KR20220102114A - A Novel Antibody Specific for CD55 and Use Thereof - Google Patents

Abstract

The present invention relates to an antibody specifically binding to CD55, or an antigen-binding fragment thereof, and a composition for prophylactic treatment and/or diagnosis of cancer comprising the same. The antibody according to the present invention may be used as an effective therapeutic composition for various CD55-mediated diseases by showing high binding to and inhibitory abilities against a CD55 protein that promotes tumor growth by suppressing the complement immune mechanism. In addition, the antibody according to the present invention may be utilized as an effective therapeutic adjuvant that fundamentally removes drug resistance and significantly improves therapeutic responsiveness in various diseases in which resistance to therapeutic agents with complement-dependent cytotoxicity (CDC) as a mechanism of action is induced due to overexpression of CD55.

Description

A Novel Antibody Specific for CD55 and Use Thereof

The present invention relates to a novel antibody or antigen-binding fragment thereof specifically recognizing CD55 and a composition for preventing, treating and/or diagnosing cancer using the same.

The global anticancer drug market has grown at a CAGR of 10 - 13% over the past decade, and is expected to reach a total of $200 billion by 2022. As of 2020, 1 in 5 men and 1 in 6 women worldwide will experience cancer at least once in their lifetime, and 1 in 8 men and 1 in 11 women will die from cancer. The morbidity and mortality rates are continuously increasing.

The global anticancer drug market is undergoing a paradigm shift from first-generation chemotherapy to second-generation targeted anticancer drugs and third-generation immune anticancer drugs. In the early stage, away from the treatment methods aimed at reducing and suppressing simple cancers, targeted anticancer treatment technologies that selectively attack only cancer cells without damaging normally rapidly dividing cells are being actively studied. Development consists of two steps: the selection of a receptor specifically expressed on the cancer cell and the development of a targeting compound that binds to the receptor, which allows for the selective targeting of the cancer cell.

On the other hand, CD55 (decay-accelerating factor, DAF) is a receptor that suppresses the complement immune mechanism, and is highly expressed in various solid cancers such as colorectal cancer, lung cancer, stomach cancer, breast cancer, ovarian cancer, leukemia, and head and neck cancer. It promotes the proliferation of cancer cells by inhibiting apoptosis of cancer cells. Therefore, CD55 has been actively studied as a target molecule for targeted anticancer therapy.

In particular, solid tumors with high CD55 expression show low therapeutic reactivity to various anticancer drugs whose mechanism of action is complement dependent cytotoxicity (CDC). There is a demand for the development of excellent antibody therapeutics capable of inhibiting.

Numerous papers and patent documents are referenced throughout this specification and citations thereof are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Patent Document 1. Republic of Korea Registration No. 10-1800774

The present inventors have dedicated themselves to developing an excellent CD55 inhibitor that can efficiently restore the immune mechanism inhibited by CD55 by specifically recognizing the CD55 protein, a receptor that suppresses the complement immune mechanism, and specifically inhibiting its activity. research efforts were made. As a result, it was found that complement-mediated cell lysis activity against cancer cells was significantly restored due to significantly increased CD55 binding and inhibitory power when using an antibody substituted with some amino acids of the key antigen recognition site in the variable region. By doing so, the present invention was completed.

Accordingly, an object of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to CD55; and to provide a composition for preventing or treating and/or diagnosing cancer comprising the same.

Another object of the present invention is to provide a bispecific antibody comprising an antigen-binding fragment of an antibody that specifically binds to CD55 and an antigen-binding fragment of an antibody that specifically binds to CD20.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides an HCDR1 region having an amino acid sequence represented by the following general formula (1); HCDR2 region having the amino acid sequence of SEQ ID NO: 1; And it provides an antibody or antigen-binding fragment thereof that specifically binds to CD55, comprising a heavy chain variable region comprising an HCDR3 region having an amino acid sequence represented by the following general formula 2:

general formula 1

DRGM- X ₁ ;

general formula 2

NA- X ₂ -AGGWHAAYIDA,

In Formula 1, X ₁ is Ala or Val, and in Formula 2, X ₂ is selected from the group consisting of Val, Ala, Ile, Leu, Phe and Met.

The present inventors have dedicated themselves to developing an excellent CD55 inhibitor that can efficiently restore the immune mechanism inhibited by CD55 by specifically recognizing the CD55 protein, a receptor that suppresses the complement immune mechanism, and specifically inhibiting its activity. research efforts were made. As a result, it was found that complement-mediated cell lysis activity against cancer cells was significantly restored due to significantly increased CD55 binding and inhibitory power when using an antibody substituted with some amino acids of the key antigen recognition site in the variable region. did.

As used herein, the term "antibody" refers to a protein molecule serving as a receptor that specifically recognizes an antigen, including immunoglobulin molecules having immunological reactivity with a specific antigen, and includes polyclonal antibodies and monoclonal antibodies. include all

Antibodies include antigen-binding fragments (antibody fragments) of antibody molecules as well as full-length antibody forms. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has a kappa (κ) and a lambda (λ) type.

As used herein, the term “antigen-binding fragment of an antibody” refers to a fragment that specifically recognizes an antigen within an entire antibody molecule and retains an antigen-antibody-binding function, and includes a single-domain antibody (sdAb), a single-chain antibody (scFv). ), Fab, F(ab'), F(ab')2 and Fv, and the like. Examples of the fragment include a monovalent fragment consisting of VL, VH, CL and CH1 domains (Fab fragment); a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (F(ab')2 fragment); Fd fragment consisting of VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of one arm of an antibody, and a disulfide-linked Fv (sdFv); a dAb fragment consisting of a VH domain; and separate complementarity determining regions (CDRs) or combinations of two or more separate CDRs optionally followed by a linker. In addition, the scFv may be linked by a linker such that the VL and VH regions are paired to form a single protein chain forming a monovalent molecule. Such single-chain antibodies are also included in antibody fragments. In addition, the antibody or fragment of the antibody may be a tetrameric antibody comprising two heavy chain molecules and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers, antibody heavy chain dimers; Intrabody; monovalent antibody; camel antibody; and single-domain antibodies (sdAbs).

Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant technology for generating Fv fragments is described in PCT International Patent Application Publications WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in a single-chain Fv (single-chain Fv), the heavy chain variable region and the single chain variable region are generally shared through a peptide linker. They are linked by a bond or are linked directly at the C-terminus to form a dimer-like structure like a double-stranded Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, by restriction digestion of the entire antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab')2 fragments), gene It can also be produced through recombinant technology.

As used herein, the term "heavy chain" refers to a variable region domain V _H comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length comprising three constant region domains C _H1 , C _H2 and C _H3 . both heavy chains and fragments thereof. In addition, as used herein, the term "light chain" refers to both a full-length light chain including a variable region domain V _L and a constant region domain _CL including an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a fragment thereof. do.

As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulin (Kabat et al . Sequences of Proteins of Immunological Interest , 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy (HCDR11, HCDR2 and HCDR3) and light chains (LCDR1, LCDR2 and LCDR3) each contain three CDRs, which provide key contact residues for antibody binding to antigen or epitope.

The scope of the antibody or antibody fragment of the present invention includes variants having conservative amino acid substitutions in the CDR regions, and to the extent that CD55 can be specifically recognized, variants to the amino acid sequences set forth in the accompanying sequence listing are included. can For example, additional changes may be made to the amino acid sequence of the antibody in order to further improve the half-life, biocompatibility, and other biological properties in addition to the binding affinity of the antibody. Considering the variation having such biological equivalent activity, it is construed that the antibody or nucleic acid molecule encoding the same of the present invention includes a sequence showing substantial identity to the sequence listed in the sequence listing. The substantial identity is at least 61% when the aligned sequence is analyzed using an algorithm commonly used in the art after aligning the sequence of the present invention and any other sequences as much as possible. homology, according to one specific example, 70% homology, according to another specific example, 80% homology, according to another specific example, 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math . (1981) 2:482 Needleman and Wunsch, J. Mol. Bio . (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol . (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al . Nuc. Acids Res . (1988) 16:10881-90; Huang et al . Comp. Appl. BioSci . (1992) 8:155-65 and Pearson et al . Meth. Mol. Biol . (1994) 24:307-31.

As used herein, the term “affinity” refers to the binding strength between an antibody or antigen-binding fragment thereof and an antigen, and may also be expressed as “binding strength”.

According to a specific embodiment of the present invention, X ₁ in Formula 1 is Ala.

According to a specific embodiment of the present invention, X ₂ of Formula 2 is Val.

More specifically, the heavy chain variable region of the present invention comprises at least one framework region (FR) selected from the group consisting of the following HFR1 to HFR4:

HFR1: EVQL Y ₁ ESGGGLVQPGGSLRLSC Y ₂ A Y ₃ GFTFS (Y ₁ is Val or Ala, Y ₂ is Val or Ala, Y ₃ is Ser or Arg)

HFR2: WVRQ Y ₄ PGKGLEWVS (Y ₄ is Ala or Ser)

HFR3: R Y ₅ TISRDNSKNTLYLQMN Y ₆ LRAEDTAVYYCAK (Y ₅ is Ala or Thr and Y ₆ is Ser or Asn)

HFR4: WGQGTTVTVSS

More specifically, the heavy chain variable region of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 22.

