab', F(ab') ₂ , Fab'-SH, Fv, single-chain scFv antibody, double-chain antibody (diabody), tri-chain antibody, four Chain antibody, micro antibody. In a preferred embodiment, the antibody molecule of the invention is a single chain scFv antibody. In a preferred embodiment, the antibody molecule of the present invention comprises a scFv and an Fc region linked to it. In a preferred embodiment, the antibody molecule of the invention is fully human.

Antibody variable region

A "variable region" or "variable domain" is a domain in the heavy or light chain of an antibody that participates in the binding of the antibody to its antigen. The heavy chain variable region (VH) and light chain variable region (VL) can be further subdivided into hypervariable regions (HVR, also known as complementarity determining regions (CDR)), with a more conservative region (ie, framework Area (FR)). Each VH and VL consists of three CDRs and 4 FRs, arranged in the following order from the amino end to the carboxyl end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some cases, a single VH or VL domain is sufficient to confer antigen-binding specificity. In addition, antibodies that bind to specific antigens can be isolated by screening complementary VL or VH domain libraries using the VH or VL domains from antibodies that bind the antigen (see, for example, Portolano, S. et al., J. Immunol. 150 (1993) ) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628). In this article, the term "VH" or "VH "Domain" encompasses the heavy chain variable region VH of a full-length antibody, Fv, scFv, dsFv, Fab, scFab, or other antibody fragments disclosed herein. In this context, the term "VL" or "VL domain" encompasses full-length antibodies, Fv , ScFv, dsFv, Fab, scFab, or the light chain variable region VL of other antibody fragments disclosed herein.

In one embodiment, the anti-CD30 antibody molecule of the present invention comprises: (i) the same antigen binding region as the antigen binding region (for example, a pair of heavy chain variable region and light chain variable region) of any antibody listed in Table A Or (ii) the antigen binding region of (i) has, for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity in the amino acid sequence Sexual antigen binding region.

In yet another embodiment, the anti-CD30 antibody molecule of the present invention comprises: (i) the same heavy chain variable region as the heavy chain variable region of any antibody listed in Table A; or (ii) and (i) The chain variable region has, for example, a heavy chain variable region with at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity in the amino acid sequence; or (iii) The variant of the heavy chain variable region of (i), wherein the variant comprises at least one and no more than 30, 20 or 10 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) , And preferably the variant contains a total of no more than 10, preferably 5-0, amino acid changes (preferably amino acid substitutions) in the 3 heavy chain complementarity determining regions (CDR) regions.

In yet another embodiment, the anti-CD30 antibody molecule of the present invention comprises: (i) the same light chain variable region as the light chain variable region of any antibody listed in Table A; or (ii) and (i) the light chain variable region The chain variable region has, for example, a light chain variable region with at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity in the amino acid sequence; or (iii) A variant of the light chain variable region of (i), wherein the variant comprises at least one and no more than 30, 20 or 10 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) And preferably the variant contains no more than 10, preferably 5-0, amino acid changes (preferably amino acid substitutions) in the 3 light chain complementarity determining regions (CDR).

In some embodiments, the present invention provides an anti-CD30 antibody comprising the amino acid sequence of a heavy chain variable region and a light chain variable region pair of any antibody listed in Table A, or a variant thereof. In a preferred embodiment, the antibody comprises amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 4/9, 14/19, 24/29, 34/39, 44/49, 54/59, 64 /69, 74/79, 84/89, 94/99, 104/109, and 114/119. In a preferred embodiment, the variant has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher in the VH and/or VL amino acid sequence Identity, or at least one and no more than 30, 20 or 10 amino acid changes in the VH and/or VL amino acid sequence (preferably amino acid substitution, preferably conservative substitution).

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 4, and the VL comprising SEQ ID NO: 9 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 14, and the VL comprising SEQ ID NO: The amino acid sequence of 19. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 24, and the VL comprising SEQ ID NO: 29 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 34, and the VL comprising SEQ ID NO: 39 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 44, and the VL comprising SEQ ID NO: 49 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 54 and the VL comprising SEQ ID NO: The amino acid sequence of 59. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 64, and the VL comprising SEQ ID NO: 69 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 74, and the VL comprising SEQ ID NO: 79 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 84, and the VL comprising SEQ ID NO: 89 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 94, and the VL comprising SEQ ID NO: 99 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 104, and the VL comprising SEQ ID NO: 109 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In some embodiments, the present invention provides an anti-CD30 antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO: 114, and the VL comprising SEQ ID NO: 119 amino acid sequence. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH and/or VL or containing no more than 10 amino acid changes.

In any of the above embodiments, preferably, relative to the reference heavy chain variable region amino acid sequence, the heavy chain variable region of the antibody of the present invention contains no more than 10 in all 3 CDR regions, Preferably, no more than 5 (for example, 3, 2, 1, or 0) amino acid changes (amino acid substitution is preferred, conservative substitution is preferred).

In any of the above embodiments, preferably, relative to the reference light chain variable region amino acid sequence, the light chain variable region VL of the antibody of the present invention contains no more than 10 CDRs in all 3 CDR regions. Preferably, no more than 5 (for example, 3, 2, 1, or 0) amino acid changes (amino acid substitution is preferred, conservative substitution is preferred).

Antibody CDR region

"Complementarity determining region" or "CDR region" or "CDR" (herein used interchangeably with hypervariable region "HVR") is an amino acid in the variable region of an antibody that is mainly responsible for binding to an epitope area. The CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.

Various schemes for determining the CDR sequence of a given VH or VL amino acid sequence are known in the art. For example, the Kabat complementarity determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991)). Chothia refers to the position of structural loops (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. "Contact" HVR is based on the analysis of available complex crystal structures. According to different CDR determination schemes, the residues of each of these HVRs/CDRs are as follows.

HVR can also be an HVR sequence located at the following Kabat residue positions according to the Kabat numbering system:

Position 24-36 or 24-34 (LCDR1) in VL, position 46-56 or 50-56 (LCDR2), and position 89-97 or 89-96 (LCDR3); and position 26-35 or in VH 26-35B (HCDR1), positions 50-65 or 49-65 (HCDR2), and positions 94-102 or 95-102 (HCDR3).

In one embodiment, the HVR of the antibody of the invention is an HVR sequence located at the following Kabat residue positions according to the Kabat numbering system:

Positions 24-34 (LCDR1), positions 50-56 (LCDR2), and positions 89-97 (LCDR3) in VL, and positions 26-35B (HCDR1), positions 50-65 (HCDR2), and positions in VH 95-102 (HCDR3).

HVR can also be determined based on the same Kabat numbering position as a reference CDR sequence (for example, any of the exemplary CDRs of the present invention).

Unless otherwise specified, in the present invention, the terms "CDR" or "CDR sequence" or "HVR" or "HVR sequence" encompass HVR or CDR sequences determined in any of the above-mentioned ways.

Unless otherwise specified, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), it refers to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

In a preferred embodiment, the CDR of the antibody of the present invention is a combined Kabat and chothia division method, and the CDR sequence determined by the union of the two is used. In another preferred embodiment, the CDR sequences of the invention are shown in Table B.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-part of the CDR. As those skilled in the art know, by using the structure of the antibody and protein folding, the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residue of the smallest binding unit can remain unchanged, while the remaining CDR residues defined by Kabat or Chothia can be replaced by conserved amino acid residues.

In some embodiments, the antibody of the invention comprises at least one, two, three, four, five, or six CDRs that are identical to the corresponding CDRs of any antibody listed in Table A, or variants thereof. In some embodiments, the antibodies of the invention comprise at least one, two, or three HCDRs that are identical to the corresponding heavy chain CDRs of any antibody listed in Table A, or variants thereof. In some embodiments, the antibodies of the invention comprise at least one, two, or three HCDRs that are identical to the corresponding light chain CDRs of any antibody listed in Table A, or variants thereof. As used herein, a CDR variant is a CDR that has been modified by at least one, for example, 1 or 2 or 3 amino acid substitutions, deletions and/or insertions, wherein the antigen-binding molecule comprising the CDR variant basically remains The biological properties of the CDR-modified antigen-binding molecule, for example, maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen-binding ability). It is understood that each CDR can be modified individually or in combination. Preferably, the amino acid modification is an amino acid substitution, especially a conservative amino acid substitution, such as the preferred conservative amino acid substitutions listed in Table X.

In some embodiments, the antibody of the present invention or an antigen-binding fragment thereof comprises a heavy chain variable region, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region:

(i) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of any antibody listed in Table A are respectively the same; or

(ii) Relative to the HCDR1, HCDR2 and HCDR3 of (i), it contains at least 1 and no more than 10, preferably no more than 5 (preferably 1, 2 or 3) amino acid changes (preferably substituted, More preferably conservative substitution), and preferably no more than 3 amino acid changes in the HCDR3 region (for example, 2, 1, or 0).

In some embodiments, the antibody of the present invention or an antigen-binding fragment thereof comprises a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein said LCDR1, LCDR2 and LCDR3:

(i) The LCDR1, LCDR2 and LCDR3 of the light chain variable region of any antibody listed in Table A are respectively the same; or

(ii) Compared with LCDR1, LCDR2 and LCDR3 of (i), it contains at least 1 and no more than 10 (preferably 1-5, preferably 1, 2 or 3) amino acid changes (preferably substituted , Better conservative substitution).

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain may The variable region includes the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the antibody:

(i) A sequence comprising all 6 CDR regions of the heavy chain and light chain variable regions of any antibody listed in Table A; or

(ii) Relative to any antibody listed in Table A, no more than 10, preferably no more than 5 (for example, 3, 2, 1, or 0) amino acid changes (compared to Preferably amino acid substitution, conservative substitution is preferred).

In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises HCDR1, 2 and 3 sequences selected from the following heavy chain variable region (VH) and light chain variable region (VL) and LCDR1, 2 and 3 sequence:

(i) VH of SEQ ID NO: 4 and VL of SEQ ID NO: 9;

(ii) The VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 19;

(iii) VH of SEQ ID NO: 24 and VL of SEQ ID NO: 29;

(iv) VH of SEQ ID NO: 34 and VL of SEQ ID NO: 39;

(v) VH of SEQ ID NO: 44 and VL of SEQ ID NO: 49;

(vi) VH of SEQ ID NO: 54 and VL of SEQ ID NO: 59;

(vii) VH of SEQ ID NO: 64 and VL of SEQ ID NO: 69;

(viii) VH of SEQ ID NO: 74 and VL of SEQ ID NO: 79;

(ix) VH of SEQ ID NO: 84 and VL of SEQ ID NO: 89;

(x) VH of SEQ ID NO: 94 and VL of SEQ ID NO: 99;

(xi) VH of SEQ ID NO: 104 and VL of SEQ ID NO: 109;

(xii) The VH of SEQ ID NO: 114 and the VL of SEQ ID NO: 119.

In some embodiments, the present invention provides a heavy chain CDR combination selected from the following amino acid sequence combinations (HCDR1, HCDR2, and HCDR3 in order): SEQ ID NOs: 1/2/3, 11/12/13, 21/22/23, 3132/33, 41/42/43, 51/52/53, 61/62/63, 71/72/73, 81/82/83, 91/92/93, 101/102/ 103, and 111/112/113. The present invention also provides variants of the heavy chain CDR combination. In a preferred embodiment, the variant includes at least one and no more than 10 or 5 amino acid changes in the three CDR regions ( Preferably amino acid substitution, conservative substitution is preferred). The present invention also provides an anti-CD30 antibody comprising the heavy chain CDR combination or the variant.

In some embodiments, the present invention provides a combination of CDR sequences selected from the following, and antibodies or antigen-binding fragments comprising the combination:

(i) HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO: 2 and HCDR3 comprising the sequence of SEQ ID NO: 3;

(ii) HCDR1 comprising the sequence of SEQ ID NO: 11, HCDR2 comprising the sequence of SEQ ID NO: 12, and HCDR3 comprising the sequence of SEQ ID NO: 13;

(iii) HCDR1 comprising the sequence of SEQ ID NO: 21, HCDR2 comprising the sequence of SEQ ID NO: 22, and HCDR3 comprising the sequence of SEQ ID NO: 23;

(iv) HCDR1 comprising the sequence of SEQ ID NO: 31, HCDR2 comprising the sequence of SEQ ID NO: 32, and HCDR3 comprising the sequence of SEQ ID NO: 33;

(v) HCDR1 comprising the sequence of SEQ ID NO: 41, HCDR2 comprising the sequence of SEQ ID NO: 42 and HCDR3 comprising the sequence of SEQ ID NO: 43;

(vi) HCDR1 comprising the sequence of SEQ ID NO: 51, HCDR2 comprising the sequence of SEQ ID NO: 52, and HCDR3 comprising the sequence of SEQ ID NO: 53;

(vii) HCDR1 comprising the sequence of SEQ ID NO: 61, HCDR2 comprising the sequence of SEQ ID NO: 62, and HCDR3 comprising the sequence of SEQ ID NO: 63;

(viii) HCDR1 comprising the sequence of SEQ ID NO: 71, HCDR2 comprising the sequence of SEQ ID NO: 72, and HCDR3 comprising the sequence of SEQ ID NO: 73;

(ix) HCDR1 comprising the sequence of SEQ ID NO: 81, HCDR2 comprising the sequence of SEQ ID NO: 82, and HCDR3 comprising the sequence of SEQ ID NO: 83;

(x) HCDR1 comprising the sequence of SEQ ID NO: 91, HCDR2 comprising the sequence of SEQ ID NO: 92, and HCDR3 comprising the sequence of SEQ ID NO: 93;

(xi) HCDR1 comprising the sequence of SEQ ID NO: 101, HCDR2 comprising the sequence of SEQ ID NO: 102, and HCDR3 comprising the sequence of SEQ ID NO: 103;

(xii) HCDR1 comprising the sequence of SEQ ID NO: 111, HCDR2 comprising the sequence of SEQ ID NO: 112, and HCDR3 comprising the sequence of SEQ ID NO: 113.

