WO2020135426A1 - Fully human anti-cd30 single chain antibodies and use thereof - Google Patents

Abstract

Novel antibodies and antibody fragments specifically binding to CD30, in particular fully human single chain antibody scFv. Nucleic acids encoding these antibodies, vectors, host cells expressing said nucleic acids, compositions comprising said antibodies, and use thereof in therapy and diagnosis.

Description

Fully human anti-CD30 single chain antibody and its application Technical field

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 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 containing the antibody of the present invention and its use in therapy and diagnosis.

Background technique

CD30, also known as Ki-1 or TNFRSF8, is a member of the tumor necrosis factor (TNF) receptor superfamily. As a type I transmembrane glycoprotein receptor of 105-120kD, CD30 has an intracellular domain, a transmembrane domain and an extracellular domain, and there are multiple cysteine-rich regions in the extracellular domain. The extracellular domain can be cleaved to produce 88kD of soluble form of CD30 (sCD30), which can be released into the serum and can be used in patients with autoimmune diseases such as rheumatoid arthritis, inflammatory conditions, and CD30-positive hematological malignancies Increased serum CD30 was detected in. Pierce JM, etc., 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's lymphoma (HL) and anaplastic large cell lymphoma (ALCL). CD30 is also expressed to varying degrees in some other lymphoproliferative disorders, including, for example, T-cell lymphoma, such as non-specific peripheral T-cell lymphoma (PTCL-NOS), immunoblastic T-cell lymphoma, vascular immunity Blast T cell lymphoma (AITL), extranodal NK/T cell lymphoma, adult T cell leukemia/lymphoma, cutaneous T cell lymphoma (CTCL); various B cell non-Hodgkin lymphomas, including diffuse Large B-cell lymphoma (DLBCL), especially EBV-positive diffuse large B-cell lymphoma; mycosis 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 and others, Cell 1993; 73:1349–1360.

In addition, the overexpression of CD30 in some lymphoma subtypes and some non-lymphoid neoplastic diseases 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 improve 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 treatment methods have been proposed, including, for example, antibody-drug conjugates ( ADC), bispecific antibodies and chimeric antigen receptors modify T cells (CAR-T cells).

For the CD30 antibody-drug conjugate (ADC), it has been reported that the chimeric CD30 monoclonal antibody is connected to the microtubule disrupting drug monomethyl auristatin E (MMAE, monomethyl auriristatin E) via a protease cleavable linker The conjugate brentuximab vedotin. The compound has been proven to be well tolerated in phase I and II clinical trials in Hodgkin lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, and even B cell lymphoma expressing CD30, and more It is important to be able to achieve disease control, including disease control in patients with multiple relapses or refractory diseases. CA, Vander, Weyden, etc., 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 most promising new development is the bispecific antibody AFM13 targeting CD30 and CD16A. These immunotherapies use bispecific antibodies as a bridge to link CD30 positive tumor cells with immune effector cells (such as NK cells/macrophages).

In terms of CAR-T cell immunotherapy, the Baylor researchers recently published a phase I clinical study of CD30 CAR-T cells in 7 patients with HL and 2 patients with ALCL, and the results showed that some patients achieved lasting complete remission (CR, complete)).

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 natural antibodies (usually through a paragraph Recombinant small-molecule antibodies made of artificially linked peptides (or "linkers"). Compared with intact antibody molecules, scFv single-chain antibodies have the following advantages: they contain intact antibody variable regions and retain the antigen specificity and binding activity of the original antibodies; they do not contain the Fc regions of antibody molecules, so they are weakly immunogenic and are used for The human body is not easy to produce immune response; the molecular weight is small, the penetration is strong, and it is easy to penetrate into the tissue. When used for imaging diagnosis or treatment, it can enter the interior of the tissue that cannot be reached by the general intact antibody; no glycosylation modification is required to form a functional Antibody molecules are conducive to mass production of prokaryotic expression systems; easy to operate, suitable for use as a genetic engineering component 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, new CD30-specific binding molecules are still needed in the art. The present invention satisfies this need by providing a fully human single-chain antibody that binds CD30 with high target specificity and high affinity, especially CD30 expressed on the surface of tumor cells, and has low side effects. The fully human single-chain antibody of the present invention is not only suitable for use in the diagnosis or treatment of tumors and cancers alone, but also, more advantageously, as a genetic engineering component to prepare other diagnostic and therapeutic molecules with high CD30 targeting For example, various forms of antibodies, scFv-Fc, and antibody-based fusions and conjugates.

Summary of the invention

The invention provides a fully human anti-human CD30 antibody and its coding gene and application. Through the use of transgenic mice and phage display technology, the inventors screened out fully human antibodies against human CD30 and obtained the variable region gene sequences, on the basis of which constructed fully human single chain scFv antibodies and scFv-hFc antibodies . 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 an antibody or antigen-binding fragment thereof that specifically binds 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 an scFv-Fc antibody. In some embodiments, the antibodies of the present invention in K _D of about 1nM to 100nM human CD30 binding protein, wherein the K _D value measurement interferometry (e.g. Fortebio assay), for example, according to the optical biofilm. In some embodiments, the human antibodies of the invention to the EC ₅₀ of about 0.1nM to 100nM binding protein cell surface expression of CD30, wherein the EC ₅₀ values for example according to flow cytometry (e.g., FACS) measurements.

In some embodiments, the antibody of the present invention comprises the VH region sequence of any of the antibodies shown in Table A or a variant thereof. In other embodiments, the antibody of the present invention comprises the VL region sequence of any of the antibodies shown in Table A or a variant thereof. In other embodiments, the antibodies of the invention comprise the VH and VL sequence pairs of any of the antibodies shown in Table A, or variants thereof. In some embodiments, the antibody of the present invention comprises the sequence of 6 CDR regions of any antibody shown in Table A, or a variant thereof, wherein the variant comprises a total of 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 changes (preferably amino acid substitutions, preferably conservative substitutions), preferably the 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 comprises VH sequence, VL sequence and linker. Preferably the scFv antibody comprises from the N-terminus to the 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 antibodies of the present invention and wild-type or altered Fc regions. In some embodiments, the Fc region of the scFv-Fc antibody of the invention is low or afucosylated. In some embodiments, the scFv antibody is fused to the Fc region through the hinge region.

In yet another aspect, the invention relates to fusions and conjugates constructed based on the antibodies of the 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 an antibody of the invention or antigen-binding fragment thereof, or a fusion or conjugate of the invention is administered to the subject. In some embodiments, the CD30-related disorder is a disorder mediated by or associated with 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 lymphoma and non-Hodgkin lymphoma, preferably selected from Hodgkin lymphoma, anaplastic large cell lymphoma (ALCL), Skin T cell lymphoma, adult T cell lymphoma (ATL), angioimmunoblastic T cell lymphoma (AITL), most preferably anaplastic large cell lymphoma and classic Hodgkin 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 radioisotope.

In yet another aspect, the present invention relates to a method and kit for detecting CD30 in a sample, wherein the method includes: (a) contacting the sample with an antibody or antigen-binding fragment, fusion or conjugate of the present invention; and ( b) Detect the formation of a complex between the antibody or antigen-binding fragment or fusion or conjugate and CD30 protein. The test 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 lymphoma patient.

Brief description of the drawings

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

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

Figure 3 shows an exemplary CDR sequence of the present invention.

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

Figure 5 shows the VL sequences of exemplary antibodies of the invention.

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

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 explicitly stated to the contrary, the practice of the present invention will employ methods of conventional chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art. A description of these methods can be found, for example, in Sambrook et al., Molecular: A Laboratory (Manual 3rd Edition, 2001); Sambrook et al., Molecular: A Laboratory (Manual 2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory (Manual (1982); Ausubel et al., Current Protocols, Molecular Biology (John Wiley and Sons, July 2008 update); Short Protocols, Molecular, Biology: A Compendium, Methods, from Current, Protocols, Green, Biology, Biology, Biology, Biology .Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis 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 Laboratory Press, Cold Spring Harbor, NY, 1998) Current Protocols Immunology QEColigan, AMKruisbeek, DHMargulies, EMShevach and W. Strober, eds., 1991); Annual Review of Immunology; and journal monographs such as Advances in Immunology.

definition

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

The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range that has a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

The term "and/or" when used to connect two or more alternatives 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 including the recited element, integer, or step, but does not exclude any other element, integer, or step. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the case consisting of the mentioned elements, integers or steps is also covered. For example, when referring to an antibody variable region that "contains" a specific sequence, it is also intended to cover the antibody variable region that consists of the specific sequence.

As used herein, the term "antigen-binding molecule" refers to a molecule, such as a protein or polypeptide, that contains an antigen-binding region or antigen-binding portion capable of binding to a target antigen. 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 will be apparent to those skilled in the art, the antigen-binding portion of an antibody generally contains amino acid residues from "complementarity determining regions" or "CDRs." In some cases, depending on the context, "CD30 binding molecule" and "antibody of the invention" or "anti-CD30 antibody" may be used interchangeably.

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

It is understood by those skilled in the art that "whole antibody" (which is used interchangeably with "full-length antibody", "complete antibody" and "whole antibody" herein) contains at least two heavy chains (H) and two light chains ( L). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) 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 binding of the antibody to its antigen. The constant region does not directly participate in the binding of the antibody to the antigen, but displays multiple effector functions. The light chain of an antibody can be assigned to 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. Editor, Second Edition, Raven Press, N.Y. (1989)) (which is hereby incorporated by reference for its entirety for all purposes).

The term "antibody fragment" refers to a molecule that is not an intact antibody, and includes a portion of the intact antibody for binding the antigen to which the intact antibody binds. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include but are not limited to Fab, scFab, Fab', F(ab') ₂ , Fab'-SH, Fv, single chain Fv, diabody, triabody, tetrachain antibody ( tetrabody), 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, Fab fragment can be obtained by papain digestion of complete antibody. The light chain (L chain) and the heavy chain (H chain) of the Fab can be fused to form a single polypeptide chain, that is, a single chain Fab (scFab) (see, for example, US20070274985A1). In addition, F(ab') ₂ , which is a dimer of Fab' and is a bivalent antibody fragment, can be produced by pepsin digesting the complete antibody under the disulfide bond in the hinge region. F(ab') ₂ can be reduced under neutral conditions by breaking the disulfide bond in the hinge region, converting from F(ab') ₂ dimer to Fab' monomer. Fab' monomers are basically Fab fragments with hinge regions. The Fv fragment consists of the VL and VH domains of one arm of the antibody. In addition, a recombinant method can be used to link the genes encoding the two domains VL and VH of the Fv fragment independently through a nucleic acid sequence encoding a connecting peptide (linker), and recombinantly express to form a single-chain Fv in the single protein chain The pairing of the VH and VL regions provides an antigen binding site. A double-chain antibody is an antibody fragment with two antigen binding sites, which contains VL and VH connected by a short linker in the same polypeptide chain. In a double-chain antibody, 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 generate two antigen binding sites. The double-chain antibody may be bivalent or bispecific. More detailed descriptions of double-chain antibodies can be found, for example, in 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 ). Three-chain antibodies and four-chain antibodies and minibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003), and Shao Rongguang et al. (eds.), 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 WE Paul, Raven Press, NY (1993).