According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof of the present invention comprises an LCDR1 region having the amino acid sequence of SEQ ID NO: 2; LCDR2 region having an amino acid sequence represented by the following general formula 3; and a light chain variable region comprising an LCDR3 region having the amino acid sequence of SEQ ID NO: 3:

general formula 3

WN- X ₃ -KRPS,

In Formula 3, X ₃ is Asn or Asp.

More specifically, X ₃ is Asp.

More specifically, the light chain variable region of the present invention comprises at least one framework region (FR) selected from the group consisting of the following LFR1 to LFR4:

LFR1: SYELTQPPSVSVSPGQTASITC

LFR2: W Y ₇ QQKPGQSPVTVIY (Y ₇ is Phe or Tyr) (F or Y)

HFR3: GIPERFSGSKSGNTATLTISGTQAMDEADYYC

HFR4: FGGGTKLTVL

More specifically, the light chain variable region of the present invention has the amino acid sequence of SEQ ID NO: 4 or 18.

According to another aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD55, comprising the following light chain variable region and heavy chain variable region:

a) the light chain variable region of SEQ ID NO: 4; Or, in the light chain variable region of SEQ ID NO: 4, the 32 amino acid sequence Y (Tyr) is substituted with F (Phe) and the 48th amino acid sequence D (Asp) is selected from the group consisting of N (Asn) substitution A light chain variable region comprising one or more substitutions, and

b) the heavy chain variable region of SEQ ID NO: 5; Or, in the heavy chain variable region of SEQ ID NO: 5, the 5th amino acid sequence V(Val) is substituted with A(Ala), the 23rd amino acid sequence A(Ala) is substituted with V(Val), the 25th amino acid sequence S(Ser) is substituted with R(Arg), 35th amino acid sequence A(Ala) is substituted with V(Val), 40th amino acid sequence A(Ala) is substituted with S(Ser), 68th amino acid The sequence A(Ala) is substituted with T(Thr), the 85th amino acid sequence S(Ser) is substituted with N(Asn), and the 101st amino acid sequence G(Gly) is V(Val), L(Leu) , I(Ile), F(Phe), M(Met), or a heavy chain variable region comprising one or more substitutions selected from the group consisting of substitutions with A(Ala).

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof of the present invention.

As used herein, the term "nucleic acid molecule" has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, include natural nucleotides as well as analogs in which sugar or base sites are modified. (analogue) (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

The nucleic acid molecule of the present invention is construed to include a nucleotide sequence exhibiting substantial identity to the above-described nucleotide sequence. The substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. of homology, in one specific example, at least 90% homology, in another specific example, at least 95% homology.

According to another aspect of the present invention, the present invention provides a composition for preventing or treating cancer comprising the above-described antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutically effective amount of the aforementioned antibody or antigen-binding fragment thereof of the present invention.

According to another aspect of the present invention, the present invention provides for use in therapy of the aforementioned antibody or antigen-binding fragment thereof of the present invention.

As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or disease in a subject who has not been diagnosed as possessing the disease or disease, but is likely to be afflicted with the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, disorder or condition; (b) alleviation of the disease, condition or condition; or (c) eliminating the disease, condition or symptom. When the composition of the present invention is administered to a subject, it restores the complement immune mechanism by inhibiting the activity of the CD55 receptor and inhibits the proliferation of cancer cells to inhibit cancer progression, or to eliminate or alleviate symptoms thereof. Therefore, the composition of the present invention may be a composition for cancer treatment by itself, or is administered with other pharmacological components, for example, a therapeutic agent having CDC (complement dependent cytotoxicity) as a mechanism of action to improve its therapeutic responsiveness. It can also be applied as an adjuvant. Accordingly, as used herein, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic adjuvant” or “therapeutic adjuvant”.

As used herein, the term “administration” or “administering” refers to administering a therapeutically effective amount of the composition of the present invention directly to a subject so that the same amount is formed in the subject's body.

In the present invention, the term "therapeutically effective amount" means the content of the composition in which the pharmacological component in the composition is sufficient to provide a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is administered, and thus " prophylactically effective amount".

As used herein, the term “subject” includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

According to a specific embodiment of the present invention, the composition further comprises at least one pharmacological component selected from the group consisting of an anti-CD20 antibody and a tyrosine kinase inhibitor.

More specifically, the anti-CD20 antibody is Rituximab.

More specifically, the tyrosine kinase inhibitor is selected from the group consisting of imatinib (Imatinib) and gefitinib (gefitinib).

According to a specific embodiment of the present invention, the cancer that can be prevented or treated by the composition of the present invention is a cancer that is CD55 positive.

As used herein, the term “CD55-positive cancer” refers to a carcinoma composed of cancer cells expressing the CD55 receptor, and specifically refers to a cancer in which CD55 is expressed enough to measurably inhibit complement immune activity in a subject. Accordingly, the term "CD55 positive cancer" is meant to include "CD55 overexpressing cancer". According to the present invention, the composition of the present invention can eliminate drug resistance and enhance treatment sensitivity in various carcinomas in which resistance to a therapeutic agent is induced due to overexpression of CD55.

CD55 positive cancers include, for example, melanoma, lymphoma, lung cancer, thyroid cancer, breast cancer, colorectal cancer, prostate cancer, head and neck cancer, stomach cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, leukemia, bone marrow cancer, ovarian cancer, uterine cancer all solid and hematological cancers expressing CD55, including, but not limited to, cervical cancer, sarcoma, cholangiocarcinoma and endometrial cell carcinoma.

According to another aspect of the present invention, the present invention provides a composition for diagnosis of cancer comprising the above-described antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

Since the antibody or antigen-binding fragment thereof used in the present invention and the target cancer have already been described above, description thereof will be omitted to avoid excessive overlap.

As used herein, the term "diagnosis" includes determination of an individual's susceptibility to a particular disease, determination of whether an individual currently has a particular disease, and determination of the prognosis of a subject with a particular disease. do.

The CD55 receptor is a typical cancer diagnostic marker showing a high expression level in cancer tissues compared to normal tissues, and the antibody of the present invention that specifically binds to CD55 can accurately determine the presence of a tumor in a biological sample. In addition, the antibody of the present invention accurately measures the expression level of CD55 in the tumor through various immunoassay methods to predict the therapeutic sensitivity of the tumor to the CDC drug, thereby establishing a customized treatment strategy according to the nature of the cancer at an early stage. can be usefully used for

As used herein, the term "diagnostic composition" refers to an integrated mixture or equipment comprising the antibody of the present invention as a means for measuring the expression level of CD55 protein in order to determine whether a subject has cancer or predict treatment sensitivity to a CDC drug. (device), which may be expressed as a “diagnostic kit”.

As used herein, the term "biological sample" means tissue, cells, whole blood, serum, plasma, saliva, urine, lymph, spinal fluid, tissue autopsy sample (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells and Bacterial expression systems include, but are not limited to.

The detection of CD55 protein in a biological sample may be performed by a colormetric method, an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment or Detection of antigen-antibody complex formation using a scintillation counting method can be performed. As used herein, the term “detection” refers to a series of processes for determining whether an antigen-antibody complex is formed. Detection can be carried out using various labels including enzymes, fluorescent substances, ligands, luminescent substances, microparticles, or radioactive isotopes.

Enzymes used as detection labels include, for example, acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-latamase, and the fluorescent substance is fluoro Contains resinein, Eu ³⁺ , Eu ³⁺ chelate or cryptate, etc., as ligands, including biotin derivatives, etc., as luminescent materials, including acridinium esters and isoluminol derivatives, and the like, and as microparticles. colloidal gold and colored latex, and the like, and radioactive isotopes may include ⁵⁷ Co, ³ H, ¹²⁵ I and ¹²⁵ I-Bonton Hunter reagents.

According to one embodiment of the present invention, the antigen-antibody complex can be detected using enzyme immunosorbent assay (ELISA). The antibody of the present invention may have a detection label, and when it does not have a detection label, the antibody of the present invention can be captured and confirmed by treating another antibody having a detection label.

According to another aspect of the present invention, the present invention provides a bispecific antibody or a functional fragment thereof comprising the antigen-binding fragment of the present invention described above and an antigen-binding fragment of an antibody that specifically binds to CD20.

As used herein, the term "bispecific antibody" refers to an antibody comprising two different antigen-binding regions defined by different amino acid sequences. Typically, the two antigen binding regions comprised in a bispecific antibody are each specific for a different antigen or epitope.

The bispecific antibody of the present invention is a single antibody (CD20 x CD55) molecule in which the aforementioned CD55-specific antigen-binding fragment of the present invention and another antigen-binding fragment that specifically recognizes CD20 are bound. , A better therapeutic effect on rituximab-resistant tumors than when each anti-CD55 antibody (eg, EP-10H in Examples described below) and an anti-CD20 antibody (eg, rituximab) were administered in combination looks like

According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD55 antibody contained in the bispecific antibody is in the form of scFv.

According to a specific embodiment of the present invention, the antigen-binding fragment of the antibody that specifically binds to CD55 includes scFv.

According to a specific embodiment of the present invention, the antigen-binding fragment of the antibody that specifically binds to CD55 is in the form of VL-linker-VH-CH2-CH3.

According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD20 antibody contained in the bispecific antibody is in the form of Fab.

According to a specific embodiment of the present invention, the antigen-binding fragment of the antibody that specifically binds to CD20 includes Fab.