In some embodiments, the present invention provides light chain CDR combinations (LCDR1, LCDR2, and LCDR3, respectively) selected from the following amino acid sequence combinations: SEQ ID NOs: 6/7/8, 16/17/18 , 26/27/28, 36/37/38, 46/47/48, 56/57/58, 66/67/68, 76/77/78, 86/87/88, 96/97/98, 106 /107/108, and 116/117/118. The present invention also provides a variant of the light chain CDR combination. In a preferred embodiment, the variant contains at least one and no more than 10 or 5 amino acid changes in the three CDR regions (preferably Amino acid substitution, conservative substitution is preferred). The present invention also provides an anti-CD30 antibody or antigen-binding fragment comprising the light chain CDR combination or the variant.

In some embodiments, the present invention provides a combination of CDRs selected from the following, and antibodies or antigen-binding fragments comprising the combination:

(i) LCDR1 comprising the sequence of SEQ ID NO: 6; LCDR2 comprising the sequence of SEQ ID NO: 7; and LCDR3 comprising the sequence of SEQ ID NO: 8;

(ii) LCDR1 comprising the sequence of SEQ ID NO:16, LCDR2 comprising the sequence of SEQ ID NO:17 and LCDR3 comprising the sequence of SEQ ID NO:18;

(iii) LCDR1 comprising the sequence of SEQ ID NO: 26, LCDR2 comprising the sequence of SEQ ID NO: 27, and LCDR3 comprising the sequence of SEQ ID NO: 28;

(iv) LCDR1 comprising the sequence of SEQ ID NO: 36, LCDR2 comprising the sequence of SEQ ID NO: 37, and LCDR3 comprising the sequence of SEQ ID NO: 38;

(v) LCDR1 comprising the sequence of SEQ ID NO: 46, LCDR2 comprising the sequence of SEQ ID NO: 47, and LCDR3 comprising the sequence of SEQ ID NO: 48;

(vi) LCDR1 comprising the sequence of SEQ ID NO:56, LCDR2 comprising the sequence of SEQ ID NO:57, and LCDR3 comprising the sequence of SEQ ID NO:58;

(vii) LCDR1 comprising the sequence of SEQ ID NO: 66, LCDR2 comprising the sequence of SEQ ID NO: 67, and LCDR3 comprising the sequence of SEQ ID NO: 68;

(viii) LCDR1 comprising the sequence of SEQ ID NO:76, LCDR2 comprising the sequence of SEQ ID NO:77, and LCDR3 comprising the sequence of SEQ ID NO:78;

(ix) LCDR1 comprising the sequence of SEQ ID NO: 86, LCDR2 comprising the sequence of SEQ ID NO: 87, and LCDR3 comprising the sequence of SEQ ID NO: 88;

(x) LCDR1 comprising the sequence of SEQ ID NO: 96, LCDR2 comprising the sequence of SEQ ID NO: 97 and LCDR3 comprising the sequence of SEQ ID NO: 98;

(xi) LCDR1 comprising the sequence of SEQ ID NO: 106, LCDR2 comprising the sequence of SEQ ID NO: 107, and LCDR3 comprising the sequence of SEQ ID NO: 108;

(xii) LCDR1 comprising the sequence of SEQ ID NO: 116, LCDR2 comprising the sequence of SEQ ID NO: 117, and LCDR3 comprising the sequence of SEQ ID NO: 118.

In still other embodiments, the present invention provides a heavy chain and light chain CDR combination selected from the following amino acid sequence combinations (HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, respectively, in order): SEQ ID NOs: 1/ 2/3/6/7/8, 11/12/13/16/17/18, 21/22/23/26/27/28, 31/32/33/36/37/38, 41/42/ 43/46/47/48, 51/52/53/56/57/58, 61/62/63/66/67/68, 71/72/73/76/77/78, 81/82/83/ 86/87/88, 91/92/93/96/97/98, 101/102/103/106/107/108, and 111/112/113/116/117/118. The present invention also provides a variant of the CDR combination. In a preferred embodiment, the variant contains at least one and no more than 20, 10, or 5 amino acid changes in the six CDR regions (preferably amine Base acid substitution, conservative substitution is preferred). The present invention also provides an anti-CD30 antibody or antigen-binding fragment comprising the heavy chain and light chain CDR combination or the variant.

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises 3 heavy chain complementarity determining regions HCDR and 3 light chain complementarity determining regions LCDR, wherein:

(a) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 3, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 6, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 8; or

(b) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 11, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 12, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 13, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 16, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 18; or

(c) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 21, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 22, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 23, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 26, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 28; or

(d) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 31, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 32, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 36, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 38; or

(e) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 41, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 42, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 43, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 46, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 47, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 48; or

(f) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 51, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 52, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 53, LCDR1 includes The amino acid sequence shown in SEQ ID NO: 56, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 57, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 58; or

(g) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 61, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 62, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 63, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 66, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 67, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 68; or

(h) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 71, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 72, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 73, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 76, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 77, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78; or

(i) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 81, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 82, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 83, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 86, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 87, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 88; or

(j) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 91, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 92, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 93, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 96, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 97, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 98; or

(k) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 101, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 102, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 103, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 106, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 107, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 108; or

(1) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 111, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 112, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 113, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 116, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 118.

In the above embodiments of the antibody of the present invention, "conservative substitution" refers to an amino acid change that causes a certain amino acid to be replaced with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables for functionally similar amino acids. In any embodiment of the present invention, in a preferred aspect, the conservatively substituted residues are derived from conservative substitution table X below, preferably the preferred substituted residues shown in Table X.

Table X

Exemplary antibody sequence

The present invention provides fully human antibodies that specifically bind to CD30 (for example, human CD30) isolated and characterized as in the Examples. Table A below lists the variable region sequences of these exemplary antibodies of the invention (see also Figures 4 and 5). Exemplary CDR sequences of these antibodies are given in Table B below (see also section 3).

Table A. The amino acid sequence and nucleotide sequence of the heavy chain variable region and the light chain variable region of an exemplary fully human antibody molecule of the present invention.

Table B. Amino acid sequences of exemplary heavy chain CDRs and light chain CDRs

The present invention also provides variants of the aforementioned antibodies. In one embodiment, the amino acid sequence of the antibody or the nucleic acid encoding the amino acid sequence has been mutated, but still has at least 60%, 65%, 70%, 75%, 80%, and the sequence described in Table A. 85%, 90%, or 95% or higher identity. In some embodiments, the antibody includes a mutated variable region amino acid sequence, wherein when compared with the variable region sequence shown in Table A, the variable region VH and/or VL has been mutated no more than 1, 2, 3, 4, 5 or 10 amino acids, but retain substantially the same antigen binding activity.

In addition, since each of the above antibodies can bind to CD30, VH and VL (the amino acid sequence and the nucleotide sequence encoding the amino acid sequence) can be "mixed and matched" to produce binding CD30 other antibodies of the invention. The binding of such "mixed and matched" antibodies to CD30 can be tested using binding assays known in the art (e.g., ELISA, and other assays described in the Examples section). When mixing and matching these chains, it is preferable to replace the VH sequence from a specific VH/VL pair with a structurally similar VH sequence. Likewise, the VL sequence from a specific VH/VL pair is preferably replaced with a structurally similar VL sequence.

In another aspect, the present invention also provides variants of the aforementioned antibodies. In one embodiment, the antibody has been mutated in one or more or all of the 6 CDR regions of the amino acid sequence or the nucleic acid encoding the amino acid sequence. In some embodiments, the amino acid sequence of the mutated CDR region, when compared with the corresponding CDR region in Table A, has mutated no more than 1, 2, 3, 4, or 5 amino acids, but retains basic The same antigen binding activity.

In addition, since each of the antibodies in Table A can bind to CD30 and the antigen binding specificity is mainly provided by the CDR1, 2 and 3 regions, it is possible to "mix the VH CDR1, 2 and 3 sequences with the VL CDR1, 2 and 3 sequences" "And match" (that is, CDRs from different antibodies can be mixed and matched, but each antibody preferably contains VH CDR 1, 2 and 3 and VL CDR 1, 2 and 3) to produce other molecules of the invention that bind CD30. The binding of such "mixed and matched" antibodies to CD30 can be tested using binding assays known in the art (e.g., ELISA, SET, Biacore) and those described in the examples. When VH CDR sequences are mixed and matched, CDR1, CDR2 and/or CDR3 sequences from a specific VH sequence are preferably replaced with structurally similar CDR sequences. Likewise, when VL CDR sequences are mixed and matched, CDR1, CDR2 and/or CDR3 sequences from a specific VL sequence are preferably replaced with structurally similar CDR sequences. Those skilled in the art understand that one or more VH and/or VL CDR region sequences can also be replaced with structurally similar CDR sequences from the antibodies shown herein to produce other antibodies of the present invention.

II. Single chain scFv antibody

In a preferred aspect, the antibody of the invention is a single chain scFv antibody.

As used herein, "single chain scFv antibody" or "scFv" or "single chain scFv" refers to a single polypeptide comprising the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin or antibody A chain in which the VH and VL regions are paired to provide an antigen binding site in a single protein chain.

In a preferred embodiment, the VH region and the VL region of the single-chain scFv antibody of the present invention are covalently linked together by a connecting peptide such as a flexible connecting peptide. The term "flexible linking peptide" is a peptide linker composed of amino acids. Through such peptide linkers, various variable domains in antibodies, such as VH and VL regions, can be connected. Peptide linkers are usually rich in glycine, which exhibits flexibility, and serine or threonine, which exhibits solubility. For example, glycine and/or serine residues can be used alone or in combination. Non-limiting examples of flexible linking peptides or peptide linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008), WO2012/138475 and WO2014/087010, the contents of which are incorporated by reference in their entirety. As known in the art, in the construction of scFv, preferably, the linker will facilitate the pairing of VH and VL, and will not interfere with the formation of a functionally effective antigen binding site between VH and VL.

In some embodiments, the scFv single-chain antibody of the present invention comprises a flexible connecting peptide or peptide linker composed of amino acid residues connected by peptide bonds. In certain embodiments, the amino acid is selected from twenty natural amino acids. In certain other embodiments, the one or more amino acids are selected from glycine, serine, threonine, alanine, proline, asparagine, glutamine, and lysine. In a preferred embodiment, the one or more amino acids are selected from Gly, Ser, Thr, Lys, Pro, and Glu.

In some embodiments, the length of the linker is about 1-30 amino acids, or about 10 to about 25 amino acids, about 15 to about 20 amino acids, or about 10 to about 20 amines Base acid or anything between The length of the intermediate amino acid. In a preferred embodiment, the linker has a length of 15-25 amino acid residues, and in a more preferred embodiment, a length of 15-18 amino acid residues. In some embodiments, the length of the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids.

Examples of peptide linkers that can be used in the present invention include: glycine polymer (G)n; glycine-serine polymer (G _1-5 S _1-5 )n, where n is at least 1, 2. , 3, 4, or 5; glycine-alanine polymer; alanine-serine polymer; and other flexible joints known in the art. Those skilled in the art can understand that, in some embodiments, the linker between VH and VL may be composed entirely of a flexible linking peptide, or the linker may be composed of a flexible linking peptide part and one or more parts that give a smaller flexible structure.

In a preferred embodiment, the peptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 157). In one embodiment, the nucleotide sequence encoding the amino acid sequence SEQ ID NO:157 is given in SEQ ID NO:158.

In one embodiment, the peptide linker is a Gly/Ser linking peptide. In one embodiment, the peptide linker is a (GxS)n linker, where G=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6 , 7 or 8), in one embodiment, x=4 and n=6 or 7. In some embodiments, the linker may include an amino acid sequence (G ₄ S)n, where n is an integer equal to or greater than 1, for example, n is an integer from 1-7. In a preferred embodiment, x=4 and n=7. In one embodiment, the linker is (G4S)3. In one embodiment, the linker is (G4S)4. In one embodiment, the linker is (G4S)6G2.

Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS; TGEKP (see, for example, Liu et al., PNAS5525-5530 (1997)); GGRR (Pomerantz et al. 1995, supra); (GGGGS) n, where n=1, 2, 3, 4 or 5 (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (Chaudhary et al., 1990, Proc.Natl.Acad.Sci.USA87: 1066-1070); KESGSVSSEQLAQFRSLD (Bird et al., 1988, Science242:423-426), GGRRGGGS; LRQRDGERP; LRQKDGGGSERP; LRQKD(GGGS)2ERP. Alternatively, you can use Computer programs for modeling DNA-binding sites and peptides themselves (Desjarlais&Berg, PNAS 90: 2256-2260 (1993), PNAS 91: 11099-11103 (1994)), or rationally design flexible linkers by phage or yeast display methods .

The VH and VL in the single chain scFv antibody of the present invention can take either direction. In some embodiments, the scFv comprises from N-terminus to C-terminus: VH-linker-VL; or VL-linker-VH. In a preferred embodiment, the single-chain scFv antibody of the present invention contains from N-terminus to C-terminus: VH-linker-VL. In a preferred embodiment, the C-terminus of the VH is covalently connected to the N-terminus of VL via a linker.

In some embodiments, in addition to the linker, other polypeptide fragments with specific functions can also be inserted between the VL and VH domains, such as a polypeptide fragment with a function of regulating immune response, or a polypeptide fragment with a cell solvent or cell killing.

In some embodiments, the single chain antibody can be stabilized by introducing disulfide bonds in the scFv. For example, the VH and VL framework regions of scFv can be connected by introducing intra-chain or inter-chain disulfide bonds. In one embodiment, one amino acid residue of each of the antibodies VH and VL can be mutated to cysteine, for example, according to the Kabat numbering system, position 44 of VH and position 100 of VL, or 105 of VH Bits and 43 bits of VL.

The single-chain scFv polypeptide antibody of the present invention can be expressed by a nucleic acid including VH- and VL- coding sequences, as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85: 5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication No. 20050196754 and 20050196754. In some embodiments, the single chain scFv antibody of the present invention is expressed in eukaryotic cells, such as yeast cells, mammalian cells such as HEK293 cells or CHO cells.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or an antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 121 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with SEQ ID NO: 121. In one embodiment, the anti-CD30 scFv is encoded by the nucleotides of SEQ ID NO:122.