The term "monoclonal antibody" as used herein means an antibody obtained from a substantially homogeneous population of antibodies, ie, except for possible variant antibodies that are usually present in very small amounts (eg, containing natural mutations or in monoclonal antibody preparations) In addition to the variant antibodies produced during the production process, the individual antibodies that make up the population are the same and/or bind the same epitope. Monoclonal antibodies can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods of using transgenic animals containing all or part of human immunoglobulin loci.

The terms "human antibody" or "fully human antibody" are used interchangeably herein to refer to antibodies that include variable regions in which both the framework 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 invention may include amino acids not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or point-specific mutagenesis in vitro or somatic mutations in vivo), for example in CDRs, especially in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which the 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) from animals (eg, mice) transgenic or transchromosomal with human immunoglobulin genes or Antibodies isolated from hybridomas prepared therefrom, (b) antibodies isolated from host cells expressing human antibodies such as transfected tumors, (c) antibodies isolated from recombinant, combined human antibody libraries such as phage display libraries, and ( d) antibodies prepared, expressed, produced or isolated by any other means including splicing human immunoglobulin genes to other DNA sequences. These recombinant human antibodies have variable regions whose framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, recombinant human antibodies can be mutagenized in vitro (or in vivo somatic mutagenesis when using human Ig sequence transgenic animals), and the amino acid sequences of the VH and VL regions of the recombinant antibody thus obtained, Although derived from and related to human germline VH and VL sequences, they are not naturally found in the human antibody germline library in vivo.

The term "chimeric antibody" refers to an antibody in which the 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 that connects CDR sequences derived from the germline of another mammalian species, such as a mouse, to a human framework sequence.

An "isolated" antibody is one 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 (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse Phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

The epitope is the antigen region to which the antibody binds. The epitope may be formed by consecutive amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of the protein.

The term "CD30" includes variants, isotypes, species homologs of human CD30, and analogs having at least one identical epitope 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 retain the ability to bind to any antibody of the 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, Approximately 5×10 ⁻⁹ M or lower K _D , which binds to human CD30, the antibody is an antibody that “specifically binds to human CD30”.

"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for 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 binding rate constant (k _dis and k _{on, respectively} ). Affinity can be measured by common methods known in the art. A specific method for measuring affinity is the ForteBio Kinetic Binding Assay herein.

A "competitively bound antibody" that refers to a reference antibody that binds to an antigen such as CD30 refers to an antibody that blocks the reference antibody from binding to an antigen (eg, CD30) by 50% or more in a competition test, and in turn, The reference antibody also blocks the antibody's binding to the antigen (eg CD30) by 50% or more in the competition test. Exemplary competition tests are 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 that contains at least a portion of a 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 noted 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 means herein, including substitutions, deletions of at least 1, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acids And/or inserted antibodies having an antibody region of interest (eg, heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region) with amino acid changes, where the variant substantially retains the antibody molecule before the change Biological characteristics. In one aspect, the invention encompasses any antibody variants described herein. In one embodiment, the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the pre-altered antibody. In some embodiments, the change does not cause the antibody variant to lose binding to the antigen, but optionally can impart properties such as increased antigen affinity and different effector functions. It can be understood that the heavy chain variable region or light chain variable region, or each CDR region of an antibody may be changed individually or in combination. In some embodiments, no more than one, two, three, four, or five amino acid changes in one or more or all three heavy chain CDRs. In some embodiments, there are no more than 1, 2, 3, 4, or 5 amino acid changes in one or more or all three light chain CDRs. In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes in all 6 CDRs. Preferably, the amino acid change is an amino acid substitution, preferably a conservative substitution. In some embodiments, the antibody variant has at least 80%, 85%, 90%, or 95% or 99% or higher amino acid identity over the target antibody sequence region. For example, in one embodiment, the antibody of the present invention has at least 80%, 85%, 90%, 91%, 92%, 93% in the heavy chain variable region compared to any antibody listed in Table A, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In yet another embodiment, the antibody of the present invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94% on the light chain variable region compared to any of the antibodies listed in Table A , 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In yet another embodiment, the antibody of the present invention has at least 80%, 85%, 90%, 91%, 92% in the heavy chain variable region and the light chain variable region compared to any of the antibodies listed in Table A , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.

As used herein, "sequence identity" refers to the degree to which sequences on a nucleotide-by-nucleotide or amino acid-by-amino acid basis are identical in the comparison window. The "percent sequence identity" can be calculated by comparing the two optimally aligned sequences in a comparison window to determine the presence of the same nucleic acid base (eg, A, T, C, G, I in both sequences) ) Or the same amino acid residue (for example, 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 produce a sequence identity percentage. The optimal alignment to determine the percent sequence identity can be achieved in various 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 suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment within the full-length sequence being compared or within the target sequence region.

In the present invention, as far as antibody sequences are concerned, the percentage of amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and in a preferred scheme after performing optimal alignment according to the Kabat numbering rule. After the alignment, the target antibody region (for example, the entire variable region of the heavy chain or light chain, or a part thereof, for example, one or more CDR regions) is compared with the same region of the reference antibody. The percent 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 (vacancies are not counted) and Multiply by 100 to get the percentage. Herein, without specifying the target antibody region, it will be suitable to perform alignment over the full length of the reference antibody sequence. In some embodiments, with respect to antibodies, sequence identity may be distributed throughout the heavy chain variable region and/or the entire light chain variable region, or the percent sequence identity may be limited to the framework region only, while corresponding to the CDR region The sequence remains 100% identical.

A. The CD30 binding molecule of the present invention

I. Anti-CD30 antibody of the present invention

In one aspect, the present invention provides antibodies that bind CD30 (especially membrane-bound CD30) with high target specificity and high affinity, especially single-chain antibodies (eg, single-chain scFv antibodies).

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

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

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

(iii) The dissociation rate constant (K _dis ) associated with 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) Binding specifically to an epitope on CD30, especially the extracellular domain ECD of CD30 (eg, recognizing the same or similar epitope as any of the antibodies listed in Table A);

(v) showing the same or similar binding affinity and/or specificity as any of the antibodies listed in Table A;

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

(vii) Binding to the same or overlapping epitope as any of the antibodies shown in Table A;

(viii) compete with any of the antibodies shown in Table A for binding to CD30 and/or to the same epitope on CD30;

(ix) has one or more biological characteristics of the antibody molecules described herein, for example, any of the antibody molecules listed in Table A;

(x) have one or more pharmacokinetic properties of the antibody molecules described herein, such as any of the antibodies shown in Table A;

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

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

In some embodiments, the anti-CD30 antibody molecules of the invention bind to human CD30 (eg, the polypeptide of SEQ ID NO: 162) with high affinity, eg, 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 bio-optical interferometry (eg, Fortebio affinity measurement).

In some embodiments, the anti-CD30 antibody molecule of the invention binds to human CD30 (eg, the polypeptide of SEQ ID NO: 162) with a dissociation rate constant (K _dis ) of less than 6×10 ⁻³ s ^-1 , such as 4-1× 10 ^-3 s ^-1 , preferably less than 8×10 ^-4 s ^-1 , for example less than 5×10 ^-4 or 3×10 ^-4 s ^-1 , for example about 2×10 ^-4 s ^-1 . In some embodiments, the binding rate constant (K _on ) of the anti-CD30 antibody molecule to human CD30 (eg, 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 molecule of the invention binds CD30-expressing cells with high affinity, preferably lymphoma cells (eg Karpas299 cells) expressing human CD30 on the cell surface, preferably, by flow cytometry (eg FACS) ) Determination, the EC50 value of the antibody binding to cells 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, more Preferably less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or 2 nM, most preferably less than 1 nM.

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), triple-chain antibody, tetra Chain antibody, mini 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 an scFv and an Fc region connected thereto. In a preferred embodiment, the antibody molecules of the invention are 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 conserved region (i.e. framework District (FR)). Each VH and VL is composed 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 a specific antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains (see, for example, Portolano, S. et al., J. Immunol. 150 (1993 ) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628). Herein, 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. As used herein, the term "VL" or "VL domain" encompasses the light chain variable region VL of a full-length antibody, Fv, scFv, dsFv, Fab, scFab, or 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 of any antibody listed in Table A (for example, a pair of heavy-chain variable region and light-chain variable region) ; Or (ii) with the antigen binding region of (i) having, for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity in amino acid sequence Antigen binding region.

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

In yet another embodiment, the anti-CD30 antibody molecule of the invention comprises: (i) the same light chain variable region as the light chain variable region of any of the antibodies listed in Table A; or (ii) and (i) the light chain variable region The chain variable region has, for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity of the light chain variable region 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 variants Include a total of no more than 10, preferably 5-0 amino acid changes (preferably amino acid substitutions) in the three light chain complementarity determining regions (CDRs).

In some embodiments, the present invention provides an anti-CD30 antibody, or a variant thereof, comprising the amino acid sequence of the heavy chain variable region and light chain variable region pair of any of the antibodies listed in Table A. In a preferred embodiment, the antibody comprises an amino acid sequence pair selected from 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 identity in the VH and/or VL amino acid sequence , Or contain 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 substitutions, preferably conservative substitutions).

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: The amino acid sequence of 9. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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 invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: The amino acid sequence of 29. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: The amino acid sequence of 39. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: Amino acid sequence of 49. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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 invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: The amino acid sequence of 69. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: The amino acid sequence of 79. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: The amino acid sequence of 89. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: The amino acid sequence of 99. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: The amino acid sequence of 109. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on 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: The amino acid sequence of 119. The invention also provides variants of the antibody, for example variants having at least 95-99% identity on VH and/or VL or containing no more than 10 amino acid changes.

In any of the above embodiments, preferably, with respect 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, preferably no more than all 3 CDR regions Five (eg, 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).

In any of the above embodiments, preferably, relative to the amino acid sequence of the reference light chain variable region, the light chain variable region VL of the antibody of the present invention contains no more than 10, preferably not More than 5 (eg, 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).

Antibody CDR region

"Complementarity determining region" or "CDR region" or "CDR" (herein interchangeably with hypervariable region "HVR") is an amino acid region in an antibody variable region that is mainly responsible for binding to an epitope. The CDRs of the heavy and light chains are commonly referred to as 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 protocols are known in the art for determining the CDR sequence of a given VH or VL amino acid sequence. 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, National Institutes of Health, Bethesda, Md. ( 1991)). Chothia refers to the position of the structural loop (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 AbM antibody modeling software. "Contact" HVR is based on the analysis of the available complex crystal structures. According to different CDR determination schemes, the residues of each of these HVR/CDRs are as follows.

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

Positions 24-36 or 24-34 (LCDR1) in VL, positions 46-56 or 50-56 (LCDR2), and positions 89-97 or 89-96 (LCDR3); and positions 26-35 or 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 the HVR sequence according to the Kabat numbering system at the following Kabat residue positions:

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

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

Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" or "HVR" or "HVR sequence" encompasses HVR or CDR sequences determined in any of the above ways.