According to a specific embodiment of the present invention, the antigen-binding fragment of the antibody that specifically binds to CD20 is in the form of VL-CK-linker-VH-CH1-CH2-CH3.

According to a specific embodiment of the present invention, the antibody that specifically binds to CD20 is Rituximab (Rituximab).

The bispecific antibody of the present invention may comprise an Fc region consisting of first and second subunits capable of stably associating. As used herein, the term “Fc region” refers to the C-terminal region of an antibody heavy chain containing at least a portion of the constant region of a full-length antibody, and includes both a wild-type sequence Fc region and a mutant Fc region. The Fc region of an IgG comprises an IgG CH2 and an IgG CH3 domain. The CH3 region of a bispecific antibody of the invention has a wild-type or mutant CH3 domain (eg, an introduced “overhang” (“knob”) on one chain thereof and an introduced “cavity” corresponding to its other chain). (“hole”).

According to a specific embodiment of the present invention, the bispecific antibody of the present invention may use a knob-into-hole method to prevent unwanted pairing of CD20 X CD55 heavy chains with each other. The “knob-into-hole” method [US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); Carter, J Immunol Meth 248, 7-15 (2001)] described a protrusion at the interface of a first polypeptide (“knob ") and a cavity ("hole") corresponding to the interface of the second polypeptide. The overhang is constructed by substituting a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (eg, tyrosine or tryptophan). By substituting a larger amino acid side chain with a smaller side chain (eg, alanine or threonine), a complementary cavity of the same or similar size as the overhang is created at the interface of the second polypeptide.

According to a specific embodiment of the present invention, the bispecific antibody of the present invention comprises an antigen-binding fragment of an anti-CD55 antibody in the form of VL-linker-VH-CH2-CH3 and VL-CK-linker-VH-CH1-CH2-CH3 It may be a form to which an antigen-binding fragment of an anti-CD20 antibody is bound.

According to a specific embodiment of the present invention, the bispecific antibody of the present invention is composed of 10 to 100, more specifically 15 to 60 Gly and Ser in order to prevent unwanted pairing of CD20 X CD55 heavy chains. A linker may be used.

Specifically, the bispecific antibody of the present invention comprises CD55-scFv-knob (VL-linker20-VH-CH2-CH3) and CD20-Fab-hole (VL-CK-linker40-VH-CH1-CH2-CH3). It may be in a combined form.

Linker20 includes 20 Glys or Sers, and examples thereof include, but are not limited to, Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser.

linker40 includes 20 Gly or Ser, for example, Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Ser Ser may include, but is not limited thereto.

The features and advantages of the present invention are summarized as follows:

(a) the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD55; And it provides a composition for prophylactic treatment and/or diagnosis of cancer comprising the same.

(b) The antibody of the present invention can be used as an effective therapeutic composition for various CD55-mediated diseases by showing high binding and inhibitory power to the CD55 protein that promotes tumor growth by suppressing the complement immune mechanism.

(c) In addition, the antibody of the present invention is effective for fundamentally eliminating drug resistance and remarkably improving treatment responsiveness in various diseases in which resistance to therapeutic agents with complement dependent cytotoxicity (CDC) as a mechanism of action is induced due to overexpression of CD55 It can be usefully used as a therapeutic adjuvant.

1 is a diagram showing the results of ELISA analysis of the affinity of 4-1H and G4-1H to CD55.
2 is a diagram showing the results of ELISA analysis of the affinity for CD55 of an antibody prepared by screening from a library in which a random mutation is introduced in the gene of the G4-1H variable region.
3 is a diagram showing the results of ELISA analysis of the affinity for CD55 of an antibody prepared by substituting a hydrophobic amino acid for a key region to improve binding.
4 is a result of a FACS assay analyzing the killing effect of BJAB cells by rituximab and EP-10H combination treatment.
5a is a result of a FACS assay comparing the expression levels of CD20 and CD55 in Ramos and Ramos-RR. Figure 5b is a result of a FACS assay analyzing the killing effect of Ramos-RR cells by the combined treatment of rituximab and EP-10H antibody.
6a is a result of a FACS assay comparing CD55 expression levels in K562 and K562-IR. Figure 6b is a FACS assay result analyzing the killing effect of 562-IR cells by the combined treatment of imatinib and EP-10H antibody.
7a is a result of a FACS assay comparing CD55 expression levels in PC9 and PC9-GR. Figure 7b is a FACS assay result analyzing the killing effect of PC9-GR cells by the combined treatment of imatinib and EP-10H antibody.
8 is a diagram showing an antibody schematic diagram comparing the diabody type reported by SBU and Macor, which is a CD20 X CD55 diabody of the present invention.
9 shows the results of SDS-PAGE analysis of the CD20 X CD55 diabody in the form of SBU of the present invention.
FIG. 10 shows the results of an ELISA assay measuring the affinity of the CD20 X CD55 diabody to CD20 ( FIG. 10A ) and CD55 ( FIG. 10B ), respectively.
11a shows the results of a FACS assay analyzing the killing effect of BJAB cells by the CD20 X CD55 diabody. FIG. 11b is a comparison result of BJAB cell death rates when treated with rituximab alone, rituximab and EP-10H antibody in combination, and CD20 X CD55 dual antibody treatment.
12A shows the results of a FACS assay analyzing the killing effect of Ramos cells by the CD20 X CD55 double antibody. 12B is a comparison result of Ramos cell death rates when treated with rituximab alone, rituximab and EP-10H antibody in combination, and CD20 X CD55 dual antibody treatment.
13A shows the results of a FACS assay analyzing the killing effect of Ramos-RR cells by the CD20 X CD55 double antibody. 13B is a comparison result of Ramos-RR cell death rates when rituximab alone, rituximab and EP-10H antibody combined, and CD20 X CD55 dual antibody treatment were performed.
14 is a result of confirming the C4d production in the supernatant in which Ramos-RR cells were treated with rituximab and CD20 X CD55 double antibody in the form of SBU.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Humanization of 4-1H

In order to reduce the immunogenicity of 4-1H disclosed in Korean Application No. 10-2016-0132333, humanization was performed in the following way:

For humanization of 4-1H, three FRs (LFR1, LFR2, LFR3) in the light chain variable region of 4-1H were substituted with FRs of IGLV3-1*01, and three FRs (HFR1, HFR2, HFR3) in the heavy chain variable region ) was substituted with FR of IGHV3-23D*02. Thereafter, in consideration of the similarity or dissimilarity in the physicochemical properties of the amino acids, the specific amino acid was substituted with the amino acid of the parent antibody (4-1H) again. The amino acids substituted with the parent antibody (4-1H) were L46T and N66K in the light chain variable region and F68A in the heavy chain variable region. The amino acid residue numbering of antibody domains is determined by the Kabat EU numbering system, Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Ed., U.S. Department of Health and Human Services, commonly used in the art. according to the EU index number as in NIH Publication No. 91-3242, 1991). The J gene of the light chain variable region was fixed with FGGGTKLTVL with reference to US2010-0056386, and the J gene of the heavy chain variable region was fixed with WGQGTTVTVSS with reference to US2014-0206849. The humanized 4-1H antibody was named G4-1H, and its base sequence is shown in Table 1.

G4-1H heavy and light chain variable region amino acid sequence light chain variable region G4-1H
(VL) SYELTQPPSVSVSPGQTASITC SGGGGSYG WYQQKPGQSPV T VIY WNDKRPS GIPERFSGS K SGNTATLTISGTQAMDEADYYC GGWDSSTYAI FGGGTKLTVL SEQ ID NO: 4 heavy chain variable region G4-1H
(VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFS DRGMA WVRQAPGKGLEWVS GISSSGRYTYYAPAVKG R A TISRDNSKNTLYLQMNSLRAEDTAVYYCAK NAGAGGWHAAYIDA WGQGTTVTVSS SEQ ID NO: 5

In the above table, underlined text is a CDR region, and bold text indicates an amino acid substituted with the parent antibody (4-1H).

Example 2: Complete antibody conversion and expression/purification of G4-1H clone

The DNA of the variable region of the G4-1H antibody prepared in Example 1 was synthesized in the form of scFv (Cosmogenetech, Korea), and converted into a full-length IgG by PCR. First, fragments of the variable and constant regions of the heavy and light chains from a pUC vector (Cosmogenetech, Korea) containing scFv were obtained through PCR using the V _H , C _H and V _L , C _K primer combinations of Table 2 below. . PCR was performed using the HC and LC primer combinations shown in Table 2 below using the variable and constant regions of the obtained antibody, and as a result, the heavy and light chains of G4-1H were obtained. The heavy chain was ligated with Eco RI and Not I (New England Biolab, UK) enzymes and pCMV vector (ThermoFisher Scientific, USA), a vector for expression of animal cells treated with the same restriction enzyme. In addition, the light chain was treated using Xba I (New England Biolab, UK) enzyme and ligated into the pCMV vector treated with the same restriction enzyme. The ligation-completed plasmid was transformed by applying heat shock to DH5α competent cells (New England Biolab, UK), and the obtained colonies were mass-cultured to obtain a plasmid.