In some embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 4 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with the amino acid sequence The heavy chain variable region of the amino acid sequence of SEQ ID NO: 9 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 9 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:4. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:9. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO: 4 and includes VL having the sequence shown in SEQ ID NO: 9. In certain embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH with the sequence shown in SEQ ID NO: 4 and/or 3 LCDR sequences of VL with the sequence shown in SEQ ID NO: 9. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:3 and LCDR3 of SEQ ID NO:8. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:1, the VH CDR2 of SEQ ID NO:2, and the VH CDR3 of SEQ ID NO:3. In some implementations In the manner, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:6, VL CDR2 of SEQ ID NO:7, and VL CDR3 of SEQ ID NO:8. In some embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 1, the VH CDR2 of SEQ ID NO: 2, and the VH CDR3 of SEQ ID NO: 3, and the VL CDR1 of SEQ ID NO: 6 VL CDR2 of SEQ ID NO:7 and VL CDR3 of SEQ ID NO:8.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or an antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 124 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity to SEQ ID NO:124. In one embodiment, the anti-CD30 scFv is encoded by the nucleotides of SEQ ID NO:125.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 14 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with it The heavy chain variable region of the amino acid sequence of SEQ ID NO: 19 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 19 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:14. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:19. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO: 14 and includes VL having the sequence shown in SEQ ID NO: 19. In some embodiments, the anti-CD30 scFv includes the 3 HCDR sequences of VH of the sequence shown in SEQ ID NO: 14 and/or the sequence of the sequence shown in SEQ ID NO: 19 3 LCDR sequences of VL. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO: 13 and LCDR3 of SEQ ID NO: 18. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 11, the VH CDR2 of SEQ ID NO: 12, and the VH CDR3 of SEQ ID NO: 13. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:16, VL CDR2 of SEQ ID NO:17, and VL CDR3 of SEQ ID NO:18. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 11, the VH CDR2 of SEQ ID NO: 12, and the VH CDR3 of SEQ ID NO: 13, and the VL CDR1 of SEQ ID NO: 16 The VL CDR2 of SEQ ID NO:17 and the VL CDR3 of SEQ ID NO:18.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 127 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with SEQ ID NO:127. In one embodiment, the anti-CD30 scFv is encoded by the nucleotides of SEQ ID NO:128.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 24 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with it The heavy chain variable region of the amino acid sequence of SEQ ID NO: 29 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 29 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes VH having the amino acid sequence of SEQ ID NO:24. In certain embodiments, the anti-CD30 scFv includes an amine group having SEQ ID NO: 29 VL of the acid sequence. In certain embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO: 24 and includes VL having the sequence shown in SEQ ID NO: 29. In some embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH of the sequence shown in SEQ ID NO: 24 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 29. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:23 and LCDR3 of SEQ ID NO:28. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:21, the VH CDR2 of SEQ ID NO:22, and the VH CDR3 of SEQ ID NO:23. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:26, VL CDR2 of SEQ ID NO:27, and VL CDR3 of SEQ ID NO:28. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 21, the VH CDR2 of SEQ ID NO: 22, and the VH CDR3 of SEQ ID NO: 23, and the VL CDR1 of SEQ ID NO: 26 VL CDR2 of SEQ ID NO:27 and VL CDR3 of SEQ ID NO:28.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 130 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with SEQ ID NO:130. In one embodiment, the anti-CD30 scFv is encoded by the nucleotide of SEQ ID NO:131.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 34 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with it The heavy chain variable region of the amino acid sequence of SEQ ID NO: 39 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 39 Of the light chain variable region, and as needed A linker between the variable region of the heavy chain and the variable region of the light chain is important, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:34. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:39. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO:34 and includes VL having the sequence shown in SEQ ID NO:39. In some embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH with the sequence shown in SEQ ID NO:34 and/or 3 LCDR sequences of VL with the sequence shown in SEQ ID NO:39. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:33 and LCDR3 of SEQ ID NO:38. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:31, the VH CDR2 of SEQ ID NO:32, and the VH CDR3 of SEQ ID NO:33. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:36, VL CDR2 of SEQ ID NO:37, and VL CDR3 of SEQ ID NO:38. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 31, the VH CDR2 of SEQ ID NO: 32, and the VH CDR3 of SEQ ID NO: 33, and the VL CDR1 of SEQ ID NO: 36 VL CDR2 of SEQ ID NO:37 and VL CDR3 of SEQ ID NO:38.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 133 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with SEQ ID NO: 133. In one embodiment, the anti-CD30 scFv is encoded by the nucleotides of SEQ ID NO:134.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 44 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The heavy chain variable region of the amino acid sequence of SEQ ID NO: 49 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 49 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:44. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:49. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO:44 and includes VL having the sequence shown in SEQ ID NO:49. In certain embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH with the sequence shown in SEQ ID NO:44 and/or 3 LCDR sequences of VL with the sequence shown in SEQ ID NO:49. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:43 and LCDR3 of SEQ ID NO:48. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:41, the VH CDR2 of SEQ ID NO:42, and the VH CDR3 of SEQ ID NO:43. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:46, VL CDR2 of SEQ ID NO:47, and VL CDR3 of SEQ ID NO:48. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 41, the VH CDR2 of SEQ ID NO: 42, and the VH CDR3 of SEQ ID NO: 43, and the VL CDR1 of SEQ ID NO: 46 VL CDR2 of SEQ ID NO:47 and VL CDR3 of SEQ ID NO:48.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or an antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 136 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In a In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with SEQ ID NO:136. In one embodiment, the anti-CD30 scFv is encoded by the nucleotide of SEQ ID NO:137.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 54 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity therewith The heavy chain variable region of the amino acid sequence of SEQ ID NO: 59 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 59 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:54. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:59. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO: 54 and includes VL having the sequence shown in SEQ ID NO: 59. In some embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH of the sequence shown in SEQ ID NO: 54 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 59. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:53 and LCDR3 of SEQ ID NO:58. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:51, the VH CDR2 of SEQ ID NO:52, and the VH CDR3 of SEQ ID NO:53. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:56, VL CDR2 of SEQ ID NO:57, and VL CDR3 of SEQ ID NO:58. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 51, the VH CDR2 of SEQ ID NO: 52, and the VH CDR3 of SEQ ID NO: 53, and the VL CDR1 of SEQ ID NO: 56 VL CDR2 of SEQ ID NO:57 and VL CDR3 of SEQ ID NO:58.

In some embodiments, the antibody of the present invention is an anti-CD30 scFv or an antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 139 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity to SEQ ID NO:139. In one embodiment, the anti-CD30 scFv is encoded by the nucleotides of SEQ ID NO:140.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 64 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with it The heavy chain variable region of the amino acid sequence of SEQ ID NO: 69 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 69 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:64. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:69. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO:64 and includes VL having the sequence shown in SEQ ID NO:69. In some embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH with the sequence shown in SEQ ID NO:64 and/or 3 LCDR sequences of VL with the sequence shown in SEQ ID NO:69. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:63 and LCDR3 of SEQ ID NO:68. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:61, the VH CDR2 of SEQ ID NO:62, and the VH CDR3 of SEQ ID NO:63. In some embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:66, VL CDR2 of SEQ ID NO:67, and VL CDR3 of SEQ ID NO:68. In some embodiments, anti CD30 scFv includes: VH CDR1 of SEQ ID NO: 61, VH CDR2 of SEQ ID NO: 62, VH CDR3 of SEQ ID NO: 63, and VL CDR1 of SEQ ID NO: 66, VL CDR2 of SEQ ID NO: 67 , And SEQ ID NO: 68 VL CDR3.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or an antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 142 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with SEQ ID NO:142. In one embodiment, the anti-CD30 scFv is encoded by the nucleotide of SEQ ID NO:143.

In certain embodiments, the anti-CD30 scFv antibody comprises: comprising the amino acid sequence of SEQ ID NO: 74 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with it The heavy chain variable region of the amino acid sequence of SEQ ID NO: 79 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 79 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:74. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:79. In certain embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO: 74 and includes VL having the sequence shown in SEQ ID NO: 79. In some embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH of the sequence shown in SEQ ID NO: 74 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 79. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:73 and LCDR3 of SEQ ID NO:78. In certain embodiments, the anti-CD30 scFv comprises: SEQ The VH CDR1 of ID NO: 71, the VH CDR2 of SEQ ID NO: 72, and the VH CDR3 of SEQ ID NO: 73. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:76, VL CDR2 of SEQ ID NO:77, and VL CDR3 of SEQ ID NO:78. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 71, the VH CDR2 of SEQ ID NO: 72, and the VH CDR3 of SEQ ID NO: 73, and the VL CDR1 of SEQ ID NO: 76 VL CDR2 of SEQ ID NO:77 and VL CDR3 of SEQ ID NO:78.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or an antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 145 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with SEQ ID NO: 145. In one embodiment, the anti-CD30 scFv is encoded by the nucleotide of SEQ ID NO:146.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 84 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity therewith The heavy chain variable region of the amino acid sequence of SEQ ID NO: 89 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 89 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:8157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:84. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:89. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO: 84 and includes VL having the sequence shown in SEQ ID NO: 89. In certain embodiments, anti-CD30 The scFv includes 3 HCDR sequences of VH shown in SEQ ID NO: 84 and/or 3 LCDR sequences of VL shown in SEQ ID NO: 89. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:83 and LCDR3 of SEQ ID NO:88. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:81, the VH CDR2 of SEQ ID NO:82, and the VH CDR3 of SEQ ID NO:83. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:86, VL CDR2 of SEQ ID NO:87, and VL CDR3 of SEQ ID NO:88. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 81, the VH CDR2 of SEQ ID NO: 82, and the VH CDR3 of SEQ ID NO: 83, and the VL CDR1 of SEQ ID NO: 86 VL CDR2 of SEQ ID NO:87 and VL CDR3 of SEQ ID NO:88.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or an antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 148 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity to SEQ ID NO: 148. In one embodiment, the anti-CD30 scFv is encoded by the nucleotides of SEQ ID NO:149.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 94 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with it The heavy chain variable region of the amino acid sequence of SEQ ID NO: 99 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 99 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:94. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:99. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO:94 and includes VL having the sequence shown in SEQ ID NO:99. In some embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH with the sequence shown in SEQ ID NO:94 and/or 3 LCDR sequences of VL with the sequence shown in SEQ ID NO:99. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:93 and LCDR3 of SEQ ID NO:98. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:91, the VH CDR2 of SEQ ID NO:92, and the VH CDR3 of SEQ ID NO:93. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:96, VL CDR2 of SEQ ID NO:97, and VL CDR3 of SEQ ID NO:98. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 91, the VH CDR2 of SEQ ID NO: 92, and the VH CDR3 of SEQ ID NO: 93, and the VL CDR1 of SEQ ID NO: 96 VL CDR2 of SEQ ID NO:97 and VL CDR3 of SEQ ID NO:98.

In some embodiments, the antibody of the present invention is an anti-CD30 scFv or an antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 151 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity to SEQ ID NO: 151. In one embodiment, the anti-CD30 scFv is encoded by the nucleotides of SEQ ID NO:152.

In certain embodiments, the anti-CD30 scFv antibody comprises: comprising the amino acid sequence of SEQ ID NO: 104 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity therewith The heavy chain variable region of the amino acid sequence of SEQ ID NO: 109 or the amino acid sequence of Less than 90%, 92%, 95%, 97%, 98%, 99% or more identical light chain variable regions, and optionally between the heavy chain variable region and the light chain variable region Linkers, such as linker peptides. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:104. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:109. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO: 104 and includes VL having the sequence shown in SEQ ID NO: 109. In some embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH of the sequence shown in SEQ ID NO: 104 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 109. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO:103 and LCDR3 of SEQ ID NO:108. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO:101, the VH CDR2 of SEQ ID NO:102, and the VH CDR3 of SEQ ID NO:103. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO:106, VL CDR2 of SEQ ID NO:107, and VL CDR3 of SEQ ID NO:108. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 101, the VH CDR2 of SEQ ID NO: 102, and the VH CDR3 of SEQ ID NO: 103, and the VL CDR1 of SEQ ID NO: 106 VL CDR2 of SEQ ID NO:107 and VL CDR3 of SEQ ID NO:108.

In certain embodiments, the antibody of the present invention is an anti-CD30 scFv or antigen-binding fragment thereof, which comprises the antigen-binding region of the amino acid sequence SEQ ID NO: 154 or a variant thereof, and specifically binds to a CD30 polypeptide (for example, The CD30 polypeptide having the amino acid sequence of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or more identity to SEQ ID NO:154. In one embodiment, the anti-CD30 scFv is encoded by the nucleotides of SEQ ID NO:155.

In certain embodiments, the anti-CD30 scFv antibody includes: comprising the amino acid sequence of SEQ ID NO: 114 or having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity with it The heavy chain variable region of the amino acid sequence of SEQ ID NO: 119 or at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 119 The variable region of the light chain, and optionally a linker between the variable region of the heavy chain and the variable region of the light chain, such as a linker peptide. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

In certain embodiments, the anti-CD30 scFv includes a VH having the amino acid sequence of SEQ ID NO:114. In certain embodiments, the anti-CD30 scFv includes a VL having the amino acid sequence of SEQ ID NO:119. In some embodiments, the anti-CD30 scFv includes VH having the sequence shown in SEQ ID NO: 114 and includes VL having the sequence shown in SEQ ID NO: 119. In some embodiments, the anti-CD30 scFv includes 3 HCDR sequences of VH shown in SEQ ID NO: 114 and/or 3 LCDR sequences of VL shown in SEQ ID NO: 119. In some embodiments, the anti-CD30 scFv comprises HCDR3 of SEQ ID NO: 113 and LCDR3 of SEQ ID NO: 118. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 111, the VH CDR2 of SEQ ID NO: 112, and the VH CDR3 of SEQ ID NO: 113. In certain embodiments, the anti-CD30 scFv includes: VL CDR1 of SEQ ID NO: 116, VL CDR2 of SEQ ID NO: 117, and VL CDR3 of SEQ ID NO: 118. In certain embodiments, the anti-CD30 scFv comprises: the VH CDR1 of SEQ ID NO: 111, the VH CDR2 of SEQ ID NO: 112, and the VH CDR3 of SEQ ID NO: 113, and the VL CDR1 of SEQ ID NO: 116 The VL CDR2 of SEQ ID NO:117 and the VL CDR3 of SEQ ID NO:118.