Unless otherwise stated, in the present invention, when referring to the residue positions in the antibody variable region (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 Immunological Interest, 5th Ed. Public Health, National Institutes of Health, Bethesda, Md. (1991)).

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

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the minimum overlap area can be determined, thereby providing a "minimal binding unit" for antigen binding. The smallest binding unit may be a sub-portion of the CDR. As is clear to those skilled in the art, through 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 contemplates any CDR variants given herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.

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

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

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

(ii) With respect to HCDR1, HCDR2 and HCDR3 of (i), a total of at least 1 and no more than 10, preferably no more than 5 (preferably 1, 2 or 3) amino acid changes (preferably substitution, more preferably conservative substitution) , And preferably no more than 3 amino acid changes in the HCDR3 region (eg, 2, 1, or 0).

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

(i) Same as LCDR1, LCDR2 and LCDR3 of the light chain variable region of any antibody listed in Table A; or

(ii) With respect to LCDR1, LCDR2 and LCDR3 of (i), a total of at least 1 and no more than 10 (preferably 1-5, preferably 1, 2 or 3) amino acid changes (preferably substitutions, more preferably conservative substitutions) ).

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

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

(ii) Containing no more than 10, preferably no more than 5 (e.g., 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions) in all 6 CDR regions relative to any antibody listed in Table A (Conservative substitutions are preferred).

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

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

(ii) VH of SEQ ID NO: 14 and 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) VH of SEQ ID NO: 114 and 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, respectively, 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 a variant of the heavy chain CDR combination. In a preferred embodiment, the variant comprises a total of at least one and no more than 10 or 5 amino acid changes (preferably amino acid substitutions) on the three CDR regions , Preferably conservative substitution). 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 group consisting of antibodies and antigen-binding fragments comprising the combination:

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, the present invention provides a combination of light chain CDRs (LCDR1, LCDR2, and LCDR3 in sequence) 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 comprises a total of at least one and no more than 10 or 5 amino acid changes (preferably amino acid substitutions) on the three CDR regions , Preferably conservative substitution). The 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 CDR combination selected from the group consisting of antibodies and antigen-binding fragments comprising the combination:

(i) LCDR1 containing SEQ ID NO: 6 sequence, LCDR2 containing SEQ ID NO: 7 sequence, and LCDR3 containing SEQ ID NO: 8 sequence;

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

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

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

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

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

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

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

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

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

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

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

In still other embodiments, the present invention provides a combination of heavy chain and light chain CDRs 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 comprises a total of at least one and no more than 20, 10, or 5 amino acid changes (preferably amino acid substitutions) on the six CDR regions. , Preferably conservative substitution). The invention also provides an anti-CD30 antibody or antigen-binding fragment comprising the heavy chain and light chain CDR combinations 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 contains the amino acid sequence shown in SEQ ID NO: 1, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR3 contains the amino acid sequence shown in SEQ ID NO: 3, and LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7 and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8; or

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

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

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

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

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

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

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

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

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

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

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

In the above embodiment of the antibody of the present invention, "conservative substitution" refers to an amino acid change that results in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. In any embodiment of the invention, in a preferred aspect, the conservative substitution residues are from the following conservative substitution table X, preferably the preferred substitution residues shown in Table X.

Table X

Original residue	Exemplary replacement	Preferred conservative amino acid substitutions
Ala(A)	Val; Leu; Ile	Val
Arg(R)	Lys; Gln; Asn	Lys
Asn(N)	Gln; His; Asp; Lys; Arg	Gln
Asp(D)	Glu; Asn	Glu
Cys(C)	Ser; Ala	Ser
Gln(Q)	Asn; Glu	Asn
Glu(E)	Asp; Gln	Asp
Gly(G)	Ala	Ala
His(H)	Asn; Gln; Lys; Arg	Arg
Ile(I)	Leu; Val; Met; Ala; Phe; norleucine	Leu
Leu(L)	Norleucine; Ile; Val; Met; Ala; Phe	Ile
Lys(K)	Arg; Gln; Asn	Arg
Met(M)	Leu; Phe; Ile	Leu
Phe(F)	Trp; Leu; Val; Ile; Ala; Tyr	Tyr
Pro(P)	Ala	Ala

Ser(S)	Thr	Thr
Thr(T)	Val; Ser	Ser
Trp(W)	Tyr; Phe	Tyr
Tyr(Y)	Trp; Phe; Thr; Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala; norleucine	Leu

Exemplary antibody sequence

The present invention provides fully human antibodies that specifically isolate and characterize CD30 (eg, human CD30) as isolated and characterized in the examples. The variable region sequences of these exemplary antibodies of the invention are listed in Table A below (see also Figures 4-5). Exemplary CDR sequences for these antibodies are given in Table B below (see also Figure 3).

Table A. Amino acid and nucleotide sequences of the heavy chain variable region and light chain variable region of exemplary fully human antibody molecules of the invention.

Antibody name	VH	VH DNA	VL	VL DNA
HB38E4	SEQ ID NO: 4	SEQ ID NO: 5	SEQ ID NO: 9	SEQ ID NO: 10
HB10F1	SEQ ID NO: 14	SEQ ID NO: 15	SEQ ID NO: 19	SEQ ID NO: 20
HB49G9	SEQ ID NO: 24	SEQ ID NO: 25	SEQ ID NO: 29	SEQ ID NO: 30
HB36F7	SEQ ID NO: 34	SEQ ID NO: 35	SEQ ID NO: 39	SEQ ID NO: 40
HB68H2	SEQ ID NO: 44	SEQ ID NO: 45	SEQ ID NO: 49	SEQ ID NO: 50
HB69G7	SEQ ID NO: 54	SEQ ID NO: 55	SEQ ID NO: 59	SEQ ID NO: 60
HB10B6	SEQ ID NO: 64	SEQ ID NO: 65	SEQ ID NO: 69	SEQ ID NO: 70
HB16H7	SEQ ID NO: 74	SEQ ID NO: 75	SEQ ID NO: 79	SEQ ID NO: 80
HB6A9	SEQ ID NO: 84	SEQ ID NO: 85	SEQ ID NO: 89	SEQ ID NO: 90
HB16H8	SEQ ID NO: 94	SEQ ID NO: 95	SEQ ID NO: 99	SEQ ID NO: 100
P5E10	SEQ ID NO: 104	SEQ ID NO: 105	SEQ ID NO: 109	SEQ ID NO: 110
P27B3	SEQ ID NO: 114	SEQ ID NO: 115	SEQ ID NO: 119	SEQ ID NO: 120

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

The invention also provides variants of the above 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%, 85%, 90 from the sequence described in Table A % Or 95% or higher identity. In some embodiments, the antibody includes a mutated variable region amino acid sequence, wherein when compared to the variable region sequence shown in Table A, no more than 1, 2, 3, or 3 mutations have been mutated in the variable region VH and/or VL 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 (amino acid sequence and nucleotide sequence encoding the amino acid sequence) can be "mixed and matched" to produce other antibodies of the invention that bind to CD30. The binding of such "mixed and matched" antibodies to CD30 can be tested using binding assays known in the art (eg, ELISA, and other assays described in the Examples section). When mixing and matching these chains, it is preferable to replace VH sequences from specific VH/VL pairs with structurally similar VH sequences. Likewise, VL sequences from a specific VH/VL pairing are preferably replaced with structurally similar VL sequences.

In another aspect, the invention also provides variants of the above antibodies. In one embodiment, the antibody has been mutated on the amino acid sequence of one or more or all 6 CDR regions or the nucleic acid encoding the amino acid sequence. In some embodiments, the amino acid sequence of the mutated CDR region, when compared to the corresponding CDR region of Table A, has been mutated by no more than 1, 2, 3, 4, or 5 amino acids, but retains substantially the same antigen binding active.

In addition, since each of the antibodies of Table A can bind to CD30 and the antigen binding specificity is mainly provided by the CDR1, 2 and 3 regions, the VHCDR1, 2 and 3 sequences and the VLCDR1, 2 and 3 sequences can be mixed And match" (ie, CDRs from different antibodies can be mixed and matched, but each antibody preferably contains VH, CDR1, 2 and 3, and VL, CDR1, 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 (eg, ELISA, SET, Biacore) and those described in the examples. When mixing and matching VH CDR sequences, the CDR1, CDR2 and/or CDR3 sequences from a specific VH sequence are preferably replaced with structurally similar CDR sequences. Likewise, when mixing and matching VL CDR sequences, the CDR1, CDR2 and/or CDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequences. Those skilled in the art understand that other antibodies of the invention can also be produced by replacing one or more VH and/or VL CDR region sequences with structurally similar CDR sequences from the antibodies shown herein.

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 In this single protein chain, the VH and VL regions are paired to provide an antigen binding site.

In a preferred embodiment, the VH and VL regions of the single chain scFv antibody of the invention are covalently linked together by a linker peptide, such as a flexible linker peptide. The term "flexible linking peptide" is a peptide linker composed of amino acids. Through such a peptide linker, various variable domains in the antibody, such as the VH and VL regions, can be connected. Peptide linkers are usually rich in glycine that exhibits flexibility and serine or threonine that 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, it is preferred that the linker will facilitate the pairing of VH and VL without interfering with VH and VL to form a functionally effective antigen binding site.

In some embodiments, scFv single chain antibodies of the invention comprise a flexible linking peptide or peptide linker consisting of amino acid residues linked 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 linker is about 1-30 amino acids in length, or about 10 to about 25 amino acids, about 15 to about 20 amino acids, or about 10 to about 20 amino acids, or any intermediate Amino acid length. In a preferred embodiment, the linker has a length of 15-25 amino acid residues, and in a more preferred embodiment, has a length of 15-18 amino acid residues. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

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 Integers; glycine-alanine polymer; alanine-serine polymer; and other flexible linkers known in the art. Those skilled in the art will 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 portion and one or more portions conferring 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, n=6 or 7. In some embodiments, the linker may include the 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 to 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; TGEPK (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. USA 87: 1066-1070 ); KESGSVSSEQLAQFRSLD (Bird et al., 1988, Science 242: 423-426), GGRRGGGS; LRQRDGERP; LRQKDGGGSERP; LRQKD (GGGS) 2ERP. Alternatively, a computer program capable of modeling DNA-binding sites and the peptide itself can be used ( Desjarlais & Berg, PNAS90: 2256-2260 (1993), PNAS91: 11099-11103 (1994)), or through phage or yeast display methods, rational design of flexible linkers.

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

In some embodiments, in addition to the linker, other polypeptide fragments having specific functions may be inserted between the VL and VH domains, for example, a polypeptide fragment having a function of modulating an immune response, or a polypeptide fragment having a cell solvent or cell killing effect.

In some embodiments, single chain antibodies can be stabilized by introducing disulfide bonds in the scFv. For example, the framework regions of the VH and VL of the scFv can be connected by introducing intra-chain or inter-chain disulfide bonds. In one embodiment, it is possible to mutate 1 amino acid residue of each of the antibodies VH and VL to cysteine, for example, according to the Kabat numbering system, VH 44 and VL 100, or VH 105 and VL Of 43.