Primers used for cloning the G4-1H complete antibody primer order SEQ ID NO: V _H forward CTT CCT GTC AGT AAC TAC AGG TGT CCA CTC CCA GGT GCA ACT GCA GCA GTC 6 reverse CCA GCG TGA CCG TAT CCA GCG CCT CCA CCA AGG GCC CCA 7 C _H forward GCC TCC ACC AAG GGC CC 8 reverse TCC CCC GGC AAG TGA GCG GCC GCT CAC 9 HC forward CAG AAT TCA CTC TAA CCA TGG AAT GGA GCT GGG TCT TTC TCT TCT TCC TGT CAG TAA CTA CAG 10 reverse TCC CCC GGC AAG TGA GCG GCC GCT CAC 9 V _L forward GGT CTT TGT ATA CAT GTT GCT GTG GTT GTC TGG TGT TGA AGG AAG CTA CGA GCT GAC AC 11 reverse CAA GCT CAC CGT CTT GCG GAC CGT GGC C 12 C _K forward CGG ACC GTG GCC GCC CCC TC 13 reverse CGG GGC GAG TGC TAG TTC TAG AAC TA 14 LC forward GGG AAT TCT AGA GGA TCG AAC CCT TTG CAA GCT TCG GCA CGA GCA GAC CAG CAT GGG CAT CAA GAT GGA GAC ACA TTC TCA GGT CTT TGT ATA CAT GTT G 15 reverse CGG GGC GAG TGC TAG TTC TAG AAC TA 14

The plasmids of the heavy and light chains converted into complete antibodies were transduced into Expi293F cells (Invitrogen, USA) using polyethyleneimine (PEI) (Polysciences, USA) and 150 mM NaCl, and in Freestyle 293 expression medium (Invitrogen, USA). Suspended culture for 7 days in Erlenmeyer flasks at 37° C., 8% CO ₂ and 55% humidity conditions. After centrifuging the expression cell culture solution at 4,000 rpm for 10 minutes, the supernatant was taken and filtered through a 0.22 μm filter. The filtered supernatant was induced to bind to 1 ml of MabSelect sure (GE Healthcare, USA) resin at 4°C. The bound resin was washed with 10 cv (column volume) of 20mM diphosphate and 500mM sodium chloride (pH 7.0) solution, and then the bound antibody was eluted using 100 mM citric acid (pH 3.0) solution, and then 1M Tris-HCL (pH 9.0) was neutralized. The size and purity of the light and heavy chains of the purified antibody were finally confirmed through SDS-PAGE after buffer exchange with pH 7.2 - 7.4 PBS using the Slide-A-Lyzer Dialysis cassette (ThermoFisher Scientific, USA). As a result, it was possible to confirm the molecular weight and high purity consistent with the theoretical calculated values.

Example 3: Affinity analysis of 4-1H and G4-1H for CD55

The binding ability of the G4-1H monoclonal antibody prepared in Example 2 to CD55 was confirmed by indirect ELISA. For indirect ELISA, recombinant human CD55 (R&D Systems, USA) was diluted at 1 μg/ml in 50 μl of PBS, placed in a 96-well immune plate (Corning, USA), and stored at 4° C. for adsorption overnight. After reacting for 1 hour at room temperature with a buffer containing 3% bovine serum albumin (Millipore, USA), washed 3 times with a buffer containing 0.5% Tween 20 (Amresco, USA), sequential concentrations (0.0001, 0.0003) , 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 nM) of each antibody was treated at 50 μl per well. After reacting for 2 hours at room temperature to allow the antibody to bind to the antigen, it was washed three times with a buffer containing 0.5% Tween 20 (Amresco, USA). Anti-human immunoglobulin Fc-HRP antibody (Jackson Immunoresearch, USA) was diluted 1:3,000 and treated at 50 μl per well, followed by reaction at room temperature for 1 hour. After the reaction, after washing 3 times with a buffer containing 0.5% Tween 20 (Amresco, USA), 3,3',5,5'-tetramethylbenzidine (TMB) (ThermoFisher Scientific, USA) 50 μl each and allowed to develop color for 10 minutes. As a result of measuring absorbance at 450 nm using a spectrophotometer (Biotek, USA), G4-1H showed a lower affinity for CD55 than 4-1H (FIG. 1).

Example 4: Construction of a library in which random mutations are introduced in the gene of the variable region

In order to improve the affinity of G4-1H for CD55, a library was constructed in which random mutations were introduced into the variable region gene of the G4-1H antibody through an error prone PCR process. Specifically, scFv sequencing primer 1 and primer 2, polymerase without proofreading function (Takara, Japan), polymerase buffer, dATP, dTTP, dCTP, and dGTP were added to pComb3x vector containing G4-1H scFv, and salt concentration and manganese were added. The mutation frequency was controlled by adjusting the amount. Error prone PCR was performed by repeating the procedure of 30 times of pre-heating at 94 ° C for 5 minutes, double helix separation at 91 ° C for 1 minute, primer binding at 55 ° C for 1 minute, and DNA synthesis reaction at 72 ° C for 3 minutes. A DNA synthesis reaction was performed at ℃ for 5 minutes. The purified PCR product and the phagemid vector, pComb3x vector, were digested using Sfi I (New England Biolab, UK) restriction enzyme, ligated, and then transformed into ER2738 E. coli and cultured.

Primers used for error prone PCR scFv sequencing primers One ACA CTT TAT GCT TCC GGC SEQ ID NO: 16 2 CAA AAT CAC CGG AAC CAG AG SEQ ID NO: 17

Example 5: Selection of clones with improved affinity for CD55 compared to 4-1H

Clones having improved affinity for CD55 compared to G4-1H and 4-1H were selected through biopanning using the library prepared in Example 4 above.

First, 2.5 μg of recombinant human CD55 (R&D Systems 2009-CD/CF, USA) was conjugated to magnetic beads (Invitrogen, USA). Phage having an affinity for CD55 was bound by reacting the phage library with CD55 conjugated to magnetic beads at room temperature for 2 hours, and then reacted with 4-1H antibody at room temperature for 30 minutes to compete with 4-1H. It was washed with a buffer containing 0.5% Tween 20 (Amresco, USA) and eluted using an acidic solution. The eluted phages were infected with E. coli ER2738 for the next round of panning, and cultured overnight to proliferate. This process was repeated 4 times and biopanning was performed. The number of washings increased as the number of panning increased by washing once in the 1st, 3rd in the 2-3rd, and 5 times in the 4th, thereby accumulating phages with high binding force.

192 clones selected from the plates of the 3rd and 4th biopanning products were placed in a 96 deep well plate with 100 μg/ml carbenicillin, 70 μg/ml kanamycin and VCSM13 helper phage, and incubated overnight at 37° C. was induced to proliferate in the expressed phage. The culture medium obtained above was centrifuged to obtain a medium supernatant containing phage, put it in an ELISA plate coated with recombinant human CD55 (R&D Systems 2009-CD/CF, USA), and incubated at 37 for 2 hours, and HRP was conjugated Anti-M13 antibody (Merck, USA) was used as a secondary antibody, and the antibody binding to CD55 was confirmed by ELISA. Using the phage of the 4-1H clone as a control, 5 clones with higher binding affinity were selected.

Example 6: Complete Antibody Conversion and Expression/Purification of Selected Clones

Cloning was performed to make EP-1E, EP-1F, EP-9B, 3R-2-1H and EP-10H in full-length full IgG form. The EP-1E, EP-1F, EP-9B, 3R-2-1H and EP-10H have the CDR sequences shown in Table 4 below.

Amino acid sequences of the light and heavy chain variable regions of the selected clones amino acid SEQ ID NO: EP-1E
light chain variable region SYELTQPPSVSVSPGQTASITC SGGGGSYG W F QQKPGQSPVTVIY WN N KRPS GIPERFSGSKSGNTATLTISGTQAMDEADYYC GGWDSSTYAI FGGGTKLTVL 18 EP-1E
heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFS DRGMA WVRQAPGKGLEWVS GISSSGRYTYYAPAVKG RATISRDNSKNTLYLQMNSLRAEDTAVYYCAK NA V AGGWHAAYIDA WGQGTTVTVSS 19 EP-1F
light chain variable region SYELTQPPSVSVSPGQTASITC SGGGGSYG WYQQKPGQSPVTVIY WNDKRPS GIPERFSGSKSGNTATLTISGTQAMDEADYYC GGWDSSTYAI FGGGTKLTVL 4 EP-1F
heavy chain variable region EVQL A ESGGGLVQPGGSLRLSCAASGFTFS DRGMA WVRQAPGKGLEWVS GISSSGRYTYYAPAVKG RATISRDNSKNTLYLQMNSLRAEDTAVYYCAK NA V AGGWHAAYIDA WGQGTTVTVSS 20 EP-9B
light chain variable region SYELTQPPSVSVSPGQTASITC SGGGGSYG WYQQKPGQSPVTVIY WNDKRPS GIPERFSGSKSGNTATLTISGTQAMDEADYYC GGWDSSTYAI FGGGTKLTVL 4 EP-9B
heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFS DRGM V WVRQ S PGKGLEWVS GISSSGRYTYYAPAVKG RATISRDNSKNTLYLQMN N LRAEDTAVYYCAK NA V AGGWHAAYIDA WGQGTTVTVSS 21 3R-2-1H
light chain variable region SYELTQPPSVSVSPGQTASITC SGGGGSYG WYQQKPGQSPVTVIY WNDKRPS GIPERFSGSKSGNTATLTISGTQAMDEADYYC GGWDSSTYAI FGGGTKLTVL 4 3R-2-1H
heavy chain variable region EVQLVESGGGLVQPGGSLRLSC V A R GFTFS DRGMA WVRQAPGKGLEWVS GISSSGRYTYYAPAVKG R T TISRDNSKNTLYLQMNSLRAEDTAVYYCAK NA V AGGWHAAYIDA WGQGTTVTVSS 22 EP-10H
light chain variable region SYELTQPPSVSVSPGQTASITC SGGGGSYG WYQQKPGQSPVTVIY WNDKRPS GIPERFSGSKSGNTATLTISGTQAMDEADYYC GGWDSSTYAI FGGGTKLTVL 4 EP-10H
heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFS DRGMA WVRQAPGKGLEWVS GISSSGRYTYYAPAVKG RATISRDNSKNTLYLQMNSLRAEDTAVYYCAK NA V AGGWHAAYIDA WGQGTTVTVSS 19

In the table above, underlined parts represent CDR regions, and bold texts represent amino acids different from those of G4-1H, respectively. Since the antibodies selected in Example 5, EP-1E, EP-1F, EP-9B, 3R-2-1H and EP-10H, were in the form of scFv, they were converted into complete antibodies, and complete antibody conversion and expression/purification are described above. It was carried out in the same manner as in Example 2.