III. scFv-Fc antibody

Antibodies with an Fc region have several advantages, including, but not limited to: the Fc region can mediate effector functions, such as CDC and ADCC immunological activities; the dimerization function of the Fc region can form a bivalent antibody, which can provide Strong antigen binding affinity, and/or change the plasma half-life and renal clearance rate; bivalent antibodies can be internalized at a rate different from monovalent Fab or scFv antibodies to change immune function or carrier function. For example, alpha emitters do not require internalization to kill target cells, but many drugs and toxins will benefit from internalization of immune complexes.

Therefore, in a preferred embodiment, a scFv-Fc antibody formed by fusion of the single-chain scFv antibody of the present invention with the Fc region of the antibody is provided. In some embodiments, the antibody comprises a single chain scFv antibody of the invention and a wild-type or altered Fc region. In a preferred embodiment, the antibody comprises from N-terminus to C-terminus: Fc-VH-linker-VL or Fc-VL-linker-VH; or preferably VH-linker-VL-Fc or VL-linker -VH-Fc. In a preferred embodiment, the Fc is linked to the variable region (VH or VL) via a hinge region. In some embodiments, Fc is an Fc region derived from a human immunoglobulin, preferably a human IgG1 or IgG4 Fc region. In a preferred embodiment, the Fc region has the amino acid sequence shown in SEQ ID NO: 161, or contains at least one, two or three, but no more than the amino acid sequence of SEQ ID NO: 161. An amino acid sequence with 20, 10 or 5 amino acid changes, or a sequence with at least 95-99% identity with the amino acid sequence of SEQ ID NO: 161. In some embodiments, the single-chain scFv antibody of the present invention is linked to the Fc region via a hinge region. In one embodiment, the hinge region is a C8 hinge region, for example, comprising the amino acid sequence shown in SEQ ID NO: 159, or at least one, two or three with respect to the amino acid sequence of SEQ ID NO: 159 , But no more than 5 amino acid changes in the amino acid sequence.

In some preferred embodiments, the present invention provides an antibody that specifically binds to a CD30 polypeptide (for example, a CD30 polypeptide having an amino acid sequence of SEQ ID NO: 162 or a fragment thereof), and comprises an antibody selected from SEQ ID NO : 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, The amino acid sequence of 153, and 156, or relative to it contains at least one, two or three, but no more than 20, 10 or 5 amino acid sequences, or the amino acid sequence with at least 80 %, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical amino acid sequences.

Table C below lists the amino acid sequences of some exemplary ScFv-Fc antibodies of the present invention and the amino acid sequences and nucleotide sequences of the single-chain scFv used to construct them. The amino acid sequences and nucleotide sequences of the linkers and hinges used in these scFv-Fc antibodies are shown in Figure 7.

Table C:

In some embodiments, the scFv-Fc antibody of the present invention has effector function. The term "effector function" refers to those biological activities attributable to the Fc-region of an antibody, which vary with antibody class. There are five main antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chains corresponding to different classes of immunoglobulins are called α , δ , ε , γ, and μ, respectively . The effector functions of antibodies include, for example, but not limited to: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptor ( For example, down-regulation of B-cell receptors; and B-cell activation. US20120014943A1 reported that the inclusion of at least one amino acid substitution selected from 239D and 332E in the Fc region can result in an antibody with enhanced Fc γ RIIIa affinity, resulting in enhanced effector function. In some embodiments, the scFv-Fc antibody of the present invention blocks or inhibits the growth of CD30-expressing cells (especially tumor cells, such as lymphoma cells) through effector cell-mediated cytotoxicity (ADCC) activity, and /Or kill the cells.

In certain embodiments, the Fc region may comprise an Fc-region with one or more amino acid substitutions that improve ADCC activity, for example, substitutions at positions 298, 333, and/or 334 of the Fc-region (residue EU Numbering). In some embodiments, changes can also be made to the Fc-region to result in altered (ie, increased or decreased) C1q binding and/or complement dependent cytotoxicity (CDC) (see, for example, US 6,194,551, WO99/51642 and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184).

In other embodiments, changes can be made to Fc to increase or decrease its degree of glycosylation and/or change its glycosylation pattern. The addition or deletion of Fc glycosylation sites can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites. For example, you can implement One or more amino acid substitutions to eliminate one or more glycosylation sites, thereby eliminating glycosylation at that site. Antibodies with altered types of glycosylation can be prepared, such as low or non-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased aliquoted GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies.

Therefore, in some preferred embodiments, the present invention provides antibodies whose Fc region is hypo- or non-fucosylated, so that the Fc domain of the antibody and the Fc γ receptor expressed on effector cells can be significantly increased. (Such as Fc γ RIIIa) binding affinity, which results in antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. For example, the amount of fucose in the antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. It can be measured by MALDI-TOF mass spectrometry to calculate the fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (such as complex, hybrid and high-mannose structures) connected to Asn 297 The average amount, from which the amount of fucose is determined, is for example described in WO 2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence changes in the antibody, Asn297 may also be located approximately ±3 upstream or downstream of position 297 There are two amino acid positions, that is, between positions 294 and 300. See, for example, US2003/0157108; US2004/0093621. Examples of publications related to "defucosylation" or "hypofucosylation" antibody variants also include: US2003/0157108; WO 2000/61739; WO2001/29246; US2003/0115614; US2002/0164328; US 2004/0093621; US2004/0132140; US2004/0110704; US2004/0110282: US2004/0109865; WO2003/085119: WO2003/084570; WO2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki, A. etc., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87:614 (2004) 614-622. Such antibody variants can be produced in cell lines capable of producing afucosylated or hypofucosylated antibodies. Examples of such cells include Lec13 CHO cells deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986): 533-545; US 2003/0157108; and WO 2004/056312, Especially Example 11); and gene knockout cell lines, such as α -1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki, N., etc., Biotech. Bioeng. 87:614 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107). For another example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 ( α (1,6)-fucosyltransferase), so that the lack of fucosyltransferase can be expressed in the Ms704, Ms705 and Ms709 cell lines. Sugar antibodies. In addition, EP 1,176,195 also describes cell lines with a functionally disrupted FUT8 gene, and antibodies expressed in such cell lines exhibit hypofucosylation. Alternatively, fucosidase can also be used to cleave fucose residues of antibodies; for example, fucosidase α -L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14: 5516-23).

In addition, the present invention also contemplates antibody variants with bisected oligosaccharides, for example, antibodies in which the biantennary oligosaccharides linked to the Fc region are equally divided by GlcNAc. These antibody variants may have reduced fucosylation and/or increased ADCC function. Examples of these antibody variants are described in, for example, WO 2003/011878; US 6,602,684; and US 2005/0123546. The present invention also considers antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region. These antibody variants may have increased CDC function. These antibody variants are described in, for example, WO 1997/30087; WO 1998/58964; and WO 1999/22764.

Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in US 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. Etc., Proc. Nat'l Acad. Sci. USA82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assay methods (see, for example, ACTI ^TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox96 ^® non-radioactive cytotoxicity assay (Promega, Madison, WI) ). Suitable effector cells for these assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be evaluated in vivo, for example, in an animal model as disclosed in Clynes, R. et al., Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656. To evaluate complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS And MJ Glennie, Blood103 (2004) 2738-2743). C1q binding assays can also be performed to determine the C1q binding and CDC activity of the antibody. See, for example, C1q and C3c binding ELISA in WO2006/029879 and WO2005/100402.

In certain embodiments, the present invention also considers antibody variants with some but not all effector functions, which makes it an ideal candidate for certain applications where the in vivo half-life of the antibody is important, but certain Some effector functions (such as complement and ADCC) are unnecessary or harmful. The in vitro and/or in vivo assays described above can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assay to ensure that the antibody lacks Fe γ R binding (hence likely lacking ADCC activity), but retain the ability to bind FcRn. For example, the Fc region may contain mutations that eliminate or reduce effector function, such as a human IgG1 Fc region with mutations P329G and/or L234A and L235A, or a human IgG4 Fc region with mutations P329G and/or S228P and L235E.

In some embodiments, the scFv-Fc region antibody of the present invention can form a bivalent antibody by dimerization of the Fc region, which can further have increased total antibody affinity and stability, Or form multispecificity such as bispecificity. For example, the Fc region may comprise i) a homodimeric Fc-region of human IgG1 subclass, or ii) a homodimeric Fc-region of human IgG4 subclass, or iii) a heterodimeric Fc-region, wherein a) an Fc-region The region polypeptide contains the mutation T366W, and another Fc-region polypeptide contains the mutations T366S, L368A, and Y407V, or b) one Fc-region polypeptide contains the mutations T366W and Y349C, and the other Fc-region polypeptide contains the mutations T366S, L368A, Y407V, S354C, or c) One Fc-region polypeptide contains the mutations T366W and S354C, while the other Fc-region polypeptide contains the mutations T366S, L368A, Y407V, and Y349C.

In some embodiments, the scFv-Fc recombinant antibody of the present invention can be directly fused or conjugated to other molecules by grouping in the Fc region portion, including but not limited to, fluorescent dyes, cytotoxins, radioisotopes, etc., for example, for antigen Quantitative research, immobilization of antibodies for affinity measurement, targeted delivery of therapeutic agents, Fc-mediated cytotoxicity testing using immune effector cells, and many other uses.

B. Polynucleotide and host

In one aspect, the present invention provides a substantially purified nucleic acid molecule that encodes a polypeptide comprising a segment or domain of the above-mentioned CD30-binding antibody chain. In some embodiments, the nucleic acid molecule of the invention encodes an antibody chain that binds to CD30 (for example, any antibody of the invention, including single chain scFv antibodies and scFv-Fc antibodies, and fragments thereof).

Some nucleic acids of the present invention comprise nucleotide sequences encoding the heavy chain variable region of any antibody shown in Table A or its variants, and/or the light chain variable region of the corresponding antibody shown in Table A or its variants. Nucleotide sequence. In a specific embodiment, the nucleic acid molecule is the DNA VH sequence and/or DNAVL sequence listed in Table A. Some other nucleic acid molecules of the present invention comprise nucleotide sequences that are substantially identical (eg, at least 65%, 80%, 95%, or 99% identical) to the nucleotide sequences of the nucleic acid molecules shown in Table A. From When expressed in an appropriate expression vector, the polypeptides encoded by these polynucleotides can display CD30 antigen binding ability.

The present invention also provides polynucleotides, which encode at least one CDR region and usually all three CDR regions from the heavy chain VH or light chain VL sequence of the above-mentioned CD30-binding antibody. In some further embodiments, the polynucleotide encodes the complete or substantially complete variable region sequence of the heavy chain and/or light chain of the CD30-binding antibody described above.

As those skilled in the art understand, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

Some nucleic acid sequences of the present invention comprise a nucleotide sequence encoding a heavy chain VH, which comprises: (i) selected from SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105 The nucleotide sequence of, and 115 or a nucleotide sequence having, for example, at least 80%, 90%, or 99% identity therewith. Some other nucleic acid sequences include the nucleotide sequence encoding the light chain VL, which includes the nucleotide sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 Or a nucleotide sequence with, for example, at least 80%, 90%, or 99% identity therewith.

In some embodiments, the nucleic acid sequence of the invention encodes any of the aforementioned single chain scFv antibodies of the invention. In some embodiments, the nucleic acid sequence of the present invention encoding a scFv antibody comprises a nucleotide sequence encoding a heavy chain VH sequence and a nucleotide sequence encoding a light chain VL sequence selected from:

(i) the sequence of SEQ ID NO: 5 or a sequence substantially the same, and the sequence of SEQ ID NO: 10 or a sequence substantially the same,

(ii) the sequence of SEQ ID NO: 15 or a sequence substantially the same, and the sequence of SEQ ID NO: 20 or a sequence substantially the same,

(iii) the sequence of SEQ ID NO: 25 or a sequence substantially the same, and the sequence of SEQ ID NO: 30 or a sequence substantially the same,

(iv) the sequence of SEQ ID NO: 35 or a sequence substantially the same, and the sequence of SEQ ID NO: 40 or a sequence substantially the same,

(v) the sequence of SEQ ID NO: 45 or a sequence substantially the same, and the sequence of SEQ ID NO: 50 or a sequence substantially the same,

(vi) the sequence of SEQ ID NO: 55 or a sequence substantially the same, and the sequence of SEQ ID NO: 60 or a sequence substantially the same,

(vii) the sequence of SEQ ID NO: 65 or a sequence substantially the same, and the sequence of SEQ ID NO: 70 or a sequence substantially the same,

(viii) the sequence of SEQ ID NO: 75 or a sequence substantially the same, and the sequence of SEQ ID NO: 70 or a sequence substantially the same,

(ix) the sequence of SEQ ID NO: 85 or a sequence substantially the same, and the sequence of SEQ ID NO: 90 or a sequence substantially the same,

(x) the sequence of SEQ ID NO: 95 or a sequence substantially the same, and the sequence of SEQ ID NO: 100 or a sequence substantially the same,

(xi) the sequence of SEQ ID NO: 105 or a sequence substantially the same, and the sequence of SEQ ID NO: 110 or a sequence substantially the same,

(xii) The sequence of SEQ ID NO: 115 or a sequence substantially the same, and the sequence of SEQ ID NO: 120 or a sequence substantially the same.

In a preferred embodiment, the nucleic acid of the present invention encoding the scFv antibody further comprises a nucleotide sequence encoding a linker, such as the sequence shown in SEQ ID NO: 158 or a sequence substantially the same.