The single-chain scFv polypeptide antibodies of the present invention can be expressed from nucleic acids including VH- and VL-coding sequences, as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85: 5879-5883, 1988). See also US Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and US Patent Publication Nos. 20050196754 and 20050196754. In some embodiments, the single chain scFv antibodies of the invention are 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-CD30scFv or 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 121. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 122.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 4 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Of the heavy chain variable region, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes VH having the amino acid sequence of SEQ ID NO:4. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO:9. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 4 and a VL including the sequence shown in SEQ ID NO: 9. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 4 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 9. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 3 and LCDR3 of SEQ ID NO: 8. In some embodiments, the anti-CD30scFv comprises: SEQ ID NO: 1 VH CDR1, SEQ ID NO: 2 VH CDR2, and SEQ ID NO: 3 VH CDR3. In certain embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 6 CDR1, VL of SEQ ID NO: 7 CDR2, and VL of SEQ ID NO: 8 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 1 VH CDR1, SEQ ID NO: 2 VH CDR2, and SEQ ID NO: 3 VH CDR3, and SEQ ID ID NO: 6 VL CDR1, SEQ The VL of ID NO: 7 is CDR2, and the VL of SEQ ID NO: 8 is CDR3.

In certain embodiments, the antibody of the present invention is an anti-CD30scFv or 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 124. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 125.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 14 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Of the heavy chain variable region, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes VH having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO:19. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 14 and a VL including the sequence shown in SEQ ID NO: 19. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 14, and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 19. In some embodiments, the anti-CD30scFv comprises SEQ ID NO: 13 HCDR3 and SEQ ID NO: 18 LCDR3. In certain embodiments, the anti-CD30scFv comprises: VH CDR1 of SEQ ID NO: 11, VH CDR2 of SEQ ID NO: 12, and VH CDR3 of SEQ ID NO: 13. In certain embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 16 CDR1, VL of SEQ ID NO: 17 CDR2, and VL of SEQ ID NO: 18 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 11 VH CDR1, SEQ ID NO: 12 VH CDR2, and SEQ ID NO: 13 VH CDR3, and SEQ ID NO: 16 VL CDR1, SEQ The VL of ID NO: 17 is CDR2, and the VL of SEQ ID NO: 18 is CDR3.

In certain embodiments, the antibody of the present invention is an anti-CD30scFv 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO:127. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 128.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 24 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Heavy chain variable region, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes VH having the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO:29. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 24 and a VL including the sequence shown in SEQ ID NO: 29. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 24 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 29. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 23 and LCDR3 of SEQ ID NO: 28. In certain embodiments, the anti-CD30scFv comprises: VH CDR1 of SEQ ID NO: 21, VH CDR2 of SEQ ID NO: 22, and VH CDR3 of SEQ ID NO: 23. In some embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 26, CDR1 of SEQ ID NO: 27, and VL CDR2 of SEQ ID NO: 28. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 21 VH CDR1, SEQ ID NO: 22 VH CDR2, and SEQ ID NO: 23 VH CDR3, and SEQ ID NO: 26 VL CDR1, SEQ The VL of ID NO: 27 is CDR2, and the VL of SEQ ID NO: 28 is CDR3.

In certain embodiments, the antibody of the invention is an anti-CD30scFv 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 130. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO:131.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 34 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Heavy chain variable region, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes a VH having the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the anti-CD30scFv includes a VL having the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the anti-CD30scFv includes VH having the sequence shown in SEQ ID NO: 34 and VL including the sequence shown in SEQ ID NO: 39. In certain embodiments, the anti-CD30scFv includes three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 34 and/or three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 39. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 33 and LCDR3 of SEQ ID NO: 38. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 31 VH CDR1, SEQ ID NO: 32 VH CDR2, and SEQ ID NO: 33 VH CDR3. In some embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 36 CDR1, VL of SEQ ID NO: 37 CDR2, and VL of SEQ ID NO: 38 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 31 VH CDR1, SEQ ID NO: 32 VH CDR2, and SEQ ID NO: 33 VH CDR3, and SEQ ID NO: 36 VL CDR1, SEQ The VL of ID NO: 37 is CDR2, and the VL of SEQ ID NO: 38 is CDR3.

In certain embodiments, the antibody of the present invention is an anti-CD30scFv 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 133. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 134.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 44 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Heavy chain variable region, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes VH having the amino acid sequence of SEQ ID NO: 44. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO: 49. In certain embodiments, the anti-CD30scFv includes VH having the sequence shown in SEQ ID NO: 44 and VL including the sequence shown in SEQ ID NO: 49. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 44 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 49. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 43 and LCDR3 of SEQ ID NO: 48. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 41 VH CDR1, SEQ ID NO: 42 VH CDR2, and SEQ ID NO: 43 VH CDR3. In certain embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 46, CDR1 of SEQ ID NO: 47, CDR2 of SEQ ID NO: 47, and VL of SEQ ID NO: 48. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 41 VH CDR1, SEQ ID NO: 42 VH CDR2, and SEQ ID NO: 43 VH CDR3, and SEQ ID NO: 46 VL CDR1, SEQ VL IDCDR NO: 47 CDR2, and SEQ ID NO NO: 48 VLCDR3.

In certain embodiments, the antibody of the present invention is an anti-CD30scFv or antigen-binding fragment thereof, which comprises the antigen-binding region of amino acid sequence SEQ ID NO: 136 or a variant thereof, and specifically binds to a CD30 polypeptide (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 136. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 137.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 54 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Of the heavy chain variable region, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 59 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes VH having the amino acid sequence of SEQ ID NO: 54. In certain embodiments, the anti-CD30scFv includes a VL having the amino acid sequence of SEQ ID NO: 59. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 54 and a VL including the sequence shown in SEQ ID NO: 59. In certain embodiments, the anti-CD30scFv includes three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 54 and/or three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 59. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 53 and LCDR3 of SEQ ID NO: 58. In some embodiments, the anti-CD30scFv comprises: SEQ ID NO: 51 VH CDR1, SEQ ID NO: 52 VH CDR2, and SEQ ID NO: 53 VH CDR3. In certain embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 56 CDR1, VL of SEQ ID NO: 57 CDR2, and VL of SEQ ID NO: 58 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 51 VH CDR1, SEQ ID NO: 52 VH CDR2, and SEQ ID NO: 53 VH CDR3, and SEQ ID NO: 56 VL CDR1, SEQ The VL of ID NO: 57 is CDR2, and the VL of SEQ ID NO: 58 is CDR3.

In certain embodiments, the antibody of the present invention is an anti-CD30scFv or 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO:139. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 140.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 64 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Of the heavy chain variable region, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 69 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes a VH having the amino acid sequence of SEQ ID NO:64. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 64 and a VL including the sequence shown in SEQ ID NO: 69. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 64 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 69. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 63 and LCDR3 of SEQ ID NO: 68. In some embodiments, the anti-CD30scFv comprises: SEQ ID NO: 61 VH CDR1, SEQ ID NO: 62 VH CDR2, and SEQ ID NO: 63 VH CDR3. In certain embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 66, CDR1 of SEQ ID NO: 67, CDR2 of SEQ ID NO: 67, and VL CDR3 of SEQ ID NO: 68. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 61 VH CDR1, SEQ ID NO: 62 VH CDR2, and SEQ ID NO: 63 VH CDR3, and SEQ ID NO: 66 VL CDR1, SEQ The VL of ID NO: 67 is CDR2, and the VL of SEQ ID NO: 68 is CDR3.

In certain embodiments, the antibody of the invention is an anti-CD30scFv or 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 142. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 143.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 74 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Heavy chain variable region, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes VH having the amino acid sequence of SEQ ID NO: 74. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO: 79. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 74 and a VL including the sequence shown in SEQ ID NO: 79. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 74 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 79. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 73 and LCDR3 of SEQ ID NO: 78. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 71 VH CDR1, SEQ ID NO: 72 VH CDR2, and SEQ ID NO: 73 VH CDR3. In some embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 76 CDR1, VL of SEQ ID NO: 77 CDR2, and VL of SEQ ID NO: 78 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 71 VH CDR1, SEQ ID NO: 72 VH CDR2, and SEQ ID NO: 73 VH CDR3, and SEQ ID NO: 76 VL CDR1, SEQ ID: NO: 77 VL2 CDR2, and SEQ ID NO: 78 VL CDR3.

In certain embodiments, the antibody of the invention is an anti-CD30scFv or 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO:145. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 146.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 84 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Of the heavy chain variable region, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 89 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes a VH having the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO:89. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 84 and a VL including the sequence shown in SEQ ID NO: 89. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 84 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 89. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 83 and LCDR3 of SEQ ID NO: 8 8. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 81 VH CDR1, SEQ ID NO: 82 VH CDR2, and SEQ ID NO: 83 VH CDR3. In certain embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 86 CDR1, VL of SEQ ID NO: 87 CDR2, and VL of SEQ ID NO: 88 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 81 VH CDR1, SEQ ID NO: 82 VH CDR2, and SEQ ID NO: 83 VH CDR3, and SEQ ID NO: 86 VL CDR1, SEQ The VL of ID NO: 87 is CDR2, and the VL of SEQ ID NO: 88 is CDR3.

In certain embodiments, the antibody of the present invention is an anti-CD30scFv or 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 148. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 149.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 94 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Heavy chain variable region, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 99 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes VH having the amino acid sequence of SEQ ID NO:94. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO: 99. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 94 and a VL including the sequence shown in SEQ ID NO: 99. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 94 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 99. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 93 and LCDR3 of SEQ ID NO: 98. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 91 VH CDR1, SEQ ID NO: 92 VH CDR2, and SEQ ID NO: 93 VH CDR3. In certain embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 96 CDR1, VL of SEQ ID NO: 97 CDR2, and VL of SEQ ID NO: 98 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 91 VH CDR1, SEQ ID NO: 92 VH CDR2, and SEQ ID NO: 93 VH CDR3, and SEQ ID NO: 96 VL CDR1, SEQ The VL of ID NO: 97 is CDR2, and the VL of SEQ ID NO: 98 is CDR3.

In certain embodiments, the antibody of the invention is an anti-CD30scFv or 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 151. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 152.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 104 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Heavy chain variable region, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 109 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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: 104. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO: 109. In certain embodiments, the anti-CD30scFv includes a VH having the sequence shown in SEQ ID NO: 104 and a VL including the sequence shown in SEQ ID NO: 109. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 104 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 109. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 103 and LCDR3 of SEQ ID NO: 108. In some embodiments, the anti-CD30scFv comprises: VH CDR1 of SEQ ID NO: 101, CDR2 of SEQ ID NO: 102, and VH CDR3 of SEQ ID NO: 103. In some embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 106 CDR1, VL of SEQ ID NO: 107 CDR2, and VL of SEQ ID NO: 108 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 101 VH CDR1, SEQ ID NO: 102 VH CDR2, and SEQ ID NO: 103 VH CDR3, and SEQ ID NO: 106 VL CDR1, SEQ The VL of ID NO: 107 is CDR2, and the VL of SEQ ID NO: 108 is CDR3.