Primers used for complete antibody cloning of the selected clones primer order SEQ ID NO: V _L forward GGT CTT TGT ATA CAT GTT GCT GTG GTT GTC TGG TGT TGA AGG AAG CTA CGA GCT GAC AC 11 reverse CAA GCT CAC CGT CTT GCG GAC CGT GGC C 12 C _K forward CGG ACC GTG GCC GCC CCC TC 13 reverse CGG GGC GAG TGC TAG TTC TAG AAC TA 14 LC forward GGG AAT TCT AGA GGA TCG AAC CCT TTG CAA GCT TCG GCA CGA GCA GAC CAG CAT GGG CAT CAA GAT GGA GAC ACA TTC TCA GGT CTT TGT ATA CAT GTT G 15 reverse CGG GGC GAG TGC TAG TTC TAG AAC TA 14 V _H forward CTT CCT GTC AGT AAC TAC AGG TGT CCA CTC CGA GGT CCA GCT GGT C 23 reverse GTG ACC GTG TCC AGC GCC TCC ACC AAG GG 24 C _H forward GCC TCC ACC AAG GGC CC 8 reverse TCC CCC GGC AAG TGA GCG GCC GCT CAC 9 HC forward CAG AAT TCA CTC TAA CCA TGG AAT GGA GCT GGG TCT TTC TCT TCT TCC TGT CAG TAA CTA CAG 10 reverse TCC CCC GGC AAG TGA GCG GCC GCT CAC 9

Example 7: Affinity Analysis for CD55 of Selected and Constructed Complete Antibodies

The affinity of the antibody prepared in Example 6 to CD55 was always confirmed by indirect ELISA. Indirect ELISA was performed in the same manner as in Example 3 described above.

As a result of ELISA, the antibody of Example 6 showed a higher affinity for CD55 compared to 4-1H ( FIG. 2 ). EP-1E, EP-1F, 3R-2-1H and EP-10H showed similar affinities, and EP-9B showed higher affinity than 4-1H but lower than other antibodies. Antibodies having a higher affinity for CD55 compared to 4-1H can be very usefully used for diagnosing various cancers or preparing pharmaceutical therapeutic compositions.

Example 8: Amino acid substitution of the binding strength improvement core region

In all of the clones listed in Table 4, G at the H100A position according to the Kabat EU numbering system was substituted with V in G4-1H. This part is contained in the HCDR3 region, which is important for antigen binding of the antibody. The present inventors identified this site as a key site for improving binding strength, and cloning was performed to replace the site with a hydrophobic amino acid other than V. 10H(A), 10H(I), 10H(L), 10H(F) and 10H(M) prepared with EP-10H different from G4-1H only at the H100A position have the following CDR sequences ( Table 6).

amino acid SEQ ID NO: 10H (A) heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSDRGMAWVRQAPGKGLEWVSGISSSGRYTYYAPAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKNA A AGGWHAAYIDAWGQGTTVTVSS 25 10H(I) heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSDRGMAWVRQAPGKGLEWVSGISSSGRYTYYAPAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKNA I AGGWHAAYIDAWGQGTTVTVSS 26 10H(L) heavy chain variable region EVQLAESGGGLVQPGGSLRLSCAASGFTFSDRGMAWVRQAPGKGLEWVSGISSSGRYTYYAPAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKNA L AGGWHAAYIDAWGQGTTVTVSS 27 10H(F) heavy chain variable region EVQLAESGGGLVQPGGSLRLSCAASGFTFSDRGMAWVRQAPGKGLEWVSGISSSGRYTYYAPAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKNA F AGGWHAAYIDAWGQGTTVTVSS 28 10H(M) heavy chain variable region EVQLAESGGGLVQPGGSLRLSCAASGFTFSDRGMAWVRQAPGKGLEWVSGISSSGRYTYYAPAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKNA M AGGWHAAYIDAWGQGTTVTVSS 29

Among the heavy chain variable regions of EP-10H, which is a complete IgG form, cloned into the pCMV vector (ThermoFisher Scientific, USA), which is a vector for animal cell expression, amino acids in the complementarity determining region of the heavy chain in which G4-1H and other mutations have occurred were substituted with hydrophobic amino acids. .

First, the variable region and constant region of the heavy chain were obtained by PCR using the HC1 and HC2 primer combinations in Table 4 from the pCMV vector containing the heavy chain variable region of EP-10H. The heavy chain variable region and constant region obtained through PCR were subjected to overlap PCR using HC3 forward primer and HC2 reverse primer to obtain VH-CH1-CH2-CH3 form. The heavy chain thus obtained was digested using Eco RI and Not I (New England Biolab, UK) restriction enzymes and ligated to pCMV vector (ThermoFisher Scientific, USA), which is a vector for expression of animal cells treated with the same restriction enzyme. The ligation-completed plasmid was transformed by applying heat shock to DH5α Escherichia coli (New England Biolab, UK), followed by mass culture to obtain a plasmid.

Primer used for amino acid substitution at the core region of improving binding strength primer order SEQ ID NO: HC1 forward CTT CCT GTC AGT AAC TAC AGG TGT CCA CTC CGA GGT CCA GCT GGT C 23 reverse GTA CTA CTG CGC CAA G 30 HC2 Forward 1 GTA CTA CTG CGC CAA GAA CGC CGC GGC CGG GGG GTG GCA CGC 31 forward 2 GTA CTA CTG CGC CAA GAA CGC CAT TGC CGG GGG GTG GCA CGC 32 forward 3 GTA CTA CTG CGC CAA GAA CGC CCT GGC CGG GGG GTG GCA CGC 33 Forward 4 GTA CTA CTG CGC CAA GAA CGC CAT GGC CGG GGG GTG GCA CGC 34 forward 5 GTA CTA CTG CGC CAA GAA CGC CTT TGC CGG GGG GTG GCA CGC 35 reverse TCC CCC GGC AAG TGA GCG GCC GCT CAC 9 HC3 forward CAG AAT TCA CTC TAA CCA TGG AAT GGA GCT GGG TCT TTC TCT TCT TCC TGT CAG TAA CTA CAG 10 reverse TCC CCC GGC AAG TGA GCG GCC GCT CAC 9

After cloning was completed, expression/purification of the complete antibody using the EP-10H light chain plasmid and the heavy chain plasmid of Table 6 was performed in the same manner as in Example 2 above.

Example 9: Affinity analysis for CD55 of a complete antibody prepared by substituting a key region for improving binding affinity

The affinity of the antibody prepared in Example 8 to CD55 was confirmed by indirect ELISA. Indirect ELISA was performed in the same manner as in Example 3 described above.

As a result of ELISA analysis, the antibody prepared by substituting a hydrophobic amino acid for the binding affinity core showed a higher affinity for CD55 than 4-1H (FIG. 3). However, 10H(A), 10H(I), 10H(L), 10H(F) and 10H(M) showed lower affinity than EP-10H. This means that the substitution of G with V at the H100A position represents the most efficient result for improving the binding force.

Example 10: Therapeutic effect of EP-10H in drug-resistant cancer cell lines overexpressing CD55

Experimental Example 10-1: Combination effect of rituximab and EP-10H in Burkitt's lymphoma cell line BJAB

BJAB is complement-resistant, due to the expression of CD55 (Golay, et al., Blood , 95(12):3900-8, 2000). For this reason, the responsiveness to rituximab, which is the main treatment mechanism for complement dependent cytotoxicity (CDC), is low. In order to confirm the possibility of improving the therapeutic effect of rituximab on Burkitt's lymphoma, which is known to be malignant among B-cell lymphomas, the apoptosis ability of BJAB, a Burkitt's lymphoma cell line, was confirmed through combination with EP-10H. 20 μg/ml of rituximab to 5x10 ⁵ BJAB cells per sample, and 50% complement of 4-1H or EP-10H and rituximab at a concentration of 50 μg/ml and 20 μg/ml, respectively, to confirm the effect of co-administration After diluting with RPMI medium to which sera human (Sigma, USA) was added, the final 100 μl was treated, and cultured at 37° C., 5% CO ₂ condition for 1 hour. Thereafter, using the FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, USA), apoptotic cells were identified with Attune NxT (ThermoFisher Scientific, USA), which is a FACS analysis equipment, and CDC was observed (FIG. 4). As previously reported, BJAB cells had low reactivity to rituximab, but their effect was increased when co-administered with anti-CD55 antibodies (4-1H and EP-10H), and the width of the increased effect was similar to that of rituximab. The combination of rituximab and EP-10H was greater than that in combination with 4-1H. These results indicate that the expression of CD55 inhibits the CDC effect of rituximab. When CD55 is inhibited using an antibody, the inhibited effect is restored, and this effect is in EP-10H, which has better affinity for CD55. shows that it is more pronounced by

Experimental Example 10-2a: Confirmation of CD55 expression in Ramos-RR, a rituximab-resistant cell line

Ramos was a rituximab-responsive B-cell lymphoma cell line, and an experiment was performed as follows to confirm CD55 expression in Ramos-RR, a rituximab-resistant cell line prepared using Ramos.