In a more preferred embodiment, the nucleic acid of the present invention encoding the scFv antibody comprises a sequence selected from SEQ ID NO: 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, and 155 , Or its basically the same sequence.

In any of the above embodiments, in a preferred aspect, the "substantially identical" nucleotide sequence refers to a nucleotide sequence that is at least 80%, 85%, 90%, 90%, 92% in sequence with the reference nucleotide sequence. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. The identity of the nucleotide sequence can be determined using various sequence alignment methods known in the art. For example, a BLAST sequence alignment search tool can be obtained from the website of NCBI (National Center for Biotechnology Information, Bethesda, MD). Typically, percent identity is performed using NCBI Blast default parameters.

These polynucleotide sequences can be generated by de novo solid-phase DNA synthesis, or by PCR mutagenesis of existing sequences encoding CD30-binding antibodies or binding fragments thereof (for example, the sequences shown in Tables A-C). The direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68: 90; Brown et al., the phosphoric acid of Meth. Enzymol. 68: 109, 1979 The diester method; Beaucage et al., Tetra. Lett., 22: 1859, 1981, the diethyl phosphatidimide method; and the solid phase support method of US Patent No. 4,458,066. The introduction of mutations into the polynucleotide sequence by PCR can be like, for example, PCR Technology: Principles and Applications for DNA Amplification, HA Erlich (eds), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis, etc. (Eds.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

C. Antibody preparation

Antibodies can be produced using recombinant methods and compositions, such as those described in US 4,816,567.

In one embodiment, a vector comprising an isolated nucleic acid encoding an antibody that binds CD30 described herein is provided. This nucleic acid may encode the amino acid sequence of the VL of the antibody and/or the amino acid sequence of the VH of the antibody. In a further embodiment, the vector is an expression vector. In a further embodiment, the invention provides a host cell comprising this nucleic acid. In one embodiment, the host cell contains (e.g., has been transformed with the following vector): (1) a vector containing a nucleic acid encoding the amino acid sequence of the antibody VL and the amino acid sequence of the antibody VH, or (2) The first vector encoding the nucleic acid containing the amino acid sequence of the antibody VL and the second vector containing the nucleic acid encoding the amino acid sequence of the antibody VH. In one embodiment, the host cell is eukaryotic, such as Chinese Hamster Ovary (CHO) cells, HEK293 cells, or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of preparing an anti-CD30 antibody is provided, wherein the method comprises culturing a host cell containing a nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression, and obtaining the host cell (or host The antibody is recovered from the cell culture medium).

For the recombinant production of anti-CD30 antibodies, nucleic acid encoding the antibody, such as the nucleic acid as described above, can be isolated and inserted into one or more vectors for further selection and/or expression in host cells. This nucleic acid can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chain variable regions).

A variety of expression vectors can be used to express polynucleotides encoding CD30-binding antibody chains (for example, any antibody of the present invention, including scFv antibodies and scFv-Fc antibodies). Both viral-based expression vectors and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems It includes plasmids, episomal vectors and artificial chromosomes, and generally contains an expression cassette for expressing protein or RNA (see, for example, Harrington et al., Nat Genet 15:345, 1997). Useful viral vectors include vectors based on retrovirus, adenovirus, adeno-associated virus, herpes virus, vectors based on SV40, papilloma virus, HBP EB virus, vaccinia virus vector, and Semliki Forest virus (SFV). See, Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Generally, an expression vector contains a promoter operably linked to a polynucleotide encoding an antibody chain or polypeptide that binds CD30. In addition to promoters, other regulatory elements may also be required or required for efficient expression of CD30-binding antibody chains or fragments. These elements usually include the ATG start codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be enhanced by incorporating enhancers suitable for the cell system used (see, for example, Scharf et al., Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al., Meth. Enzymol., 153 : 516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

The expression vector can also provide a secretion signal sequence to form a fusion protein containing a CD30 binding polypeptide. Alternatively, the CD30 binding antibody/polypeptide sequence can also be linked to the signal sequence before being inserted into the vector. In a preferred embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:164. The vector used to receive the sequence encoding the CD30-binding antibody light chain variable domain and heavy chain variable domain may sometimes also encode a constant region or part thereof. Such vectors allow the variable region to be expressed as a fusion protein with the constant region, thereby leading to the production of whole antibodies or fragments thereof. Generally, such constant regions are human constant regions such as human IgG1 Fc region. In a preferred embodiment, the Fc region fused to the variable region contains the amino acid sequence shown in SEQ ID NO:161.

Suitable host cells for the selection or expression of the vector include prokaryotic or eukaryotic cells. For example, especially when glycosylation and Fc effector functions are not required, antibodies can be produced in bacteria. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US5,648,237, US5,789,199 and US5,840,523. (See also Charlton, KA, in: Methods in Molecular Biology, Volume 248, Lo, BKC (Editor), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli) . After expression, the antibody can be separated from the bacterial cell paste in the soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable selection or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", which results in some Or the production of antibodies with fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech (2006) 24: 210-215. Suitable host cells for expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified, which can be used in combination with insect cells, especially for the transfection of Spodopterafrugiperda cells. Plant cell cultures can also be used as hosts. See, for example, US5,959,177, US6,040,498, US6,420,548, US7,125,978 and US6,417,429 (description of PLANTIBODIES ^™ technology for producing antibodies in transgenic plants). Vertebrate cells that can be used as hosts include, for example, suspension growth-adapted mammalian cell lines can be useful. Other examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or, for example, Graham, FL et al., J. GenVirol. 36 (1997) 59 described in 293 Cells); Baby Hamster Kidney Cells (BHK); Mouse Sertoli Cells (e.g. TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-251); Monkey Kidney Cells (CV1); Africa Green Monkey Kidney Cells (VERO-76); Human Cervical Cancer Cells (HELA); Canine Kidney Cells (MDCK); Buffalo Rat Hepatocytes (BRL3A); Human Lung Cells (W138); Human Hepatocytes (HepG2); Mice Breast tumors (MMT 060562); TRI cells such as those described in Mather, JP et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR ^. CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of some mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methodsin Molecular Biology, Vol. 248, Lo. BKC (editor), HumanaPress, Totowa, NJ (2004) pp.255-268. In some preferred embodiments, mammalian host cells are used to express and produce CD30-binding antibody polypeptides of the invention.

D. Screening, identification and characterization of antibodies

The anti-CD30 antibodies provided herein can be screened, identified, or characterized by various tests known in the art for their physical/chemical properties and/or biological activity.

Phages that bind to the target antigen of interest with high affinity can be selected from a phage display library expressing human antibodies. There are a variety of methods for displaying antibodies or antibody fragments on the surface of phage and screening libraries, see, for example, Shao Rongguang et al. (Editor), Antibody Drug Research and Application, People's Medical Publishing House (2013). For example, phage display library screening can be performed by magnetic bead sorting (MACS). For example, a group of phage presenting intact antibodies or antibody fragments can be exposed to biotinylated target antigens and streptavidin magnetic beads, incubated in the liquid phase for a period of time, then washed, and enriched with phage bound to the target antigen by the action of a magnetic field. , Then the phage is eluted and amplified. After several times of "adsorption-elution-amplification", the obtained phages that specifically bind to the antigen are selected and sequenced.

It is also possible to use transgenic animals expressing human immunoglobulin libraries, such as transgenic mice, to screen for fully human antibodies that bind to the target antigen with high affinity. For example, the transgenic mouse can be immunized with the target antigen, and then the spleen of the mouse can be obtained and fused with myeloma cells, and the fusion tumor producing the desired antibody can be screened and sequenced.

For the identification of antibodies, the antigen binding activity can be identified or characterized by known methods such as ELISA, α LISA, Western blot, antibody or reversed-phase array, etc., as well as the methods described in the examples. antibody.

For example, antibodies can be spotted on glass or nitrocellulose chips. Block and incubate the slides with a solution containing CD30, wash to remove unbound antibody, and detect the bound antibody with the corresponding fluorescently labeled secondary antibody. The fluorescent signal is measured by a fluorescent slide scanner. Similarly, for reversed-phase arrays, spot recombinant CD30, cell supernatant, cell or tissue lysate, body fluids, etc., on glass or nitrocellulose chips. The slides were blocked and the array was incubated with antibodies against specific epitopes on CD30. The unbound antibody is washed away, and the bound antibody is detected with the corresponding fluorescently labeled secondary antibody. The fluorescent signal is measured by a fluorescent slide scanner (Dernick, G. et al., J. Lipid Res., 52 (2011) 2323-2331).

The ForteBio assay can also be used to detect antibodies. ForteBio affinity determination can be carried out according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p.270-8). For example, an AHC (anti-hIgG-Fc capture surface) sensor can be used to perform kinetic analysis of the interaction between the scFv-hFc antibody and the antigen molecule.

The binding of antibodies to cells expressing CD30 on the surface can also be detected by flow cytometry. For example, Karpas299 cells expressing CD30 can be incubated with serially diluted antibodies for a period of time (for example, 4°C, 30 minutes). Then incubate with a secondary antibody (such as a secondary antibody labeled with phycobilichrome) for a period of time (such as 4 ℃, 30 minutes). After washing the cells, the cells were analyzed by flow cytometry. Flow cytometry can be performed on the Accuri C6 system (BD Biosciences), and the EC50 value can be calculated using Graphpad software.

E. Fusions and conjugates

In yet another aspect, the invention provides fusions or conjugates comprising the antibodies of the invention. Fusions or conjugates can be produced by fusing or conjugating the antibodies of the invention to heterologous molecules. In some embodiments, the antibody polypeptide of the present invention can be fused or conjugated with one or more heterologous molecules, where the heterologous molecules include but are not limited to proteins/polypeptides/peptides, markers, drugs, and cytotoxic agents. Methods of fusion or conjugation of proteins, polypeptides or peptides or chemical molecules with antibodies are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929 and EP 367,166.

In one embodiment, the antibody of the present invention is recombinantly fused with a heterologous protein or polypeptide or peptide to form a fusion protein. In yet another embodiment, the antibody of the invention is conjugated to a protein molecule or a non-protein molecule to produce a conjugate.

In some embodiments, the antibodies of the invention may be fused or conjugated to heterologous molecules in the form of full-length antibodies or antibody fragments. In a preferred embodiment, the single chain scFv antibody of the present invention is used for fusion or conjugation. In a further preferred embodiment, a fusion protein comprising the single chain scFv of the present invention is provided. Such fusion proteins can be easily prepared by recombinant methods known in the art. In yet another preferred embodiment, a conjugate comprising the single-chain scFv of the present invention is provided, for example, a conjugate comprising the scFv of the present invention and a non-protein drug molecule.

Linkers can be used to covalently link different entities in the fusions and/or conjugates of the invention. The linker includes a chemical linker or a single-chain peptide linker. In some embodiments, the single-chain antibody of the present invention, such as scFv antibody, is fused to other peptides or proteins via a peptide linker. In some embodiments, The single-chain antibody of the present invention, such as scFv antibody, is conjugated to other molecules such as markers or drug molecules via chemical linkers.

Peptide linkers that can be used to form the present invention include peptides composed of amino acid residues. Such linker peptides are generally flexible, allowing the antigen binding moiety such as scFv to be linked to move independently. The length of the linker peptide can be easily determined by those skilled in the art according to the actual situation, for example, at least 4-15 amino acids in length, or longer, for example, about 20-25 amino acids.

The chemical linker that can be used to form the present invention includes, for example, various coupling agents. Examples of coupling agents are N-succinimidyl-3-(2-pyridinedisulfanyl) propionate (SPDP), succinimidyl-4-(N-maleimidinyl)cyclohexane Alkyl-1-carboxylate (SMCC), iminosulfurane (IT), bifunctional derivatives of imidate (such as dimethyl adipimide HCl), active ester (such as suberic acid) Disuccinimidyl ester), aldehydes (such as glutaraldehyde), diazide compounds (such as bis(p-azidobenzoyl) hexamethylene diamine), bis-diazo derivatives (such as bis-(p- Diazobenzyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and double-reactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In addition, the linker may be a "cleavable linker" that facilitates the release of the polypeptide after delivery to the target site. For example, acid-labile linkers, peptidase-sensitive linkers, light-labile linkers, dimethyl linkers or disulfide-containing linkers can be used (Chari et al., Cancer Research 52 (1992) 127-131; US 5,208,020) .

F. Methods and compositions for diagnosis and testing

In one aspect, the present invention provides the use of the anti-CD30 antibody, fusion or conjugate of the present invention in diagnosis and detection. Any of the anti-CD30 antibodies, fusions or conjugates provided herein can be used to detect the presence of human CD30 in a biological sample. The term "detection" as used herein includes quantitative or quantitative Sexual testing. Exemplary detection methods include, but are not limited to, immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody molecule complexed magnetic beads, ELISA assays, PCR-technology (e.g., RT-PCR). In some embodiments, the biological sample includes body fluids, cells, or tissues. In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin.

In one embodiment, an anti-CD30 antibody, fusion or conjugate is provided for use in a diagnostic, prognostic or detection method. In a further aspect, a method of detecting the presence of CD30 in a biological sample is provided. In some embodiments, the method includes contacting the biological sample with the anti-CD30 antibody, fusion or conjugate described herein under conditions that allow the anti-CD30 antibody, fusion or conjugate to bind to CD30, and detecting the anti-CD30 antibody, fusion or conjugate. Whether a complex is formed between CD30 antibody, fusion or conjugate and CD30. Such methods can be in vitro or in vivo methods.

In some embodiments, the antibodies of the present invention can be used to diagnose or prognose CD30-related disorders, where exemplary disorders include, for example, classical Hodgkin's lymphoma, anaplastic large cell lymphoma, and the like. Reed-Sternberg cells of classic Hodgkin's lymphoma express CD30 and CD15. The lack of CD30 and CD15 expression can separate nodular lymphocyte dominant Hodgkin's lymphoma (NLPHL) from classic Hodgkin's lymphoma. CD30 expression is also an essential feature of ALCL, including ALK-positive and ALK-negative ALCL. In addition, Lymphomatoid Papulosis (Lymphomatoid Papulosis, LyP) also has the characteristic expression of CD30. It has been reported that in the serum from patients with anaplastic large cell lymphoma and Hodgkin’s disease, increased soluble CD30 levels are associated with poor prognosis (Younes & Kadin, 2003, Journal of Clinical Oncology, 21(18): 3526- 3534; Al-Shamkhani, 2004, Current Opinion in Pharmacology, 4:355-359).