In certain embodiments, the antibody of the invention is an anti-CD30scFv 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 (eg, having an amino acid sequence CD30 polypeptide of SEQ ID NO: 162 or a fragment thereof). In some embodiments, the variant has at least 90%, 92%, 95%, 97%, 98%, 99%, or higher identity with SEQ ID NO: 154. In one embodiment, the anti-CD30scFv is encoded by the nucleotide of SEQ ID NO: 155.

In certain embodiments, the anti-CD30scFv antibody comprises: an amino acid sequence comprising SEQ ID NO: 114 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it Of the heavy chain variable region, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 119 or having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , And optionally a linker between the heavy chain variable region and the light chain variable region, 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-CD30scFv includes a VH having the amino acid sequence of SEQ ID NO: 114. In certain embodiments, the anti-CD30scFv includes VL having the amino acid sequence of SEQ ID NO: 119. In certain embodiments, the anti-CD30scFv includes VH having the sequence shown in SEQ ID NO: 114 and VL including the sequence shown in SEQ ID NO: 119. In certain embodiments, the anti-CD30scFv includes the three HCDR sequences of the VH of the sequence shown in SEQ ID NO: 114 and/or the three LCDR sequences of the VL of the sequence shown in SEQ ID NO: 119. In some embodiments, the anti-CD30scFv comprises HCDR3 of SEQ ID NO: 113 and LCDR3 of SEQ ID NO: 118. In certain embodiments, the anti-CD30scFv comprises: VH CDR1 of SEQ ID NO: 111, VH CDR2 of SEQ ID NO: 112, and VH CDR3 of SEQ ID NO: 113. In certain embodiments, the anti-CD30scFv includes: VL of SEQ ID NO: 116 CDR1, VL of SEQ ID NO: 117 CDR2, and VL of SEQ ID NO: 118 CDR3. In certain embodiments, the anti-CD30scFv comprises: SEQ ID NO: 111 VH CDR1, SEQ ID NO: 112 VH CDR2, and SEQ ID NO: 113 VH CDR3, and SEQ ID NO: 116 VL CDR1, SEQ ID: NO: 117 VL2 CDR2, and SEQ ID: NO: 118 VL3 CDR3.

III. scFv-Fc antibody

Antibodies with an Fc region have several advantages, including, but not limited to: effector functions can be mediated through the Fc region, such as CDC and ADCC immunological activity; divalent antibodies formed through the dimerization function of the Fc region can provide strong Antigen binding affinity, and/or changes in plasma half-life and renal clearance; bivalent antibodies can be internalized at a rate different from monovalent Fab or scFv antibodies, changing 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, the scFv-Fc antibody formed by fusion of the single chain scFv antibody of the present invention and 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-terminal to C-terminal: 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 connected to the variable region (VH or VL) via a hinge region. In some embodiments, the Fc is an Fc region from 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 not more than 20, 10 relative to the amino acid sequence of SEQ ID NO: 161 Or an amino acid sequence with 5 amino acid changes, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 161. In some embodiments, the single chain scFv antibodies of the invention are connected to the Fc region via a hinge region. In one embodiment, the hinge region is a C8 hinge region, for example, including the amino acid sequence shown in SEQ ID NO: 159, or the amino acid sequence relative to SEQ ID NO: 159 contains at least one, two or three, but not more than Amino acid sequence with 5 amino acid changes.

In some preferred embodiments, the present invention provides an antibody that specifically binds to a CD30 polypeptide (eg, a CD30 polypeptide having an amino acid sequence of SEQ ID NO: 162 or a fragment thereof), and includes an antibody selected from SEQ ID NO: 123, Amino acid sequences of 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, and 156, or contain at least one, two or three, but not more than 20, 10 or 5 Amino acid sequence with an amino acid change, or an amino acid sequence with at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity to it.

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 are listed in Table C below. The amino acid sequence and nucleotide sequence of the linker and hinge used in these scFv-Fc antibodies are shown in FIG. 7.

Table C:

Antibody name	scFv amino acid sequence	scFv DNA sequence	scFv-hFc amino acid sequence
HB38E4scFv-Fc	SEQ ID NO: 121	SEQ ID NO: 122	SEQ ID NO: 123
HB10F1scFv-Fc	SEQ ID NO: 124	SEQ ID NO: 125	SEQ ID NO: 126
HB49G9scFv-Fc	SEQ ID NO: 127	SEQ ID NO: 128	SEQ ID NO: 129
HB36F7scFv-Fc	SEQ ID NO: 130	SEQ ID NO: 131	SEQ ID NO: 132
HB68H2scFv-Fc	SEQ ID NO: 133	SEQ ID NO: 134	SEQ ID NO: 135
HB69G7scFv-Fc	SEQ ID NO: 136	SEQ ID NO: 137	SEQ ID NO: 138
HB10B6scFv-Fc	SEQ ID NO: 139	SEQ ID NO: 140	SEQ ID NO: 141
HB16H7scFv-Fc	SEQ ID NO: 142	SEQ ID NO: 143	SEQ ID NO: 144
HB6A9scFv-Fc	SEQ ID NO: 145	SEQ ID NO: 146	SEQ ID NO: 147
HB16H8scFv-Fc	SEQ ID NO: 148	SEQ ID NO: 149	SEQ ID NO: 150
P5E10scFv-Fc	SEQ ID NO: 151	SEQ ID NO: 152	SEQ ID NO: 153
P27B3scFv-Fc	SEQ ID NO: 154	SEQ ID NO: 155	SEQ ID NO: 156

In some embodiments, the scFv-Fc antibodies of the invention have effector functions. The term "effector function" refers to those biological activities attributable to the Fc-region of an antibody, which vary with the 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 heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Antibody effector functions include, for example and without limitation: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors ( For example, down-regulation of B cell receptors; and B-cell activation. US20120014943A1 reports that the inclusion of at least one amino acid substitution selected from 239D and 332E in the Fc region can result in the antibody having enhanced affinity for FcγRIIIa, thereby causing enhanced effector function. In some embodiments, the scFv-Fc antibody of the present invention blocks, inhibits the growth of CD30 expressing cells (especially tumor cells such as lymphoma cells) through effector cell-mediated cytotoxicity (ADCC) activity, and/or Or kill the cells.

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

In other embodiments, the Fc can be altered to increase or decrease its degree of glycosylation and/or change its glycosylation pattern. The addition or deletion of glycosylation sites to Fc can be conveniently achieved by changing the amino acid sequence in order to create or remove one or more glycosylation sites. For example, one or more amino acid substitutions can be made 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 no fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased aliquot GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies.

Therefore, in some preferred embodiments, the present invention provides antibodies whose Fc region is low or afucosylated, so that the antibody Fc domain and Fcγ receptor expressed on effector cells (such as The binding affinity of FcyRIIIa), thereby resulting in antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. For example, the amount of fucose in the antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. It can be measured by MALDI-TOF mass spectrometry, and the average of fucose in the sugar chain at Asn297 is calculated relative to the sum of all sugar structures (such as complex, hybrid, and high-mannose structures) connected to Asn297 The amount, thereby determining the amount of fucose, for example as described in WO2008/077546. Asn297 refers to asparagine residues located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, due to minor sequence changes in the antibody, Asn297 may also be located approximately ±3 upstream or downstream of position 297 Amino acid position, that is between position 294 and 300. See for example US2003/0157108; US2004/0093621. Published examples related to "defucosylated" or "low fucosylated" antibody variants also include: US2003/0157108; WO2000/61739; 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 defucosylated or hypofucosylated antibodies. Examples of such cells include Lec13CHO cells that are deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986): 533-545; US 2003/0157108; and WO 2004/056312, in particular Is Example 11); and gene knockout cell lines, such as the CHO cells knocked out of the α-1,6-fucosyltransferase gene FUT8 (see, eg, Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 : 614 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107). As another example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), so that the lack of fucoids can be expressed in the Ms704, Ms705, and Ms709 cell lines Sugar antibodies. In addition, EP 1,176,195 also describes a cell line with a disrupted FUT8 gene, and antibodies expressed in such cell lines exhibit low fucosylation. 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 biantennary oligosaccharides linked to the Fc region are bisected 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 invention also contemplates antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region. These antibody variants may have improved CDC function. These antibody variants are described in, for example, WO1997/30087; WO1998/58964; and WO1999/22764.

Non-limiting examples of in vitro assays to evaluate 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. et al., Proc. Nat'l Acad. Sci. USA 82 (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 (CellTechnology, Inc. Mountain View, CA) for flow cytometry and

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 animal models as disclosed in Clynes, R. et al., Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656 Evaluation. 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., Blood101 (2003) 1045-1052; and Cragg, MS And MJ Glennie, Blood 103 (2004) 2738-2743). C1q binding assays can also be performed to determine the antibody's C1q binding and CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.

In certain embodiments, the present invention also considers antibody variants with some but not all effector functions, which makes them ideal candidates for certain applications in which the in vivo half-life of the antibody is important, but Certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo assays as described above can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. For example, the Fc region may contain mutations that eliminate or weaken effector functions, such as the human IgG1 Fc region with mutations P329G and/or L234A and L235A, or the 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, and thus can further have increased total antibody affinity and stability, or form multispecificity such as bispecific Sex. For example, the Fc region may comprise i) a homodimeric Fc-region of the human IgG1 subclass, or ii) a homodimeric Fc-region of the human IgG4 subclass, or iii) a heterodimeric Fc-region, where a) one Fc- The region polypeptide comprises the mutation T366W, and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or c) One Fc-region polypeptide contains mutations T366W and S354C, while the other Fc-region polypeptide contains 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, including but not limited to, fluorescent dyes, cytotoxins, radioisotopes, etc., for example, for antigen quantification Research, immobilization of antibodies for affinity measurement, for 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 invention provides a substantially purified nucleic acid molecule that encodes a polypeptide comprising the segment or domain of the CD30-binding antibody chain described above. In some embodiments, the nucleic acid molecule of the invention encodes an antibody chain that binds CD30 (eg, 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 a nucleotide sequence encoding the heavy chain variable region or variant of any antibody shown in Table A, and/or the light chain variable region or variant of the corresponding antibody shown in Table A Nucleotide sequence. In a specific embodiment, the nucleic acid molecule is the DNA VH sequence and/or DNA VL sequence listed in Table A. Some other nucleic acid molecules of the present invention comprise a nucleotide sequence that is substantially identical (eg, at least 65%, 80%, 95%, or 99% identical) to the nucleotide sequence of the nucleic acid molecule shown in Table A. When expressed from a suitable expression vector, the polypeptides encoded by these polynucleotides can exhibit CD30 antigen binding ability.

The present invention also provides polynucleotides that encode at least one CDR region and usually all three CDR regions from the heavy chain VH or light chain VL sequence of the CD30-binding antibody described above. 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 is clear to those skilled in the art, because of the degeneracy of the codons, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences.