5x10 ⁵ Ramos and Ramos-RR per sample were suspended in PBS with or without EP-10H monoclonal antibody at a concentration of 10 μg/ml, and incubated at 4° C. for 1 hour. The culture solution was centrifuged at 4,000 rpm for 5 minutes, washed with 100 μl of PBS, and centrifuged again at 4,000 rpm for 5 minutes. The cells were treated with the Goat anti-human IgG antibody, Alexa Fluor 488 (ThermoFisher Scientific, USA), diluted in PBS at a ratio of 1:100, and incubated at 4° C. for 30 minutes while blocking light. The fluorescently stained cells were washed with PBS and then suspended in 400 μl of PBS and analyzed using Attune NxT (ThermoFisher Scientific, USA), which is a FACS analysis equipment, and the results are shown in FIG. 5a.

As a result, it was confirmed that the CD55 expression of Ramos-RR was increased compared to that of Ramos. This means that CD55 is involved in drug resistance of rituximab.

Experimental Example 10-2b: Combination effect of rituximab and EP-10H on rituximab-resistant cell line Ramos-RR

In Ramos-RR, which overexpressed CD55 and exhibited resistance to rituximab, apoptosis ability was confirmed by co-administration of rituximab and EP-10H by the method of Experimental Example 10-1 (FIG. 5b). Rituximab induced apoptosis in Ramos, whereas the degree of apoptosis induction was lower in Ramos-RR than in Ramos. However, the combination of EP-10H and 4-1H increased apoptosis in Ramos-RR, and as in BJAB, the combination with EP-10H significantly increased the apoptosis of rituximab compared to the combination with 4-1H. did it These results show that EP-10H can exhibit therapeutic effects not only in B-cell lymphomas (BJAB cell line is the primary cancer cell line) but also in carcinomas that have acquired resistance to conventional therapies.

Experimental Example 10-3a: Confirmation of CD55 expression in K562-IR, an imatinib-resistant cell line

K562 is a leukemia cell line that is responsive to imatinib, a tyrosine kinase inhibitor. CD55 expression in K562-IR, an imatinib-resistant cell line prepared using K562, was confirmed in the same manner as in Experimental Example 10-2a. As a result, it was confirmed that the CD55 expression of K562-IR was increased compared to that of K562 (FIG. 6a). This means that CD55 is involved in drug resistance of imatinib.

Experimental Example 10-3b: Confirmation of the combined effect of imatinib and EP-10H in K562-IR cell line

Immediately after dispensing 2 x 10 ⁵ cells per well in a 12-well cell culture plate with K562-IR overexpressing K562 and CD55 showing resistance to imatinib, 2 μM of imatinib, and 2 μM of imatinib and 4-1H Alternatively, each of EP-10H was diluted with RPMI medium to a concentration of 10 μg/ml, treated with a final 500 μl, and cultured at 37° C., 5% CO ₂ conditions for 24 hours. Thereafter, apoptotic cells were analyzed with Attune NxT, a FACS analysis equipment, using 7-AAD and FITC Annexin V Apoptosis Detection Kit (FIG. 6b).

While imatinib induced apoptosis in K562 cell line, it had no effect in K562-IR, but co-administered with EP-10H or 4-1H induced apoptosis in K562-IR. In addition, the combination of EP-10H and imatinib showed a greater apoptosis effect than the combination of 4-1H and imatinib. These results prove that CD55 is the cause of resistance to imatinib, a tyrosine kinase inhibitor, together with the results of Experimental Example 10-3a, and inhibition of CD55 using EP-10H means that it is an effective strategy to overcome the resistance of imatinib. Imatinib is a long-term drug widely used for the treatment of leukemia, and a strategy to overcome drug resistance that may be induced by long-term administration is urgently needed. Accordingly, the strategy of overcoming the resistance of imatinib using EP-10H is expected to be very effective in treating leukemia.

Experimental Example 10-4a: Confirmation of CD55 expression in PC9-GR, a gefitinib-resistant cell line

PC9 is a non-small cell lung cancer (NSCLC) cell line that is responsive to the tyrosine kinase inhibitor gefitinib. As a result of confirming CD55 expression in the gefitinib-resistant cell line PC9-GR prepared using PC9 by the method of Experimental Example 10-2a, it was confirmed that the CD55 expression of PC9-GR was increased compared to PC9 ( FIG. 7a ). This means that CD55 is involved in drug resistance of gefitinib.

Experimental Example 10-4b: Confirmation of the combined effect of gefitinib and EP-10H in PC9-GR

NSCLC cell lines PC9 and CD55 overexpressed PC9-GR, which exhibits resistance to gefitinib, were aliquoted into 1 x 10 ⁵ per well in a 12-well cell culture plate, and then cultured at 37° C. and 5% CO ₂ conditions. 24 hours after dispensing, 5 μg/ml of gefitinib, and 5 μg/ml of gefitinib and 4-1H or EP-10H, respectively, were diluted with RPMI medium to 5 μg/ml to confirm the effect of co-administration, and the final 500 μl After treatment, it was cultured again at 37° C., 5% CO ₂ conditions for 24 hours. Thereafter, apoptotic cells were analyzed with Attune NxT, a FACS analysis equipment, using 7-AAD and FITC Annexin V Apoptosis Detection Kit (FIG. 7b).

Gefitinib induced apoptosis in the NSCLC cell line, PC9 cell line, but had no effect in PC9-GR, but the combination of EP-10H or 4-1H and gefitinib induced apoptosis in PC9-GR. In addition, the combination of EP-10H and gefitinib showed a greater apoptosis effect than the combination of 4-1H and gefitinib. These results, together with the results of Experimental Example 10-4a, demonstrate that CD55 is the cause of resistance to gefitinib, a tyrosine kinase inhibitor, among targeted anticancer drugs, and inhibition of CD55 using EP-10H is an effective It means strategy. Gefitinib is an effective drug for NSCLC for which there are not many therapeutic agents, and when drug resistance develops in a situation where there are not many other therapeutic options, it is difficult to select a second drug, so a strategy to overcome this drug resistance is urgently needed. The strategy of overcoming resistance to gefitinib using EP-10H is expected to be very effective in treating NSCLC.

Example 11: Production of dual antibodies using EP-10H and confirmation of efficacy

In Example 10, the combined effect of EP-10H and various drugs was confirmed. Based on the results of the combination effect with rituximab confirmed in Experimental Examples 10-1 and 2 of Example 10, a method of using two antibodies targeting different antigens in combination and a method of using one antibody to simultaneously target two antigens In order to compare the cancer cell killing ability of the double antibody, a CD20 x CD55 double antibody was prepared using rituximab and EP-10H.

Experimental Example 11-1. Dual antibody (CD20 X CD55) cloning for animal cell expression

In the present invention, since a novel diabody different from the previously reported diabodies targeting both CD20 and CD55 (Macor, et al., Leukemia , 29(2):406-14, 2014) was developed, the present inventors developed a diabody type In order to compare the efficacy according to the present invention, a dual antibody targeting both CD20 and CD55 was cloned using the genes of rituximab and EP-10H. The bi-antibody produced in the present invention was named SBU (Specific Bi-functional Unit) (FIG. 8).

The SBU-type CD20 X CD55 diabody used a knob-into-hole technique to prevent undesirable binding of heavy chains to each other, and a linker composed of Gly and Ser was used to prevent erroneous binding between light chains. Cloning was carried out in the form of CD55-scFv-knob (VL-linker20-VH-CH2-CH3) and CD20-Fab-hole (VL-CK-linker40-VH-CH1-CH2-CH3), respectively, and CD55-scFv-knob In the case of EP-10H prepared in Example 6 was used as a template, and PCR was performed using the primer combinations in Table 8. The amplified genome was treated with Bss HII and Xba I (New England Biolab, UK) enzymes, and was ligated to pMAZ vector, a vector for expression of animal cells treated with the same restriction enzyme. The ligation-completed plasmid was transformed by applying heat shock to DH5α competent cells (New England Biolab, UK), and the obtained colonies were mass-cultured to obtain a plasmid.

For the construction of CD20-Fab-hole (VL-CK-linker40-VH-CH1-CH2-CH3), PCR was performed using the primer combinations in Table 9 based on the rituximab sequence published in the Drug Data Bank. The amplified genome was treated with Bss HII and Xba I enzymes, and was ligated to pMAZ vector, a vector for expression of animal cells treated with the same restriction enzyme. The ligation-completed plasmid was transformed by applying heat shock to a DH5α competent cell (New England Biolab, UK), and the obtained colonies were mass-cultured to obtain a plasmid and sequence analysis (Table 10) was completed.