In some embodiments, anti-CD30 antibodies, fusions or conjugates can be used to select subjects suitable for treatment with anti-CD30 antibodies. In some embodiments, the use of the present invention is provided A method for stratifying a patient with antibodies, fusions or conjugates, the method comprising determining whether cells of the patient, such as tumor cells, express CD30 protein on the surface of the cells, wherein the cells are on the surface If the CD30 protein is expressed, the patient will likely respond and be treated with a therapeutic agent targeting CD30 (for example, an anti-CD30 antibody).

In some embodiments, the anti-CD30 antibody can be conjugated to a diagnostic or detectable agent. In some embodiments, the present invention provides a kit for diagnosis or detection, which comprises any anti-CD30 antibody, fusion or conjugate of the present invention.

G. Methods and compositions for treatment

In yet another aspect, the present invention relates to a method for treating a CD30-related disorder, which comprises administering to the subject an effective amount of the antibody or antigen-binding fragment thereof, or the conjugate or fusion of the present invention.

The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and large animals). mouse). In particular, the subject is a human.

The term "treatment" refers to a clinical intervention intended to alter the natural course of disease in an individual being treated. The desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.

CD30-related disorders are disorders mediated or related to CD30 or CD30-expressing cells (especially neoplastic cells), including, but not limited to, CD30-positive tumors and autoimmune diseases. Can make Exemplary disorders treated with CD30 antibodies include, for example, Hodgkin's disease, anaplastic large cell lymphoma (ALCL), adult T cell leukemia/lymphoma (ATL), extranodal NK/T cell lymphoma, skin T cell lymphoma Tumor (CTCL), serum immunoblastic lymphadenopathy-like T-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), granuloma fungoides (MF), lymphomatoid papulosis (LyP), immunoblast Lymphoma, multiple myeloma.

As shown in the examples, the present inventors constructed the antibodies of the present invention based on antibody sequences screened from the human antibody library. Therefore, advantageously, in some embodiments, the antibody of the present invention is a fully human antibody comprising a fully human VH region and a fully human VL region amino acid sequence, such as the antibodies shown in Table A and Table C The single chain scFv shown and the scFv-Fc antibody containing human hFc fragment constructed therefrom. In some embodiments, the conjugates and fusions of the invention are conjugates and fusions comprising fully human antibodies, such as fully human single chain scFv. Therefore, in a preferred aspect, the antibodies, fusions and conjugates of the present invention are particularly suitable for human therapeutic applications. In some preferred embodiments, the antibodies, conjugates and fusions of the present invention are used to treat human CD30-related disorders, such as CD30-positive tumors or autoimmune diseases involving immune cells expressing CD30, preferably, Hodge Gold lymphoma (HL) and non-Hodgkin lymphoma (NHL).

In some embodiments, the administration of the anti-CD30 antibody, conjugate or fusion of the present invention results in growth inhibitory or killing effect on CD30-expressing tumor cells in vivo. Such tumor cells include, for example, Hodgkin cells, Reed-Sternberg cells, HRS cells, anaplastic large cell lymphoma (ALCL) cells, skin T cell lymphoma cells, pleomorphism and Immunoblastic lymphoma cells. In a specific embodiment, the human antibody of the present invention or a conjugate or fusion containing it is used in the treatment of Hodgkin’s lymphoma (especially classic Hodgkin’s lymphoma) to inhibit/mediate the killing of Hodgkin cells And Rees' cells. In another specific embodiment, the human antibody of the present invention or a conjugate or fusion comprising the same is used to inhibit/mediate the killing of CD30-positive ALCL tumor cells in the treatment of ALCL.

It is understood that the CD30 antibody of the present invention, or the conjugate or fusion of the present invention can be administered in combination with other treatment modalities for the treatment of the aforementioned diseases such as tumors. The other forms of treatment include therapeutic agents, radiotherapy, chemotherapy, transplantation, immunotherapy and the like. In some embodiments, the antibody molecule of the invention, or the conjugate or fusion of the invention is used in combination with other therapeutic agents. Exemplary therapeutic agents include cytotoxic agents, radiotoxins, immunosuppressants, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutics, radiotherapy agents, therapeutic antibodies or other active agents and adjuvants, For example, anti-tumor drugs. In some embodiments, the antibody molecule of the invention is conjugated to a therapeutic agent, for example, to a cytotoxin or a radioisotope.

H. Composition and preparation

The invention also contemplates a composition comprising any one or more CD30 binding antibody molecules, fusions, conjugates, polynucleotides, vectors, or host cells herein. The composition includes, but is not limited to, a pharmaceutical composition. The pharmaceutical composition may be used for administration to cells or animals alone or in combination with one or more other treatment modalities.

Pharmaceutical formulations of the antibody or fusion or conjugate of the present invention can be prepared, for example, by making the antibody, fusion or conjugate of the desired purity together with one or more pharmaceutically acceptable carriers (Renmington's Pharmaceutical Science, 16th edition, Osol, A. (Editor) (1980)) mixed and prepared in the form of a lyophilized preparation or an aqueous solution. The pharmaceutically acceptable carrier is generally non-toxic to the recipient at the dose and concentration used, and includes but is not limited to: buffers, such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionamine Acid; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethylene chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; Methyl or Propyl Hydroxybenzoate; Catechol; Resorcinol Phenol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as poly(ethylene Pyrrolidone); amino acids such as glycine, glutamine, aspartame, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or Dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as Zn-protein complexes); and/or non- Ionic surfactants, such as polyethylene glycol (PEG).

Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US6,171,586 and WO2006/044908, the latter formulations include histidine-acetate buffer.

The formulations herein can also contain more than one active ingredient as needed for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

The following examples are described to assist the understanding of the present invention. The examples are not intended and should not be construed in any way to limit the scope of protection of the present invention.

Example Example 1. Screening of anti-CD30 fully human antibodies using transgenic mice

Using fusion tumor technology, use recombinant human CD30 protein (Acro, article number: CD0-H5229) to immunize Harbour H2L2 transgenic mice (manufacturer: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.; license number: SCXK (京) 2016-0006 ), and then fusion of mouse spleen cells and myeloma cells to obtain fusion tumor cells capable of expressing CD30 antibody. The specific process is as follows.

Immunized transgenic mice: Ribi adjuvant (manufacturer: Sigma; NO: S6322; Lot: 077M4061V), proteins, etc. mixed with a volume huCD30 per mice (total of 5 known) 50 μ g / 200 μ l immunization, wherein 100 μl was injected subcutaneously at two points, and 100 μl was injected intraperitoneally. I will be immunized every two weeks for a total of 5 immunizations. Three days before the fusion, the dose of 50 μg/300 μl huCD30 protein per mouse was intraperitoneally injected for subsequent fusion experiments.

Preparation of the electrofusion dish: Thoroughly soak the electrofusion dish with 70% ethanol, and dry it in a clean bench for later use.

Isolation of spleen cells: the mice were sacrificed by cervical dislocation, the body surface was disinfected with 75% alcohol for 5 minutes, and then placed on the mouse dissecting board in the ultra-clean bench, lying on the left side, and fixing the limbs with a 7-gauge needle. Open the abdominal cavity aseptically to take out the spleen, wash with basal medium (preparation method as shown in Table 1), and carefully remove the surrounding connective tissue. The spleen was then transferred to another petri dish containing basal medium. Press the spleen with an elbow needle, insert a hole on the spleen with a small needle, and squeeze it with forceps to fully release the spleen cells to make a spleen cell suspension. The cell suspension was filtered through a 70μM cell sieve and washed with 30ml basal medium and centrifuged at 1200rpm for 6min.

Table 1: Basic medium

Lysis of red blood cells: remove the supernatant after centrifugation of the spleen cell suspension, and resuspend the cells with 10ml RBC Lysis Buffer (GIBCO). Then add 20ml RBC lysis buffer. The suspension was allowed to stand for 5 minutes and then centrifuged at 1100 rpm for 6 minutes. After removing the supernatant, resuspend the cells with 10ml of basal medium, then add 30ml of basal medium and centrifuge at 1100rpm for 6min. After removing the supernatant, the cells were resuspended in 20 ml of basal medium and counted.

Electrofusion: Resuspend mouse myeloma SP2/0 cells (ATCC) in 20ml basal medium and count them. Mix SP2/0 and spleen cells after lysis of red blood cells at a ratio of 1:2 to 1:1, and centrifuge at 1000rpm for 6min. After removing the supernatant, the mixed cells were resuspended in 10 ml of fusion buffer (BTXpress). Then add 15 ml of fusion buffer, centrifuge at 1000 rpm for 5 min, and remove the supernatant. After repeating the above steps, add an appropriate volume of fusion buffer to resuspend the cells, and adjust the mixed cell density to 1×10 ⁷ cells/ml. The parameter settings of the electrofusion instrument are shown in Table 6. Add 2ml of the above cell suspension to each electrofusion dish for electrofusion.

Electrofusion instrument parameter setting

Plating after electrofusion: cells are allowed to stand for 5 min at room temperature in an electrofusion dish. Transfer the cells into a centrifuge tube, and dilute the cells to 1~2×10 ⁴ cells/ml with the selection medium (the preparation method is shown in Table 2 below). Add 100μl of cell suspension to each well of 96-well plate. The selection medium was replaced on the 7th day after fusion. After the 10th day of culture (or longer, according to the cell growth status), the fusion tumor cells expressing specific anti-CD 30 antibodies were screened by flow cytometry (FACS).

Table 2 Screening medium

Sub-selection of positive fusion tumor cells:

Sub-selection step: prepare a 96-well plate, and add 200 μl of the basal medium as described above to each well of rows 2 to 8. Add 300ul of the cells in the positive wells selected by the above fusion to each well in the first row at a density of about 1×10 ⁵ cells/ml. Using the row grab, add 100 μl of the cell suspension from the first row to the second row, mix well and add 100 μl to the next row. Repeat the above steps until the volume of the last column becomes 300μl; let the 96-well plate stand for 15min, observe and count under the microscope. Take the volume corresponding to 100 cells and add 20ml of the basal medium as described above, and mix and spread the plate, 200μl per well. Observe under the microscope one week later, judge and mark simple plant holes, and pick out positive holes to be tested.

Cryopreservation of cells: Observe the cell status, and when the cells grow well and their viability is >90%, centrifuge at 1000 rpm for 5 minutes to remove the supernatant. Resuspend the cells with freezing solution (45.5% FBS (Hyclone), 44.5% RPMI-1640 (Hyclone), 10% DMSO) to 1×10 ⁷ cells/ml, aliquot them into cryopreservation tubes, and put them into the program cooling box Medium, freeze at -80℃.

Fusion tumor sequencing:

Use NucleoSpin RNA Plus (Macherey-Nagel, catalog number 740984.250) kit for RNA extraction: Take fresh cells and centrifuge at 1000 rpm for 5 min, remove the supernatant, add 350 μl of cell lysis buffer LBP to the pellet, and mix until clear. Add to the DNA removal tube, centrifuge at 11000rpm for 30s, and collect the flow-through. Add 100 μl of binding solution BS to the flow-through solution, and mix until clear. Add the clear solution to the RNA collection tube, centrifuge at 11000 rpm for 1 min, remove the liquid, add 200 μl washing solution WB1, centrifuge at 11,000 rpm for 15 seconds, discard the liquid; open the lid of the RNA column and move it to a new collection tube, add 600 μl washing solution WB2, centrifuge at 11,000 rpm for 15 seconds, discard the liquid; add 250 μl washing solution WB2, centrifuge at 11,000 rpm for 2 min, discard the liquid; after centrifugation to completely volatilize the ethanol, add 30 μl DEPC water to the collection column Centrifuge at 12000g for 2min, and collect the eluate. Determine the RNA concentration.

Use PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) to obtain cDNA by reverse transcription. The steps are as follows (all the reagents mentioned are from this kit):

Prepare reaction system I as shown in Table 3

Table 3: Reaction System I

After incubating at 65°C for 5 min, quickly cool on ice. Add reaction system I to the following reverse transcription system (Table 4), the total amount is 20 μl :

Table 4: Reverse transcription system

After mixing slowly, perform reverse transcription and translation according to the following conditions: 42°C 60min→95°C 5min, then cool on ice to obtain cDNA.

Use Mighty TA-cloning Kit (Takara) to connect cDNA to T vector, the steps are as follows:

PCR amplifies the variable regions of the heavy and light chains respectively. The PCR reaction system is shown in Table 5 below.

Table 5: PCR reaction system

The PCR reaction conditions are shown in Table 6 below.

Table 6: PCR reaction conditions

The primers used in the PCR reaction are shown in Tables 7 and 8 below.

Table 7. CD30 antibody heavy chain variable region (VH) primer (Primer Mix 1)

After the above primers are mixed in equal proportions, Primer Mix1 is obtained for PCR amplification of VH.

Table 8. CD30 antibody light chain variable region (VL) primers (Primer Mix 2):

After the above primers are mixed in equal proportions, Primer Mix 2 is obtained for PCR amplification of VL.

Take 4.5 μl of the PCR product obtained in the above PCR reaction, add 0.5 μl pMD20-T vector (Takara), 5 μl Ligation Mighty Mix (Takara), gently mix, and react at 37° C. for 2 hours to obtain the ligation product.