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

In some embodiments, the nucleic acid sequences of the invention encode any single chain scFv antibody of the invention described above. In some embodiments, the nucleic acid sequence of the invention encoding an 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 its substantially identical sequence, and the sequence of SEQ ID NO: 10 or its substantially identical sequence,

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

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

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

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

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

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

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

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

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

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

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

In a preferred embodiment, the nucleic acid of the 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 as it.

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

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

These polynucleotide sequences can be generated by de novo solid phase DNA synthesis, or by PCR mutagenesis of existing sequences encoding antibodies or binding fragments thereof that bind CD30 (eg, the sequences shown in Tables A-C). 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., phosphoric acid of Meth. Enzymol. 68:109, 1979 Diester method; Beaucage et al., Tetra. Lett., 22: 1859, 1981 diethylphosphimide method; and US Patent No. 4,458,066 solid phase support method. Introducing mutations to the polynucleotide sequence by PCR can be like, for example, PCR Technology: Principles and Applications for DNA Amplification, HAErlich (edited), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods Methods and Applications, Innis and others (Editor), 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. Preparation of antibodies

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

In one embodiment, a vector comprising an isolated nucleic acid encoding an antibody described herein that binds CD30 is provided. This nucleic acid may encode the amino acid sequence of the VL containing the antibody and/or the amino acid sequence of the VH containing the antibody. In a further embodiment, the vector is an expression vector. In a further embodiment, the present invention provides a host cell containing this nucleic acid. In one embodiment, the host cell comprises (e.g., it has been transformed with the following vector): (1) a vector comprising a nucleic acid encoding an amino acid sequence containing antibody VL and an amino acid sequence containing an antibody VH, or (2) a vector encoding an antibody containing VL The first vector of nucleic acids of the amino acid sequence of the amino acid 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 (eg, 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 comprising a nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression, and optionally from the host cell (or host Cell culture medium) to recover antibodies.

For the recombinant production of anti-CD30 antibodies, nucleic acids encoding antibodies, such as those described above, can be isolated and inserted into one or more vectors for further cloning 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 antibody chains that bind CD30 (such as any antibodies of the 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 include plasmids, episomal vectors, and artificial chromosomes, and generally contain expression cassettes for expressing proteins or RNA (see, for example, Harrington et al., Nat Genet 15:345, 1997). Useful viral vectors include vectors based on retroviruses, adenovirus, adeno-associated virus, herpes virus, vectors based on SV40, papilloma virus, HBP Epstein-Barr 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 antibody chains or fragments that bind CD30. 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 including 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 may be linked to the signal sequence before insertion into the vector. In a preferred embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 164. Vectors for accepting sequences encoding the light chain variable domain and heavy chain variable domain of an antibody that binds to CD30 may sometimes encode a constant region or a portion thereof. Such vectors allow the variable region to be expressed as a fusion protein with the constant region, thereby leading to the production of intact antibodies or fragments thereof. Generally, such constant regions are human constant regions such as human IgG1 Fc regions. In a preferred embodiment, the Fc region fused to the variable region comprises the amino acid sequence shown in SEQ ID NO: 161.

Suitable host cells for cloning or expression of vectors 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, US 5,648,237, US 5,789,199 and US 5,840,523. (See also Charlton, KA, see: Methods in Molecular Biology, Volume 248, Lo, BKC (Editor), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes antibody fragments in E. coli expression). 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 cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", which results in partial or Production of antibodies in fully human glycosylation mode. See Gerngross, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech (2006) 24:210-215. Suitable host cells for glycosylated antibody expression 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 transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describe the PLANTIBODIES ^TM technology for producing antibodies in transgenic plants). Vertebrate cells that can be used as hosts include, for example, mammalian cell lines adapted for suspension growth, which may be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or described in, for example, Graham, FL et al., J. Gen Virol. 36 (1997) 59 293 cells); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2) ; Mouse breast tumor (MMT 060562); TRI cells, such as those described in Mather, JP et al., Annals NYAcad. Sci. 383 (1982) 44-68; MRC 5 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. USA 77 (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, Methods in Molecular Biology, Vol. 248, Lo. BKC (Editor), Humana Press, 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 for their physical/chemical properties and/or biological activity through various tests known in the art.

Phages that bind to the target antigen of interest can be selected from phage display libraries that express human antibodies. There are various methods for displaying antibodies or antibody fragments on the surface of phage and screening libraries. For example, see 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) method. For example, a phage group presenting intact antibodies or antibody fragments can be incubated with biotinylated target antigen and streptavidin magnetic beads in the liquid phase for a period of time, then washed, and enriched with the target antigen by the action of a magnetic field. Phage, after which the phage are eluted and amplified. After several times of "adsorption-elution-amplification", the obtained antigen-specific phage were cloned and sequenced.

Transgenic animals expressing human immunoglobulin libraries, such as transgenic mice, can also be used 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 is obtained and fused with myeloma cells, and the hybridoma producing the desired antibody is selected and sequenced.

For the identification of antibodies, the antibodies of the present invention can be identified or characterized by known methods, such as ELISA, αLISA, Western blot, antibody or reverse phase array, and the methods described in the examples.

For example, the antibody can be spotted on a glass or nitrocellulose chip. The slides were blocked and incubated with a solution containing CD30, washed to remove unbound antibody, and the bound antibody was detected with a fluorescently labeled corresponding secondary antibody. The fluorescence signal is measured by a fluorescence slide scanner. Similarly, for reversed-phase arrays, recombinant CD30, cell supernatant, cell or tissue lysate, body fluids, etc., are spotted on a glass or nitrocellulose chip. The slides were blocked and the array was incubated with antibodies against specific epitopes on CD30. Wash off unbound antibody and detect the bound antibody 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 antibody can also be detected using the ForteBio assay. ForteBio affinity determination can be performed 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, AHC (anti-hIgG-Fc capture surface) sensors can be used to perform kinetic analysis of the interaction of scFv-hFc antibody and antigen molecules.

The binding of the antibody 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 (eg, 4°C, 30 minutes). Afterwards, it is incubated with a secondary antibody (for example, a secondary antibody labeled with phycobilisome pigment) for a period of time (for example, 4°C, 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 is 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 antibodies of the invention to heterologous molecules. In some embodiments, the antibody polypeptides of the invention can be fused or conjugated to one or more heterologous molecules, where the heterologous molecules include, but are not limited to proteins/polypeptides/peptides, labels, drugs, and cytotoxic agents. Methods for fusion or conjugation of proteins, polypeptides or peptides or chemical molecules with antibodies are known in the art. See, for example, US 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, antibodies of the invention can 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 antibodies of the invention are 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. Linkers include chemical linkers or single-chain peptide linkers. In some embodiments, single chain antibodies of the invention, such as scFv antibodies, are fused to other peptides or proteins via peptide linkers. In some embodiments, single chain antibodies of the invention, such as scFv antibodies, are conjugated to other molecules such as labels 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 portion to which they are attached, such as scFv, to move independently. The length of the linker peptide can be easily determined by those skilled in the art according to actual conditions, for example, at least 4-15 amino acids in length, or longer, for example, about 20-25 amino acids.

The chemical linkers of the present invention can be used to include, for example, various coupling agents. Examples of coupling agents are N-succinimide-3-(2-pyridinedithio) propionate (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexyl Alkyl-1-carboxylate (SMCC), iminosulfane (IT), bifunctional derivatives of imidoesters (such as adipic acid dimethyl ester HCl), active esters (such as suberic acid bis Succinimide ester), aldehydes (such as glutaraldehyde), diazide compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), bis-diazo derivatives (such as bis-(p (Nitrobenzoyl)-ethylenediamine), diisocyanate (such as toluene 2,6-diisocyanate), and dual-active 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, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52 (1992) 127-131; US 5,208,020) .

F. Methods and compositions for diagnosis and detection

In one aspect, the invention provides the use of the anti-CD30 antibody, fusion or conjugate of the invention in diagnosis and detection. Any anti-CD30 antibody, fusion or conjugate 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 qualitative detection. Exemplary detection methods include, but are not limited to, immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-technology (eg, RT-PCR). In some embodiments, the biological sample includes body fluids, cells, or tissues. In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin.

In one embodiment, anti-CD30 antibodies, fusions or conjugates are provided for diagnostic, prognostic or detection methods. 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 the 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, classic Hodgkin lymphoma, anaplastic large cell lymphoma, and the like. Reed-Sternberg cells of classic Hodgkin lymphoma express CD30 and CD15. The lack of CD30 and CD15 expression can separate nodular lymphocyte-dominant Hodgkin lymphoma (NLPHL) from classical Hodgkin lymphoma. CD30 expression is also a necessary feature of ALCL, including ALK positive and ALK-negative ALCL. In addition, Lymphomatoid Papulosis (LyP) also has the characteristic expression of CD30. It has been reported that in 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 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, there is provided a method of stratifying a patient with the antibody, fusion or conjugate of the present invention, the method comprising determining whether a cell of the patient, such as a tumor cell, is on the surface of the cell Expressing the CD30 protein, where the cell expresses the CD30 protein on its surface, the patient will likely respond and use a therapeutic agent targeting CD30 (eg, anti-CD30 antibody) for treatment.

In some embodiments, the anti-CD30 antibody can be conjugated with a diagnostic agent or a 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 of treating CD30-related disorders, comprising administering to the subject an effective amount of an antibody of the invention or antigen-binding fragment thereof, or a conjugate or fusion of the invention.

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

The term "treatment" refers to a clinical intervention that is intended to alter the natural course of the disease in the individual being treated. Desirable therapeutic effects include but are not limited to preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, improving or alleviating the disease state, and relieving 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, autoimmune diseases. Exemplary conditions that can be 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 cells Lymphoma (CTCL), serum immunoblastic lymphadenopathy-like T-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mycosis granulomatosis (MF), lymphoma-like papulosis (LyP), immune mother Cellular lymphoma, multiple myeloma.

As shown in the examples, the inventors constructed antibodies of the invention based on antibody sequences screened from human antibody libraries. Therefore, advantageously, in some embodiments, the antibody of the invention is a fully human antibody comprising the amino acid sequences of a fully human VH region and a fully human VL region, such as the antibodies shown in Table A, and shown in Table C Single-chain scFv and scFv-Fc antibody constructed from human hFc fragments. 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 invention are used to treat CD30-related disorders in humans, such as CD30 positive tumors or autoimmune diseases involving CD30 expressing immune cells, preferably, Hodgkin lymphoma Tumor (HL) and non-Hodgkin's lymphoma (NHL).

In some embodiments, administration of an anti-CD30 antibody, conjugate or fusion of the invention results in growth inhibition or killing effects 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, cutaneous T-cell lymphoma cells, pleomorphic and Immunoblastic lymphoma cells. In a specific embodiment, the human antibody of the present invention or a conjugate or fusion containing the same is used to inhibit/mediated killing of Hodgkin cells in the treatment of Hodgkin lymphoma (especially classic Hodgkin lymphoma) And Rees' cells. In another specific embodiment, the human antibodies of the invention or conjugates or fusions comprising the same are used in the treatment of ALCL to inhibit/mediate the killing of CD30 positive ALCL tumor cells.