Primers used for CD55-scFv-knob cloning primer order SEQ ID NO: V _L forward CAG CGC GCA CTC CAG CTA CGA GCT GAC ACA GCC 36 reverse GAC CAA GCT CAC CGT CTT GGG CGG AGG CGG GAG TGG TGG TGG CGG TAG CGG TGG AGG AGG C 37 V _H forward GTG GAG GAG GCA GTG GAT CTG GCG GCT CTG AGG TCC AGC TGG TCG 38 reverse GGC ACA ACC GTG ACC GTG TCC AGC GGC GGC TCA GAC AAA ACT CAC AC 39 C _H forward CGT GTC CAG CGG CGG CTC AGA CAA AAC TCA CAC ATG C 40 reverse CTC TCC CTG TCC CCG GGT AAA TGA CTA GAA CTA 41

Primers used for CD20-Fab-hole cloning primer order SEQ ID NO: V _L- C _K forward CGC AGC GAG CGC GCA CTC CCA GAT TGT CCT GTC TCA GTC TCC TGC 42 reverse CGG CCG CCG TGC GAG ATC TTT TGA TTT CCA GTT TAG TTC CGC CG 43 V _H forward CAA GTC CAA CTG CAA CAA CCG GG 44 reverse GGA AGA CCG ATG GGC CCT TGA AGC TAG CGG CGG AAA CCG TTG TGC CAG AG 45 C _H forward GCA AGC TTC AAG GGC 46 reverse CTC TCC CTG TCC CCG GGT AAA TGA CTA GAA CTA 41

Variable region amino acid sequences of SBU-form CD55-scFv-knob and CD20-Fab-hole CD55-
scFv-knob SYELTQPPSVSVSPGQTASITCSGGGGSYGWYQQKPGQSPVTVIYWNDKRPSGIPERFSGSKSGNTATLTISGTQAMDEADYYCGGWDSSTYAIFGGGTKLTVLGGGGSGGGGSGGGGSGSGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDRGMAWVRQAPGKGLEWVSGISSSGRYTYYAPAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKNAVAGGWHAAYIDAWGQGTTVTVSSGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:
47 CD20-Fab-hole QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRSRTAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGSGGSGGGGSGGGGSGGGGSGSGSSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASFKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 48

Experimental Example 11-2. Expression and purification in animal cells

Expression of the DNA prepared in Experimental Example 11-1 was performed in the same manner as in Example 2 described above. CD20 X CD55 diabodies in the form of SBU were transduced into HEK293F cells (Invitrogen, USA) using polyethyleneimine (PEI) (Polysciences, USA) and 150 mM NaCl and plasmids of SEQ ID NOs: 47 and 48, respectively, in Freestyle 293 expression medium. (Invitrogen, USA) at 37 °C temperature, 8% CO ₂ and 55% humidity conditions were cultured for 7 days. A diabody similar to SBU has been reported (Moore, et al., A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats. Methods, 154:38-50), but In this case, three types of DNA are required for expression. Unlike the conventional method for preparing a double antibody that requires three types of DNA, the SBU of the present invention uses a linker composed of Gly and Ser to prevent erroneous binding of light chains, so that double antibody expression is possible with two types of DNA. After centrifuging the expression cell culture solution at 4,000 rpm for 10 minutes, the supernatant was taken and filtered through a 0.22 μm filter. The filtered supernatant was induced to bind to 1 ml of KappaSelect (GE Healthcare, USA) resin at 4°C. The bound resin was washed with a PBS solution of 10 cv (column volume), and then the bound antibody was eluted using a 100 mM glycine-HCl (pH 2.7) solution, and then neutralized with 1 M Tris-HCL (pH 9.0). . After buffer exchange with PBS at pH 7.2-7.4, the size and purity of the light and heavy chains of the purified antibody were confirmed through SDS-PAGE. As a result, molecular weight and high purity consistent with the theoretical calculated values were confirmed (FIG. 9). The SBU-type diabody is a hetero form in which the CD20 binding site is Fab and the CD55 binding site is scFv. Knob-knob homodimer or knob monomer known to be most likely to be generated during dual antibody purification when purified with protein L using the C kappa portion of the Fab (Giese, et al., Biotechnol Prog , 34(2)397-404) It is possible to suppress the production of , and only the heterodimer in the desired form can be obtained.

Experimental Example 11-3. Confirmation of binding affinity of the double antibody through ELISA

Affinity to CD20 and CD55, each antigen targeted by the CD20 X CD55 diabody prepared in Experimental Example 11-2, was confirmed by indirect ELISA. Indirect ELISA was performed by the method of Example 3, and showed excellent binding to both CD20 and CD55 ( FIGS. 10a and 10b ). In particular, the SBU form of the CD20 X CD55 diabody showed a higher affinity for CD20 than the CD20 X CD55 diabody disclosed in Macor et al. It showed similar affinity to the X CD55 diabody. As for the CD20 X CD55 diabody, which can be used as a treatment for hematologic cancers in which CD20 is overexpressed, the CD20 X CD55 diabody in the form of SBU, which has a higher affinity for CD20, will be more effective.

Experimental Example 11-4. Confirmation of apoptosis activity of CD20 X CD55 diabodies in BJAB

The apoptosis ability of the CD20 X CD55 double antibody was confirmed by the method of Experimental Example 10-1. 20 μg/ml of rituximab for 5x10 ⁵ BJAB cells per sample, 20 μg/ml of EP-10H and rituximab, respectively, to confirm the effect of co-administration, and the CD20 X CD55 diabody, the form disclosed in the prior literature written by Macor and SBU forms were diluted with RPMI medium supplemented with 20% complement sera human (Sigma, USA) at a concentration of 20 μg/ml, respectively, and the final 100 μl was treated. Others were performed in the same manner as in Experimental Example 10-1.

As a result, in BJAB cells with low reactivity to rituximab, the CD20 X CD55 diabody showed a significant difference in apoptosis compared to the control treated with complement sera human only, and SBU showed a significant difference in cell death compared to rituximab. showed apoptosis ( FIGS. 11A and 11B ). These results show that, compared to co-administration of two antibodies targeting different antigens, one diabody that can simultaneously target both antigens is more effective in inducing CDC against cancer cells, and a representative CD20 X CD55 diabody It means that the SBU of the present invention is more suitable for inducing CDC against cancer cells than the Macor diabody. In addition, the CD20 X CD55 diabody in the form of SBU could be an effective treatment for Burkitt's lymphoma, which is known to be malignant among B-cell lymphomas.

Experimental Example 11-5. Confirmation of apoptosis of CD20 X CD55 diabodies in Ramos

The apoptosis ability of the CD20 X CD55 dual antibody was confirmed in the Ramos cell line by the method of Experimental Example 11-4 ( FIGS. 12A and 12B ). As a result, in rituximab-responsive Ramos cells, the CD20 X CD55 double antibody showed a significant difference in apoptosis compared to rituximab. In addition, the SBU of the present invention showed superior apoptosis ability than the dual antibody of Macor. These results indicate that the CD20 X CD55 diabody can exhibit more effective anticancer effects than rituximab in rituximab-responsive carcinoma, and that SBU can be applied as an effective therapeutic agent among the CD20 X CD55 diabodies in CD20-expressing hematologic cancers. do.

Experimental Example 11-6. Confirmation of apoptosis of CD20 X CD55 diabodies in Ramos-RR

The apoptosis ability of the CD20 X CD55 dual antibody was confirmed in the Ramos-RR cell line by the method of Experimental Example 11-4 ( FIGS. 13a and 13b ). As a result, in Ramos-RR cells that did not respond to rituximab, the CD20 X CD55 dual antibody showed a significant difference in apoptosis compared to rituximab. Similar to the results of the BJAB cell line, the SBU of the present invention showed a significantly superior apoptosis activity than the Macor double antibody among the diabodies. These results suggest that the CD20 X CD55 diabody in the form of SBU can be applied as an effective therapeutic agent for rituximab-resistant carcinomas among hematologic cancers expressing CD20.