Transformed cells:

Take out TOP10 competent (Tiangen Biochemical Technology (Beijing) Co., Ltd.) at -80°C, melt on ice, take 5 μl of the ligation product obtained above and add to the melted TOP10 competent, mix well and incubate on ice for 30 min. After a heat shock at 42°C for 90 seconds, it was quickly cooled on ice for 2 minutes, and 900 μl of LB medium (Sanggong Bioengineering (Shanghai) Co., Ltd.) was added to the EP tube, and cultured on a shaker at 37°C at 220 rpm for 1 hour. Centrifugation at 3000g for 2min, 800 μ l supernatant was removed by suction, the remaining bacteria were resuspended with culture medium and plated on ampicillin-resistant plates amine. Incubate overnight at 37°C, select pure strains for sequencing, and obtain the VH and VL sequences in the fusion tumor after analysis. Finally, 10 antibodies were obtained, HB38E4, HB10F1, HB49G9, HB36F7, HB68H2, HB69G7, HB10B6, HB16H7, HB6A9, HB16H8, and their sequences are shown in Table A.

Example 2. Screening of fully human antibodies against CD30 by phage display technology

In addition to using transgenic mice to produce fully human antibodies against CD30, phage display technology is also used to prepare antibodies. Screen the fully human antibodies that specifically bind to CD30 from 6 synthetic antibody libraries with a total diversity greater than 1×10 ¹⁰ (for library design and construction, please refer to Raffi Tonikian et al. Nature Protocols, 2007 and Thomas A. Kunkel .Current Protocols in Molecular Biology, 1987).

Screening was done by magnetic bead sorting (MACS) and using biotin-labeled recombinant human CD30 fused with Fc to screen 6 different synthetic antibody libraries. The specific process is as follows:

1. Preparation of Input Phage for Screening

Add 50-100 times the storage volume of the frozen bacteria. When inoculating, bring the OD value to 0.05. When the OD value reaches 0.5 at 37℃ 200rpm, add 5×10 ⁹ cfu/ml helper phage (helper phage) M13K07, shake well Firstly, let it stand at 37° for 20-30min, then at 37°, 200rpm for 30min, and finally at 30°C for 20h. Collect the phage as the input phage in the first round.

2.Panning (panning)

Take 50ul magnetic beads (Thermo, catalog number 815-968-0747), wash 4 times with PBS; rotate and seal with 3% BSA in PBS at room temperature for 2h. Combine the magnetic beads with biotin-labeled CD30/Avitag protein (Acro, catalog number CD0-H82E6), rotate at room temperature, and incubate for 1 hour.

Combine the magnetic beads processed above with the input phage of step 1, rotate at room temperature, and incubate for 1.5-2 hours. Then wash with 0.05% PBST 8-10 times, and finally eluted with 0.5ml 0.1M hydrochloric acid-glycine eluent, and then neutralized with 2M Tris.

3. Phage infection

Take half volume of phage eluate to infect logarithmic growth phase of Escherichia coli XL1-Blue bacterial solution, incubate at 37°C, 200 rpm for 20 minutes, and then spread agar plates. The next day, scrape, centrifuge at 3000rpm for 5min, then resuspend in 20% glycerol-2×YT medium, and measure the OD value.

2×YT medium (1L) is prepared in Table 9.

Table 9: 2×YT medium

Then produce phage overnight, the method is the same as step 1 above. Steps 1-3 are repeated for four rounds.

After completing the above 4 rounds of panning (panning), the positive signal was found to be significantly enriched after the fourth round by Elisa detection. The enriched phage population containing the specific antibody sequence was spread on the agar plate to obtain the specific antibody gene Colonies of pure strains of bacteriophages. Pick simple strains and carry out Elisa detection of pure strains of single phage. The Elisa-positive pure strain was sequenced, and there were about 37 antibodies with different sequences.

Subsequently, the 37 antibodies were constructed in the form of scFv-hIgG antibody (the method is the same as in Example 3), and the resulting plasmid was transiently transfected into a 6-well plate, and the expression supernatant was taken 5 days later for Elisa verification. Elisa-positive pure strains were tested by Fortebio (the method is the same as in Example 5). Two pure strains P5E10 and P27B3 with good affinity were selected, and the 10 pure candidate strains produced by fusion tumors using transgenic mice in Example 1 were expressed and purified in large quantities.

The amino acid sequences and corresponding nucleotide sequences of the two antibody molecules P5E10 and P27B3 are given in Table A above.

Example 3. Construction of scFv-hFc recombinant single chain antibody expression vector

In order to verify the affinity of the candidate antibody in scFv format with the target antigen CD30, recombinant protein expression vectors of the single-chain antibody variable region (scFv) and human Fc fragment of the antibody obtained in Example 1 and Example 2 were constructed. At the same time, two positive control antibodies V2AC10 and XL in the same form were also constructed. Table C shows the amino acid sequence of the constructed scFv-Fc recombinant protein and its corresponding coding nucleotide sequence. The amino acid sequences of the variable regions of the two positive control antibodies are shown in Figure 8.

Figure 1 shows a schematic diagram of vector construction. The specific construction process is exemplified by one of the antibody (HB69G7) sequences as follows:

Using the upstream and downstream primer sequences shown in Table 10 and the PCR reaction system shown in Table 11, the VH and VL regions were amplified by PCR.

Table 10. Primers used in PCR amplification:

Table 11. PCR system

The PCR amplified products were subjected to nucleic acid electrophoresis, the target bands were harvested, and overlap PCR was performed using the reaction system shown in Table 12.

Table 12. Overlap PCR system:

The PCR amplified product is subjected to nucleic acid electrophoresis, and the target band is recovered for subsequent homologous recombination.

Using the homologous recombination system shown in Table 13, the homologous recombination reaction was carried out.

Table 13. Homologous recombination system

React at 37°C for 30 min to obtain a recombinant product. The recombinant product was transformed into TOP10 competent, and simple strains were selected for sequencing, and the pure strain containing the plasmid with the correct insertion direction was selected as the positive pure strain, and the positive pure strain was stored in the glycerol tube.

Example 4. Expression of scFv-hFc recombinant single chain antibody

1. Passage HEK293 cells according to the required transfection volume, and adjust the cell density to 1.2×10 ⁶ /ml the day before transfection.

2. Take 3mL OptiMEM medium (Gibco, 31985-070) as transfection buffer, respectively, corresponding to the above-described plasmid carrying a gene encoding an antibody scFv-hFc recombinant single chain 30 μ g, filtered aside for 5min after mixing.

3. Add 90 μ L of 1mg / rnL polyethyleneimine (PEI) (Polysciences, 23966) to the mixture of plasmid -Opti MEM, incubated at room temperature after mixing 15min. Pour the mixture gently into the cells and incubate at 36.5°C with 8% CO2.

After 4.20h additional 0.6mL of 200g / L of the FEED (soy peptone Phytone Peptone (BD, 211906) and plant peptone Phytone (BD, 210931) ratio, etc.), 0.3mL of 200g / L glucose stock solution, 30 μ L 2.2M valproate sodium salt (VPA) (Sigma, P4543).

5. Continue to cultivate until the vitality is less than 60%, collect the supernatant, filter and purify by affinity chromatography.

Example 5. Purification of scFv-hFc recombinant single chain antibody by protein A method

1. Rinse the packing and gravity column with ultrapure water to remove the packing protection liquid.

2. 0.1M NaOH filler soaking 2h, add 300 μ L after soaking Protein A affinity chromatography medium (Mabselect sure) (GE Healthcare, 17-5438-03) column packing each of gravity.

3. The cell material solution was centrifuged at 8000r/min for 40min, then filtered with a 0.45 μm filter, and stored at 4°C for later use.

4. Wash the gravity column and packing with a large amount of ultrapure water to remove the lye.

5. Equilibrate the filler with 10ml binding/washing buffer (20mM Tris+150mM NaCl (pH 7.2)) before purification.

6. Load the sample and pass the supernatant to be purified through the column.

7. Rinse, use 5~10ml binding/washing buffer (20mM Tris+150mM NaCl(pH 7.2)) to wash the packing material to remove non-specific binding protein.

8. Elution, wash the packing with 1 mL of elution buffer (100 mM sodium citrate/citrate buffer, pH 3.5) to collect specific binding proteins.

9. Add neutralization buffer (2M Tris) to the collection solution at a ratio of 85 μl /ml, and adjust the pH to 6~7.

Example 6. Fortebio detection of scFv-hFc recombinant single chain antibody

Biofilm-based optical interference technology (BLI) based on optical fiber biosensors measures the dynamic constants of antibody molecules.

The basic principle of BLI is: when biomolecules bind to the surface of the sensor, a biofilm is formed. The biofilm causes interference to the waveform of the light passing through the sensor. The interference is detected by phase shifting, so that the molecules bound to the sensor can be detected Quantity changes: Fit the kinetic curve according to the changes in the real-time response value, and calculate the binding constant (Kon), dissociation constant (Kdis), and affinity (KD).

The model of the Fortebio device we chose is Octet Red96. ForteBio affinity determination is based on the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs, 2013.5(2): p.270- 8) Proceed. The specific process is:

1. Half an hour before the start of the experiment, take an appropriate amount of AHC Sensor and soak it in SD buffer (50ml PBS+0.1% BSA+0.05% Tween-20) according to the number of samples.

2. Take 100 μl of SD buffer, scFv-hFc antibody, and human CD30-His antigen (ACRO BIOSYSTEMS, BCA-H522Y) and add them to a 96-well black polystyrene half-volume microplate.

3. According to the sample position layout, select the sensor position, set the running steps and time: Baseline, Loading~1nm, Baseline, Association and Dissociation time depends on the sample binding and dissociation speed; the speed is 1000rpm, the temperature is 30℃.

4. See Table 14 for the affinity detection results of 12 scFv-hFc recombinant single-chain antibodies with human CD30-His.

In addition, ForteBio was used for epitope determination. First, fix biotinylated CD30 on the surface of the SA sensor, then use a high concentration (300-500nM) control antibody to bind to CD30 to saturation, and finally use 100nM of other competing antibodies to bind to CD30. If the competing antibody has a binding signal, it indicates that the binding epitope of the competing antibody and the control antibody is different from that of CD30. If the competing antibody has no binding signal, it indicates that the binding epitope of the competing antibody and the control antibody is the same as that of CD30.

The two control antibodies XL and V2AC10 were first competitively combined, and the results showed that they were not the same epitope (Bin). The epitope binding between the control antibody XL and the antigen was defined as Bin1, and the epitope binding between the control antibody V2AC10 and the antigen was defined as Bin2. The results are shown in Table 14. P5E10 and P27B3 antibodies are Bin1 as the control antibody XL; HB38E4, HB10F1 and HB49G9 antibodies and the control antibody -V2AC10 are the same as Bin2; HB36F7, HB68H2, HB69G7, HB10B6, HB16H7 and HB6A9 antibodies and the two control antibodies are not the same antigen The binding epitope is defined as Bin3. The binding epitopes of the HB16H8 antibody and the two control antibodies have a certain overlap, which is defined as 1/2Bin. The results of epitope detection are shown in Table 14.

Table 14. Fortebio detection results of scFv-hFc and human CD30-His affinity and Bin

It can be seen from the data in the above table that the affinity (KD value) of 12 single-chain antibodies to monovalent human CD30 (CD30-His) ranges from 30 nM to 1 nM. Among them, the affinity of HB49G9 and P5E10 single-chain antibodies to CD30-His is stronger than that of the two positive control antibodies. The experimental data of epitope competitive binding shows that the antibodies we screened cover not only the binding epitopes of the control antibody, but also other epitopes, indicating that the antibody diversity obtained by the screening is high.

Example 7. Detection of affinity between scFv-hFc recombinant single-chain antibody and Karpas299

After the scFv-hFc fusion antibody was prepared, CD30 ⁺ human anaplastic large cell lymphoma cell Karpas299 was used to further verify the cell binding of the single chain antibody. The specific method is as follows:

1. Take human Karpas299 (biofeng.com, Cat#3135) cells, adjust the cell density to 2×10 ⁶ /ml, and centrifuge at 100 μl per well in a 96-well microtiter plate, centrifuge at 400G for 5 min, and remove the supernatant.

2. Start the scFv-hFc antibody at a concentration of 400nM, and dilute it in a 3-fold gradient in PBS containing 0.1% bovine serum albumin (BSA) for a total of 12 points. Add 100 μl of the diluted antibody to each well at 4°C Incubate for 30min;

3. Centrifuge at 400G for 5 min, add PBS and wash twice, add 100 μl of secondary antibody ((Phycoerythrin (Phycoerythrin, PE) labeled goat anti-human IgG antibody, SouthernBiotech) diluted in PBS (1% BSA) to each well Final concentration 5 μ g/ml), incubate at 4°C for 30 min (protect from light);

4. Centrifuge at 400G for 5 minutes, add PBS and wash twice, and resuspend the cells in 100 μl PBS per well. Flow cytometry was performed on the Accuri C6 system (BD Bioscience) to detect the PE positive signal and calculate the MFI based on the C6 software. Use GraphPad software to calculate EC50 value.

The test results are shown in Figure 2 and Table 15 below. It can be seen from the results in the figure that, except for the negative control IgG4 antibody, all 12 single-chain antibodies showed binding to Karpas299 cells, and the EC50 value of affinity ranged from 70 nM to 0.4 nM.