It can be 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 treatment modalities include therapeutic agents, radiotherapy, chemotherapy, transplantation, immunotherapy and the like. In some embodiments, antibody molecules of the invention, or conjugates or fusions of the invention are used in combination with other therapeutic agents. Exemplary therapeutic agents include cytotoxic agents, radiotoxins, immunosuppressive agents, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, 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 radioisotope.

H. Compositions and preparations

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

Pharmaceutical preparations of antibodies or fusions or conjugates of the invention can be prepared, for example, by making antibodies, fusions or conjugates of the desired purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical) Science, 16th edition, Osol, A. (Editor) (1980)) mixed and prepared in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methylthio Amino acid; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; chlorhexidine; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as Methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein, such as serum white Protein, gelatin, or immunoglobulin; hydrophilic polymers, such as poly(vinylpyrrolidone); amino acids, such as glycine, glutamine, asparagine, 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 nonionic surfactants, such as polyethylene glycol (PEG).

An exemplary lyophilized antibody preparation is described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO2006/044908, the latter formulations include histidine-acetate buffer.

The formulations herein may 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 in the understanding of the present invention. It is not intended and should not be construed in any way to limit the scope of protection of the present invention.

Examples

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

Using hybridoma technology, immunize Harbour H2L2 transgenic mice with recombinant human CD30 protein (Acro, catalog number: CD0-H5229) (manufacturer: Beijing Viton Lihua Experimental Animal Technology Co., Ltd.; license number: SCXK (京) 2016- 0006), and then the mouse spleen cells and myeloma cells are fused to obtain hybridoma cells capable of expressing CD30 antibody. The specific process is as follows.

Immunization of transgenic mice: adjuvant Ribi (manufacturer: Sigma; article number: S6322; batch number: 077M4061V), mixed with huCD30 protein in equal volumes, immunized at 50μg/200μl per mouse (total 5), of which 100μl are subcutaneously Injection, 100 μl intraperitoneal injection. Immunize once every two weeks for a total of 5 times. Three days before fusion, each mouse was injected intraperitoneally at a dose of 50 μg/300 μl of huCD30 protein for subsequent fusion experiments.

Preparation of the electric fusion dish: Soak the electric fusion dish thoroughly with 70% ethanol, and blow dry in an ultra-clean stand for 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 anatomical plate of the mouse in the ultra-clean table, lying on the left side, and the limbs were fixed with a 7-gauge needle. Aseptically open the abdominal cavity to remove the spleen, wash with basal medium (preparation method as shown in Table 1 below), and carefully remove the surrounding connective tissue. The spleen was then transferred to another dish containing basal medium. Squeeze the spleen with an elbow needle, use a small needle to insert the spleen into the spleen, and squeeze it with tweezers to fully release the spleen cells to make a spleen cell suspension. The cell suspension was filtered through a 70 μM cell sieve, washed with 30 ml of basal medium, and centrifuged at 1200 rpm for 6 min.

Table 1: Basal medium

Lysis of red blood cells: remove the supernatant after centrifuging the spleen cell suspension and resuspend the cells with 10ml of RBC lysis buffer (GIBCO). Then add 20ml of RBC lysis buffer. After the suspension was allowed to stand for 5 minutes, it was 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 cells SP2/0 cells (ATCC) with 20 ml of basal medium and count. SP2/0 and spleen cells after lysis of red blood cells were mixed at a ratio of 1:2 to 1:1, and centrifuged at 1000 rpm for 6 minutes. After removing the supernatant, the mixed cells were resuspended in 10 ml of fusion buffer (BTXpress). Then add 15ml of fusion buffer, centrifuge at 1000rpm for 5min, and remove the supernatant. After repeating the above steps once, 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 electric fusion meter are shown in Table 6 below. Add 2ml of the above cell suspension to each electrofusion dish for electrofusion.

Electric fusion instrument parameter setting

condition:	Mouse (SP2/0-ECF-F)
Alignment:	60v, 30sec
Membrane breaking:	1500V, 30μs, 3X
Post-fusion pulse:	60V, 3sec

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

Table 2 Screening media

Subcloning of positive hybridoma cells:

Subcloning procedure: Prepare a 96-well plate and add 200 μl of basal medium as described above to each well in rows 2 to 8. The cells in the positive wells selected by the above fusion were taken at a density of about 1×10 ⁵ cells/ml, and 300 ul was taken into each well of the first row. Grab the row, add 100 μl of the cell suspension in the first row to the second row, and mix well after taking 100 μl to the next row. Repeat the above steps until the volume of the last column becomes 300μl; stand for 96 minutes in a 96-well plate and observe the count under the microscope. Take the volume corresponding to 100 cells and add it to 20ml of the basal medium as described above, and mix and spread the plate, 200μl per well. After a week, observe under the microscope, determine and mark the monoclonal wells, and select the positive wells to be tested.

Cell cryopreservation: Observe the state of the cells and wait for the cells to grow well and viability >90%, centrifuge at 1000 rpm for 5 min, and remove the supernatant. Resuspend cells to 1×10 ⁷ cells/ml in cryopreservation solution (45.5% FBS (Hyclone), 44.5% RPMI-1640 (Hyclone), 10% DMSO), aliquot into cryotubes, and place in a program cooling box Stored at -80℃.

Hybridoma sequencing:

RNA extraction was performed using the NucleoSpin RNA Plus (Macherey-Nagel, Catalog No. 740984.250) kit: fresh cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, 350 μl of cell lysis buffer LBP was added to the pellet, and mixed 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 of washing solution WB1, centrifuge at 11,000 rpm for 15 s, discard the liquid; remove the lid of the RNA column and move to a new collection tube, add 600 μl of washing Solution WB2, centrifuge at 11,000 rpm for 15 s, discard the liquid; add 250 μl of washing solution WB2, centrifuge at 11,000 rpm for 2 min, discard the liquid; after centrifugation to completely evaporate the ethanol, add 30 μl of DEPC water to the collection column, centrifuge at 12000 g for 2 min, collect and elute liquid. Measure RNA concentration.

Use PrimeScript IIst Strand cDNA Synthesis Kit (Takara) to reverse transcribe to obtain cDNA, the steps are as follows (the reagents mentioned are from the kit):

Prepare the reaction system I as shown in Table 3 below

Table 3: Reaction System I

After incubating at 65°C for 5 minutes, it was quickly placed on ice to cool. The reaction system I was added to the following reverse transcription system (Table 4), the total amount was 20 μl:

Table 4: Reverse transcription system

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

Using Mighty TA-cloning Kit Kit (Takara), the cDNA is connected to the 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. Primers for the heavy chain variable region (VH) of CD30 antibody (Primer Mix 1)

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

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

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

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

Transformed cells:

Remove TOP10 competent state (Tiangen Biochemical Technology (Beijing) Co., Ltd.) at -80°C, melt on ice, add 5 μl of the ligation product obtained above to the melted TOP10 competent state, mix well and incubate on ice for 30 min. After heat shock at 42°C for 90s, it was quickly cooled on ice for 2min, and 900μl of LB medium (Shenggong Biological Engineering (Shanghai) Co., Ltd.) was added to the EP tube at 37°C and cultured on a shaker at 220rpm for 1h. Centrifuge at 3000g for 2min, aspirate 800μl of supernatant, resuspend the bacteria with the remaining medium and spread on ampicillin-resistant plates. Incubate at 37°C overnight, pick clones for sequencing, and obtain VH and VL sequences in hybridomas after analysis. Finally, 10 antibodies were obtained, HB38E4, HB10F1, HB49G9, HB36F7, HB68H2, HB69G7, HB10B6, HB16H7, HB6A9, HB16H8, the sequence of which is shown in Table A.

Example 2. Phage display technology to screen fully human antibodies against CD30

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

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

1. Preparation of Input Phage for Screening

Put in 50-100 times the storage capacity of frozen bacteria, inoculate the OD value to 0.05, incubate at 37 ℃ 200rpm until the OD value reaches 0.5, add 5×10 ⁹ cfu/ml helper phage (helper phage) M13K07, shake First, let stand at 37° for 20-30min, then incubate at 37°, 200rpm for 30min, and finally incubate at 30°C for 20h. Collect phages as the first input phage.

2.Panning (Panning)

Take 50ul magnetic beads (Thermo, Catalog No. 815-968-0747), wash 4 times with PBS; block with 2% BSA in PBS at room temperature for 2h. The magnetic beads were combined with the biotin-labeled CD30/Avitag protein (Acro, Catalog No. CD0-H82E6), room temperature, rotation, and incubation 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 elute with 0.5ml 0.1M hydrochloric acid-glycine eluent, and then neutralize with 2M Tris.

3.Phage infection

Take half the volume of phage eluent to infect the E. coli XL1-Blue bacterial solution in the logarithmic growth phase, incubate at 37 ℃, 200 rpm for 20 min, and then spread on an agar plate. The next day the blade was scraped, centrifuged at 3000 rpm, 5 min, and then resuspended in 20% glycerol-2×YT medium, and the OD value was measured.

See Table 9 for the preparation of 2×YT medium (1L).

Table 9: 2×YT medium

Then phage were produced overnight, in the same way as in step 1 above. Steps 1-3 do this 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, and the enriched phage population containing specific antibody sequences was coated on agar plates to obtain specific antibody genes. Phage clone colonies. Pick single clones for Elisa detection of single phage clones. The Elisa-positive clones were sequenced and there were about 37 antibodies with different sequences.

Subsequently, these 37 antibodies were constructed in scFv-hIgG antibody format (the method is the same as in Example 3), and the resulting plasmid was transiently transfected into a 6-well plate. After 5 days, the expression supernatant was taken for Elisa verification. Elisa-positive clones were tested by Fortebio (method same as Example 5). Two clones with good affinity, P5E10 and P27B3, were selected and expressed and purified together with the 10 candidate clones generated by hybridoma using transgenic mice in Example 1.

The amino acid sequences and corresponding nucleotide sequences of the two strains of 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 scFv-form candidate antibodies to target antigen CD30, recombinant protein expression vectors of the single-chain antibody variable region (scFv) and human Fc fragments of the antibodies obtained in Examples 1 and 2 above were constructed, respectively. At the same time, two positive control antibodies V2AC10 and XL of 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.

A schematic diagram of vector construction is shown in Figure 1. The specific construction process is illustrated 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 PCR amplified.

Table 10. Primers used for PCR amplification:

Table 11. PCR system

*For VH chain amplification, apply Prime F1, Primer R1; for VL chain amplification, apply Prime F2, Prime R2.

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

Table 12. Overlap PCR system:

The PCR amplification 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, a homologous recombination reaction was carried out.

Table 13. Homologous recombination system

*: Constructed based on pTT5 vector, inserted with hFc coding sequence.

The reaction was carried out at 37°C for 30 min to obtain the recombinant product. The recombinant product was transformed into TOP10 competent state, and the monoclonal sequence was picked. The clone containing the plasmid with the correct insertion direction was selected as the positive clone, and the positive clone was stored in the glycerol tube.

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

1. Pass 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 Opti MEM medium (Gibco, 31985-070) as transfection buffer, respectively 30μg of the above plasmids carrying scFv-hFc recombinant single-chain antibody encoding gene, mix and filter, and let stand for 5min.

3. Add 90 μL of 1 mg/mL polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid-OptiMEM mixture, mix well and incubate at room temperature for 15 min. The mixture was gently poured into the cells and cultured at 36.5°C and 8% CO2.

4. After 20h, add 0.6mL of 200g/L FEED (soy peptone Phytone Peptone (BD, 211906) to plant peptone Phytone (BD, 210931) etc. ratio), 0.3mL of 200g/L mother glucose solution, 30μL of 2.2M Valproic acid sodium salt (VPA) (Sigma, P4543).

5. Continue to cultivate until the viability is less than 60%, collect the supernatant, and filter for affinity chromatography purification.

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. Soak the packing with 0.1M NaOH for 2h, add 300μL of protein A affinity chromatography medium (Mabselect) (GE Healthcare, 17-5438-03) packing to each gravity column after soaking.

3. Centrifuge the cell feed at 8000r/min for 40min, and then filter it using a 0.45μm filter, and store at 4℃ for later use.

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

5. Before purification, equilibrate the filler with 10 ml of binding/washing buffer (20 mM Tris + 150 mM NaCl (pH 7.2)).

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

7. Rinse, rinse the filler with 5-10ml binding/washing buffer (20mM Tris+150mM NaCl (pH7.2)) to remove non-specific binding protein.

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

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

The optical interference technology (BLI) of the biofilm layer based on optical fiber biosensors determines the kinetic constants of antibody molecules.

The basic principle of BLI is that when a biomolecule is bound to the sensor surface, a layer of biofilm is formed. The biofilm causes an interference phenomenon to the waveform of light passing through the sensor. The interference phenomenon is detected in a phase shift manner, so that it can be detected to bind to Change in the number of sensor molecules; fit the kinetic curve according to the change in real-time response value, and calculate the binding constant (Kon), dissociation constant (Kdis), affinity (KD).

The model of Fortebio equipment we chose is Octet Red96, and the affinity determination of ForteBio is based on the existing methods (Estep, P, et al., High Throughput, Solution, Based Measurement, of antibody-antigen, affinity, and epitope, winning.MAbs, 2013.5(2): p.270 -8) proceed. The specific process is:

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

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

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

4. The results of affinity detection of 12 scFv-hFc recombinant single-chain antibodies with human CD30-His are shown in Table 14.

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

First, two control antibodies XL and V2AC10 were competitively bound, and the results showed that they were not the same epitope (Bin). The binding epitope of the control antibody XL and the antigen was defined as Bin1, and the binding epitope of the control antibody V2AC10 and the antigen was defined as Bin2. The results are shown in Table 14. The P5E10 and P27B3 antibodies are the same as the control antibody XL Bin1; HB38E4, HB10F1 and HB49G9 antibodies and the control antibody -V2AC10 are the same Bin2; HB36F7, HB68H2, HB69G7, HB10B6, HB16H7 and HB6A9 antibodies are not the same antigen as the two control antibodies Bind to epitope, 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. See Table 14 for the epitope detection results.

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

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

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

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

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

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

3. Centrifuge at 400G for 5 minutes, add PBS to wash twice, add 100 μl of secondary antibody ((Phycoerythrin, PE) labeled goat anti-human IgG antibody diluted in PBS (1% BSA) to each well, Southern Biotech, final Concentration 5μg/ml), incubate at 4 ℃ for 30min (protected from light);

4. Centrifuge at 400G for 5min, add PBS to wash twice, and resuspend cells in 100μl per well of PBS. 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. GraphPad software was used to calculate the EC50 value.

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

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

Antibody name	EC50(nM)
HB38E4	17.64
HB10F1	2.891
HB49G9	4.22
HB36F7	13.12
HB68H2	7.316
HB69G7	28.88
HB10B6	0.4311
HB16H7	7.497
HB6A9	13.8
HB16H8	9.789
P5E10	7.06
P27B3	63.23
Control antibody-V2AC10	7.687
Control antibody-XL	1.105

Sequence listing description

Claims (27)

1. 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 heavy chain variable region and the light chain variable region selected from:

   (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 heavy chain variable region shown in SEQ ID NO: 114 and the light chain variable region shown in SEQ ID NO: 119.
2. An isolated antibody or antigen-binding fragment thereof that specifically binds to CD30. The antibody comprises three heavy chain complementarity determining regions HCDR and three light chain complementarity determining regions LCDR, wherein:

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

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

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

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

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

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

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

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

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

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

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

   (l) HCDR1 contains the amino acid sequence shown in SEQ ID NO: 111, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 112, HCDR3 contains the amino acid sequence shown in SEQ ID NO: 113, and LCDR1 contains the amino acid sequence shown in SEQ ID NO: 116 LCDR2 contains the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 118.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody comprises a heavy chain variable region VH selected from:

   (a) The amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (b) an amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (c) an amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (d) an amino acid sequence 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 , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (e) an amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (f) an amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (g) an amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (h) an amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (i) The amino acid sequence 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 , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (j) an amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (k) an amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (l) An amino acid sequence 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 with it , Or VH with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes.
4. The antibody or antigen-binding fragment thereof of claim 1 or 2, the antibody comprising a light chain variable region VL, the VL selected from:

   (a) An amino acid sequence comprising 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (b) an amino acid sequence 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (c) an amino acid sequence comprising 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (d) an amino acid sequence 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (e) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 49, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (f) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 59, or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (g) an amino acid sequence comprising 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (h) The amino acid sequence comprising 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (i) The amino acid sequence comprising 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (j) an amino acid sequence comprising 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (k) an amino acid sequence comprising 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 , Or VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes;

   (l) An amino acid sequence 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 Or a VL with respect to an amino acid sequence that contains no more than 10 (preferably no more than 5) amino acid changes.
5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH and VL are selected from:

   (a) an amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO:9 VL;

   (b) an amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO:19 VL;

   (c) an amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 29 VL;

   (d) an amino acid sequence 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 VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 39 VL;

   (e) an amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 49 VL;

   (f) an amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 59 VL;

   (g) an amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 69 VL;

   (h) An amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 79 VL;

   (i) an amino acid sequence 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 VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO:89 VL;

   (j) an amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 99 VL;

   (k) an amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 109 VL;

   (l) The amino acid sequence 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 with it VH, and an amino acid sequence comprising or having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO:119 VL.
6. An isolated antibody or antigen-binding fragment thereof that specifically binds CD30. The antibody has one or more of the following characteristics:

   (i) shows the same or similar binding affinity and/or specificity as any of the antibodies listed in Table A;

   (ii) inhibit (eg, competitively inhibit) the binding of any of the antibodies listed in Table A to CD30;

   (iii) Binding to the same or overlapping epitope as any of the antibodies shown in Table A;

   (iv) compete with any of the antibodies shown in Table A for binding to CD30;

   (v) has one or more biological properties of any of the antibody molecules listed in Table A.
7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody has one or more of the following characteristics:

   (i) With high affinity, for example, with a K _D value of less than 100 nM, such as less than 50 nM, such as 1-30 nM, preferably less than 10 nM, binds to human CD30 (such as the polypeptide of SEQ ID NO: 162);

   (ii) With high affinity, for example, less than 100 nM, for example less than 70 nM, for example 0.1-30 nM, preferably less than 20 nM, more preferably less than 10 or 5 nM, most preferably less than 1 nM EC ₅₀ value, and cell surface expressed human CD30 (such as SEQ ID NO: 162 polypeptide) binding;

   (iii) The dissociation rate constant (K _dis ) associated with 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) Binding specifically to an epitope on the extracellular domain ECD of human CD30.
8. The antibody or antigen-binding fragment thereof of any preceding claim, wherein the antibody is a fully human antibody.
9. The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody is a single chain antibody.
10. The antibody of any one of the preceding claims, 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 domain, or VH domain- Linker-VL domain.
11. The antibody of claim 10, wherein the linker comprises 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20, preferably 15-20 amino acids.
12. The antibody of claim 11, wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 157.
13. The antibody of any one of claims 10-12, wherein the single chain scFv antibody comprises amino acids selected from SEQ ID NO: 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, and 154 Sequence, or an amino acid sequence having at least 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity with, or containing at least one, for example 1-5, but Amino acid sequence with no more than 30, 20 or 10 amino acid changes.
14. An isolated antibody that specifically binds CD30, wherein the antibody comprises the single chain scFv antibody of any one of claims 10-13 and an Fc region.
15. The antibody of claim 14, wherein the single chain scFv is connected to the Fc region through a hinge region, preferably, the hinge region is a CD8 hinge region, and more preferably, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 159 or relative to The amino acid sequence of SEQ ID NO: 159 contains at least one, two or three, but no more than 5 amino acid changes.
16. The antibody of any one of claims 14-15, wherein the Fc region is a human IgG1 or IgG4 Fc region, preferably the Fc region is low or afucosylated.
17. The antibody of any one of claims 14-16, wherein the antibody comprises an amino acid sequence selected from SEQ ID NO: 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, and 156, or The amino acid sequence containing at least one, such as 1-5, but no more than 30, 20, or 10 amino acid changes, or having at least 80%, 85%, 90%, 92%, 95%, Amino acid sequences of 97%, 98%, 99% or higher identity.
18. An isolated nucleic acid encoding the isolated antibody or antigen-binding fragment of any one of claims 1 to 17.
19. A vector containing the nucleic acid of claim 18, preferably the vector is an expression vector.
20. A host cell comprising the vector of claim 19, preferably, the host cell is selected from yeast cells and mammalian cells.
21. A method of preparing the isolated antibody or antigen-binding fragment of any one of claims 1 to 17, comprising: cultivating the host cell of claim 20 under conditions suitable for expressing the antibody or antigen-binding fragment thereof.
22. A conjugate or fusion comprising the antibody of any one of claims 1-17.
23. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 17, or the conjugate or fusion of claim 22, and optionally a pharmaceutically acceptable carrier, and optionally further Contains one or more other therapeutic agents.
24. A method for detecting CD30 in a sample, including:

    (a) contacting the sample with the isolated antibody or antigen-binding fragment of any one of claims 1 to 17, or the conjugate or fusion of claim 22; and

    (b) Detecting the formation of a complex between the antibody or its antigen-binding fragment or conjugate or fusion and CD30 protein.
25. A method for treating CD30-related disorders, comprising administering to the subject an effective amount of the isolated antibody or antigen-binding fragment of any one of claims 1-17, the conjugate or fusion of claim 22, Or the composition of claim 23.
26. The method of claim 25, wherein the CD30-related disorder is a CD30-expressing tumor, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma.
27. The method of claim 26, wherein the CD30-related disorder is selected from Hodgkin's 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 cutaneous T cell lymphoma.

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