Table 15: Affinity (EC50 value) of single-chain antibody to Karpas299 cells

Sequence Listing Description

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<210> 98

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 98

<210> 99

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 99

<210> 100

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 100

<210> 101

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 101

<210> 102

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 102

<210> 103

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 103

<210> 104

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 104

<210> 105

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 105

<210> 106

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 106

<210> 107

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 107

<210> 108

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 108

<210> 109

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 109

<210> 110

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 110

<210> 111

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 111

<210> 112

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<2 23> Antibody sequence

<400> 112

<210> 113

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 113

<210> 114

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 114

<210> 115

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 115

<210> 116

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 116

<210> 117

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 117

<210> 118

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 118

<210> 119

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 119

<210> 120

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 120

<210> 121

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 121

<210> 122

<211> 732

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 122

<210> 123

<211> 516

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 123

<210> 124

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 124

<210> 125

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 125

<210> 126

<211> 513

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 126

<210> 127

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 127

<210> 128

<211> 732

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 128

<210> 129

<211> 516

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 129

<210> 130

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 130

<210> 131

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 131

<210> 132

<211> 513

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 132

<210> 133

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 133

<210> 134

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 134

<210> 135

<211> 517

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 135

<210> 136

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 136

<210> 137

<211> 732

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 137

<210> 138

<211> 516

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 138

<210> 139

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 139

<210> 140

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 140

<210> 141

<211> 514

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 141

<210> 142

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 142

<210> 143

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 143

<210> 144

<211> 514

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 144

<210> 145

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 145

<210> 146

<211> 741

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 146

<210> 147

<211> 519

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 147

<210> 148

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 148

<210> 149

<211> 732

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 149

<210> 150

<211> 516

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 150

<210> 151

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 151

<210> 152

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 152

<210> 153

<211> 515

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 153

<210> 154

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 154

<210> 155

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 155

<210> 156

<211> 515

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 156

<210> 157

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 157

<210> 158

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Adaptor DNA

<400> 158

<210> 159

<211> 45

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge

<400> 159

<210> 160

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Hinge DNA

<400> 160

<210> 161

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 Fc

<400> 161

<210> 162

<211> 595

<212> PRT

<213> Artificial sequence

<220>

<223> Human CD30 full-length sequence

<400> 162

<210> 163

<211> 361

<212> PRT

<213> Artificial sequence

<220>

<223> Human CD30 ECD

<400> 163

<210> 164

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse IgGκ signal peptide

<400> 164

<210> 165

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse IgGκ signal peptide DNA

<400> 165

<210> 166

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 166

<210> 167

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 167

<210> 168

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 168

<210> 169

<211> 143

<212> PRT

<213> Artificial sequence

<220>

<223> Antibody sequence

<400> 169

Claims (27)

An isolated antibody or antigen-binding fragment thereof that specifically binds to CD30, the antibody comprising HCDR1, 2 and 3 sequences and LCDR1, 2 and 3 sequences selected from the group consisting of heavy chain variable regions and light chain variable regions: (i) The heavy chain variable region shown in SEQ ID NO: 4, and the light chain variable region shown in SEQ ID NO: 9, (ii) The heavy chain variable region shown in SEQ ID NO: 14, and the light chain variable region shown in SEQ ID NO: 19, (iii) the heavy chain variable region shown in SEQ ID NO: 24, and the light chain variable region shown in SEQ ID NO: 29, (iv) The heavy chain variable region shown in SEQ ID NO: 34, and the light chain variable region shown in SEQ ID NO: 39, (v) The heavy chain variable region shown in SEQ ID NO: 44, and the light chain variable region shown in SEQ ID NO: 49, (vi) The heavy chain variable region shown in SEQ ID NO: 54 and the light chain variable region shown in SEQ ID NO: 59, (vii) The heavy chain variable region shown in SEQ ID NO: 64, and the light chain variable region shown in SEQ ID NO: 69, (viii) the heavy chain variable region shown in SEQ ID NO: 74, and the light chain variable region shown in SEQ ID NO: 79, (ix) the heavy chain variable region shown in SEQ ID NO: 84, and the light chain variable region shown in SEQ ID NO: 89, (x) The heavy chain variable region shown in SEQ ID NO: 94, and the light chain variable region shown in SEQ ID NO: 99, (xi) the heavy chain variable region shown in SEQ ID NO: 104, and the light chain variable region shown in SEQ ID NO: 109, (xii) The variable region of the heavy chain shown in SEQ ID NO: 114, and the variable region of the light chain shown in SEQ ID NO: 119. An isolated antibody or antigen-binding fragment thereof that specifically binds to CD30, the antibody comprising 3 heavy chain complementarity determining regions HCDR and 3 light chain complementarity determining regions LCDR, wherein: (a) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 3, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 6, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 8; or (b) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 11, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 12, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 13, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 16, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 18; or (c) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 21, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 22, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 23, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 26, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 28; or (d) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 31, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 32, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 36, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 38; or (e) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 41, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 42, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 43, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 46, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 47, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 48; or (f) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 51, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 52, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 53, LCDR1 includes The amino acid sequence shown in SEQ ID NO: 56, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 57, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 58; or (g) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 61, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 62, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 63, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 66, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 67, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 68; or (h) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 71, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 72, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 73, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 76, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 77, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78; or (i) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 81, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 82, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 83, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 86, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 87, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 88; or (j) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 91, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 92, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 93, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 96, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 97, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 98; or (k) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 101, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 102, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 103, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 106, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 107, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 108; or (1) HCDR1 includes the amino acid sequence shown in SEQ ID NO: 111, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 112, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 113, and LCDR1 includes The amino acid sequence shown in SEQ ID NO: 116, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 118. The antibody or antigen-binding fragment thereof according to item 1 or 2 of the scope of patent application, wherein the antibody comprises a heavy chain variable region VH, and the VH is selected from: (a) Containing the amino acid sequence shown in SEQ ID NO: 4, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (b) Containing the amino acid sequence shown in SEQ ID NO: 14, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (c) Containing the amino acid sequence shown in SEQ ID NO: 24, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (d) Comprising the amino acid sequence shown in SEQ ID NO: 34, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (e) Containing the amino acid sequence shown in SEQ ID NO: 44, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (f) Containing the amino acid sequence shown in SEQ ID NO: 54, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (g) Containing the amino acid sequence shown in SEQ ID NO: 64, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (h) Containing the amino acid sequence shown in SEQ ID NO: 74, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (i) Comprising the amino acid sequence shown in SEQ ID NO: 84, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (j) Containing the amino acid sequence shown in SEQ ID NO: 94, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (k) Containing the amino acid sequence shown in SEQ ID NO: 104, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH of the amino acid sequence containing no more than 10 (preferably no more than 5) amino acid changes relative to it; (1) Containing the amino acid sequence shown in SEQ ID NO: 114, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or the VH containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence. The antibody or antigen-binding fragment thereof according to item 1 or 2 of the scope of patent application, the antibody comprises a light chain variable region VL, and the VL is selected from: (a) Containing the amino acid sequence shown in SEQ ID NO: 9, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (b) Comprising the amino acid sequence shown in SEQ ID NO: 19, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (c) Containing the amino acid sequence shown in SEQ ID NO: 29, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (d) Comprising the amino acid sequence shown in SEQ ID NO: 39, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (e) Contains the amino acid sequence shown in SEQ ID NO: 49, or has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (f) Contains the amino acid sequence shown in SEQ ID NO: 59, or has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (g) Containing the amino acid sequence shown in SEQ ID NO: 69, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (h) Containing the amino acid sequence shown in SEQ ID NO: 79, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (i) Containing the amino acid sequence shown in SEQ ID NO: 89, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (j) Containing the amino acid sequence shown in SEQ ID NO: 99, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (k) Containing the amino acid sequence shown in SEQ ID NO: 109, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it An amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence; (1) Comprising the amino acid sequence shown in SEQ ID NO: 119, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it The amino acid sequence, or a VL containing no more than 10 (preferably no more than 5) amino acid changes relative to the amino acid sequence. For example, the antibody or antigen-binding fragment thereof according to any one of items 1 to 4 in the scope of patent application, the antibody comprises a heavy chain variable region VH and a light chain variable region VL, and the VH and VL are selected from: (a) An amine comprising the amino acid sequence shown in SEQ ID NO: 4 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 9 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (b) An amine comprising the amino acid sequence shown in SEQ ID NO: 14 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith Base acid sequence VH, and contains The amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity with it VL; (c) An amine comprising the amino acid sequence shown in SEQ ID NO: 24 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 29 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (d) An amine comprising the amino acid sequence shown in SEQ ID NO: 34 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 39 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (e) An amine comprising the amino acid sequence shown in SEQ ID NO: 44 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 49 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (f) An amine comprising the amino acid sequence shown in SEQ ID NO: 54 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 59 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (g) An amine comprising the amino acid sequence shown in SEQ ID NO: 64 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 69 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (h) An amine comprising the amino acid sequence shown in SEQ ID NO: 74 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith Base acid sequence VH, and contains The amino acid sequence shown in SEQ ID NO: 79 or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it VL; (i) An amine comprising the amino acid sequence shown in SEQ ID NO: 84 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 89 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (j) An amine comprising the amino acid sequence shown in SEQ ID NO: 94 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 99 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (k) An amine comprising the amino acid sequence shown in SEQ ID NO: 104 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 109 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher VL of identical amino acid sequence; (1) An amine comprising the amino acid sequence shown in SEQ ID NO: 114 or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity therewith The VH of the base acid sequence, and the amino acid sequence shown in SEQ ID NO: 119 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher The VL of the amino acid sequence of identity. An isolated antibody or antigen-binding fragment thereof that specifically binds CD30, the antibody has one or more of the following characteristics: (i) Showing the same or similar binding affinity and/or specificity as any antibody listed in Table A; (ii) Inhibit (for example, competitively inhibit) the binding of any antibody listed in Table A to CD30; (iii) It binds the same or overlapping epitope with any antibody shown in Table A; (iv) Compete with any antibody shown in Table A for binding to CD30; (v) Having one or more biological properties of any antibody molecule listed in Table A. For example, the antibody or antigen-binding fragment thereof according to any one of items 1 to 6 in the scope of patent application, wherein the antibody has one or more of the following characteristics: (i) with high affinity, e.g. less than 10OnM, e.g. less than 50 nM, such as 1-30 nm, preferably less than 10nM of K _D value, of CD30 and human (such as SEQ ID NO: 162 polypeptide) binding; (ii) high affinity, e.g. less than 10OnM, e.g. less than 70nM, e.g. 0.1-30 nm, preferably less than 20 nM, more preferably of 5 nM or less than 10, most preferably less than EC ₅₀ values of 1nM human cell surface expression of CD30 (Such as the polypeptide of SEQ ID NO: 162) binding; (iii) The dissociation rate constant (K _dis ) of binding to human CD30 (such as the polypeptide of SEQ ID NO: 162) is less than 60×10 ^-4 s ^-1 , for example, 30-10×10 ^-4 s ^-1 , preferably 5-1×10 ^-4 s ^-1 ; (iv) It specifically binds to the epitope on the ECD of the extracellular domain of human CD30. Such as the antibody or antigen-binding fragment thereof according to any one of items 1 to 7 in the scope of patent application, wherein the antibody is a fully human antibody. The antibody or antigen-binding fragment thereof according to any one of items 1 to 8 in the scope of patent application, wherein the antibody is a single-chain antibody. The antibody of any one of items 1 to 9 in the scope of patent application, wherein the antibody is a single-chain scFv antibody, preferably the single-chain scFv comprises: from N-terminus to C-terminus, VL domain-linker-VH structure Domain, or VH domain-linker-VL domain. The antibody of claim 10, wherein the linker contains 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20, preferably 15- 20 amino acids. The antibody according to claim 11, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:157. The antibody according to any one of the 10th to 12th patents, wherein the single-chain scFv antibody comprises SEQ ID NO: 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151 And the amino acid sequence of 154, or an amino acid sequence with at least 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it, or at least one , For example, 1-5, but not more than 30, 20 or 10 amino acid sequences with altered amino acids. An isolated antibody that specifically binds to CD30, wherein the antibody comprises a single-chain scFv antibody and an Fc region as in any one of items 10 to 13 in the scope of the patent application. The antibody according to claim 14, wherein the single-chain scFv is connected to the Fc region by a hinge region. Preferably, the hinge region is a CD8 hinge region, and more preferably, the hinge region includes the hinge region of SEQ ID NO: 159 The shown amino acid sequence or the amino acid sequence relative to SEQ ID NO: 159 contains at least one, two or three, but no more than 5 amino acid sequences with altered amino acids. The antibody according to item 14 or 15 of the scope of patent application, wherein the Fc region is a human IgG1 or IgG4 Fc region, and preferably the Fc region is low or non-fucosylated. Such as the antibody of any one of the 14th to 16th items in the scope of the patent application, wherein the antibody comprises an antibody selected from SEQ ID NO: 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153 and 156 The amino acid sequence, or an amino acid sequence containing at least one, such as 1-5, but no more than 30, 20, or 10 amino acid changes, or at least 80%, 85% of the amino acid sequence , 90%, 92%, 95%, 97%, 98%, 99% or higher identity of amino acid sequences. An isolated nucleic acid that encodes the isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 17 in the scope of the patent application. A vector containing the nucleic acid described in item 18 of the scope of the patent application, preferably the vector is an expression vector. A host cell containing the vector described in item 19 of the scope of the patent application, preferably, the host cell is selected from yeast cells and mammalian cells. A method for preparing the isolated antibody or antigen-binding fragment thereof as described in any one of the scope of the application for patent application, comprising: culturing the antibody or antigen-binding fragment thereof under conditions suitable for expression of the antibody or antigen-binding fragment thereof The host cell described in item 20 of the scope of the patent. A conjugate or fusion comprising the antibody described in any one of items 1 to 17 in the scope of the patent application. A pharmaceutical composition, which comprises the isolated antibody or antigen-binding fragment thereof as described in any one of the scope of patent application 1 to 17, or the conjugate or fusion as described in the scope of patent application 22, And optionally a pharmaceutical carrier, and optionally one or more other therapeutic agents. A method for detecting CD30 in a sample, including: (a) Combining the sample with the isolated antibody or antigen-binding fragment thereof as described in any one of the scope of patent application 1 to 17, or the conjugate or fusion as described in the scope of patent application 22 Contact; and (b) Detecting the formation of a complex between the antibody or its antigen-binding fragment or conjugate or fusion and the CD30 protein. A method for treating a CD30-related disorder, comprising administering to the subject an effective amount of the isolated antibody or antigen-binding fragment thereof as described in any one of the scope of patent application 1 to 17, such as item 22 in the scope of patent application The conjugate or fusion, or the composition as described in item 23 of the scope of the patent application. The method according to claim 25, wherein the CD30-related disorder is a tumor expressing CD30, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma. The method according to item 26 of the scope of patent application, wherein the CD30-related disorder is selected from Hodgkin lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T cell lymphoma, adult T cell lymphoma (ATL), Angioimmunoblastic T cell lymphoma (AITL), preferably, anaplastic large cell lymphoma, Hodgkin lymphoma, and skin T cell lymphoma.

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