Experimental Example 11-7. CDC activation analysis of CD20 X CD55 diabodies in the form of SBU

In Experimental Example 11-5, CDC activation was analyzed by taking the supernatant treated with rituximab and CD20 X CD55 dual antibody in the form of SBU before flow cytometry analysis of the Ramos-RR cell pellet (FIG. 14). The CDC mechanism is a reaction that occurs through the binding of C1q to an antibody, and activation analysis is possible at an intermediate stage in the entire CDC process based on the production of C4d, a by-product thereof. When analyzed through an ELISA kit (Quidel, USA) that can confirm C4d production in each supernatant, more C4d was produced in the supernatant treated with the CD20 X CD55 double antibody in the form of SBU compared to the supernatant treated with rituximab. The difference was significant. These results clearly show that the excellent cell killing ability of the SBU CD20 X CD55 diabody is due to the CDC activation mechanism.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> SG medical <120> A Novel Antibody Specific for CD55 and Use Thereof <130> HPC-10439 <150> KR 10-2021-0004196 <151> 2021-01-12 <160> 48 <170> KoPatentIn 3.0 < 210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 1 Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val Lys 1 5 10 15 Gly <210 > 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 2 Ser Gly Gly Gly Gly Ser Tyr Gly 1 5 <210> 3 <211> 10 <212> PRT <213 > Artificial Sequence <220> <223> LCDR3 <400> 3 Gly Gly Trp Asp Ser Ser Thr Tyr Ala Ile 1 5 10 <210> 4 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> G4-1H_VL <400> 4 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Gly Gly Gly Ser Tyr Gly Trp Tyr 20 25 30 Gln Gln Lys Pro Gly Gln Ser Pro Val Thr Val Ile Tyr Trp Asn Asp 35 40 45 Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Lys Ser Gly 50 55 60 Asn Thr A la Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala 65 70 75 80 Asp Tyr Tyr Cys Gly Gly Trp Asp Ser Ser Thr Tyr Ala Ile Phe Gly 85 90 95 Gly Gly Thr Lys Leu Thr Val Leu 100 <210> 5 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> G4-1H_VH <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Gly Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 <210> 6 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> G4-1H VH F primer <400> 6 cttcctgtca gtaacta cag gtgtccactc ccaggtgcaa ctgcagcagt c 51 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> G4-1H VH R primer <400> 7 ccagcgtgac cgtatccagc gcctccacca agggcccca 39 <210> 8 <211 > 17 <212> DNA <213> Artificial Sequence <220> <223> G4-1H CH F primer <400> 8 gcctccacca agggccc 17 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> G4-1H CH R primer <400> 9 tccccccggca agtgagcggc cgctcac 27 <210> 10 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> G4-1H HC F primer <400> 10 cagaattcac tctaaccatg gaatggagct gggtctttct cttcttcctg tcagtaacta 60 cag 63 <210> 11 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> G4-1H VL F primer <400> 11 ggtctttgta tacatgttgc tgtggttgtc tggtgttgaa ggaagctacg agctgacac 59 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> G4-1H VL R210 primer <400> 12 caagctcacc gtcttgcgga < ccgt211 > 20 <212> DNA <213> Artificial Sequence <220> <223> G4-1H CK F primer <400> 13 cggaccgtgg ccgccccctc 20 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> G4-1H CK R primer <400> 14 cggggcgagt gctagttcta gaacta 26 <210> 15 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> G4-1H LC F primer <400> 15 gggaattcta gaggatcgaa ccctttgcaa gcttcggcac gagcagacca gcatgggcat 60 caagatggag acacattctc aggtctttgt atacatgttg 100 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 17 16 acactttag cttcc Artificial Sequence <220> <223> scFv < 1 <400 211> 20 <212> DNA <213> Artificial Sequence <220> <223> scFv primer 2 <400> 17 caaaatcacc ggaaccagag 20 <210> 18 <211> 104 <212> PRT <213> Artificial Sequence <220> <223 > EP-1E_VL <400> 18 Ser Tyr Glu Leu Thr Gln Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Gly Gly Gly Ser Tyr Gly Trp Phe 20 25 30 Gln Gln Lys Pro Gly Gln Ser Pro Val Thr Val Ile Tyr Trp Asn Asn 35 40 45 Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Lys Ser Gly 50 55 60 Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala 65 70 75 80 Asp Tyr Tyr Cys Gly Gly Gly Trp Asp Ser Ser Thr Tyr Ala Ile Phe Gly 85 90 95 Gly Gly Thr Lys Leu Thr Val Leu 100 <210> 19 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> EP-1E_VH <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Val Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 20 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> EP-1F_VH <400> 20 Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Val Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 21 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> EP-9B_VH <400> 21 Glu Val Gln Leu Val Glu Ser G ly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Val Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Val Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gly Gly Thr Thr Val Thr Val Ser 115 120 <210> 22 <211> 123 < 212> PRT <213> Artificial Sequence <220> <223> 3R-2-1H_VH <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Arg Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Thr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Val Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gly Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 23 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> VH F primer <400> 23 cttcctgtca gtaactacag gtgtccactc cgaggtccag ctggtc 46 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> VH R primer <400> 24 gtgaccgtgt ccagcgcctc caccaaggg 29 <210> 25 <211> 123 <212 > PRT <213> Artificial Sequence <220> <223> 10H(A)_VH <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Il e Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Ala Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 26 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 10H(I)_VH <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Ile Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 27 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 10H(L)_VH <400> 27 Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Leu Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 28 <211> 123 <212> PRT <213> Artificial Sequence <220 > <223> 10H(F)_VH <400> 28 Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Tr p Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Phe Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 29 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 10H(M)_VH <400> 29 Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 A la Lys Asn Ala Met Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 30 <211> 16 <212> DNA <213> Artificial Sequence < 220> <223> HC1 R primer <400> 30 gtactactgc gccaag 16 <210> 31 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> HC2 F1 primer <400> 31 gtactactgc gccaagaacg ccgcggccgg ggggtggcac gc 42 <210> 32 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> HC2 F2 primer <400> 32 gtactactgc gccaagaacg ccattgccgg ggggtggcac gc 42 <210> 33 <211> 42 <212> DNA < 213> Artificial Sequence <220> <223> HC2 F3 primer <400> 33 gtactactgc gccaagaacg ccctggccgg ggggtggcac gc 42 <210> 34 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> HC2 F4 primer < 400> 34 gtactactgc gccaagaacg ccatggccgg ggggtggcac gc 42 <210> 35 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> HC2 F5 primer <400> 35 gtactactgc gccaagaacg cctttgccgg ggggtggcac 36 <gc 42 <210> 211> 33 <21 2> DNA <213> Artificial Sequence <220> <223> CD55-scFv-knob_VL F primer <400> 36 cagcgcgcac tccagctacg agctgacaca gcc 33 <210> 37 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> CD55-scFv-knob_VL R primer <400> 37 gaccaagctc accgtcttgg gcggaggcgg gagtggtggt ggcggtagcg gtggaggagg 60 c 61 <210> 38 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> CD55-scFv- knob_VH F primer <400> 38 gtggaggagg cagtggatct ggcggctctg aggtccagct ggtcg 45 <210> 39 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> CD55-scFv-knob_VH R primer <400> 39 ggcacaaccg tgaccgtgtc tcagacaaaa ctcacac 47 <210> 40 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> CD55-scFv-knob_CH F primer <400> 40 cgtgtccagc ggcggctcag acaaaactca cacatgc 37 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> CD55-scFv-knob_CH R primer <400> 41 ctctccctgt ccccgggtaa atgactagaa cta 33 <210> 42 <211> 45 <212> DNA <213> Artificial Sequence <220 > <223> CD20-Fab-hole VL-CK F primer <400> 42 cgcagcgagc gcgcactccc agattgtcct gtctcagtct cctgc 45 <210> 43 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> CD20-Fab-hole VL-CK R primer <400> 43 cggccgccgt gcgagatctt ttgatttcc gccg 44 210> 44 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CD20-Fab-hole VH F primer <400> 44 caagtccaac tgcaacaacc ggg 23 <210> 45 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> CD20-Fab-hole VH R primer <400> 45 ggaagaccga tgggcccttg aagctagcgg cggaaaccgt tgtgccagag 50 <210> 46 <211> 15 <212> DNA <213> Artificial Sequence <220> < 223> CD20-Fab-hole CH F primer <400> 46 gcaagcttca agggc 15 <210> 47 <211> 477 <212> PRT <213> Artificial Sequence <220> <223> CD55-scFv-knob <400> 47 Ser Tyr Glu Leu Thr Gln Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Gly Gly Gly Ser Tyr Gly Trp Tyr 20 25 30 Gln Gln Lys Pro Gly Gln Ser Pro Val Thr Val Ile Tyr Trp Asn Asp 35 40 45 Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Lys Ser Gly 50 55 60 Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala 65 70 75 80 Asp Tyr Tyr Cys Gly Gly Trp Asp Ser Ser Thr Tyr Ala Ile Phe Gly 85 90 95 Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Arg Gly Met Ala Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser 165 170 175 Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val Lys Gly Arg Ala 180 185 190 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys A sn Ala 210 215 220 Val Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala Trp Gly Gin Gly 225 230 235 240 Thr Thr Val Thr Val Ser Ser Gly Gly Ser Asp Lys Thr His Thr Cys 245 250 255 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 260 265 270 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 275 280 285 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 290 295 300 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 305 310 315 320 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 325 330 335 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 340 345 350 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu L ys Thr Ile Ser Lys 355 360 365 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 370 375 380 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys 385 390 395 400 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 405 410 415 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 420 425 430 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 435 440 445 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 450 455 460 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 48 <211> 706 <212> PRT <213> Artificial Sequence <220> <223> CD20-Fab-hole <400> 48 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ser Arg Thr Ala Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Le u Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Ser Ser Gln 245 250 255 Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser 260 265 270 Val Lys Met Ser Cys Ly s Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn 275 280 285 Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly 290 295 300 Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 305 310 315 320 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 325 330 335 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 340 345 350 Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala 355 360 365 Gly Thr Thr Val Thr Val Ser Ala Ala Ser Phe Lys Gly Pro Ser Val 370 375 380 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 385 390 395 400 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 405 410 415 Trp Asn Se r Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 420 425 430 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 435 440 445 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 450 455 460 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 465 470 475 480 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 485 490 495 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 500 505 510 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 515 520 525 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 530 535 540 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 545 550 555 56 0 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 565 570 575 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 580 585 590 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 595 600 605 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 610 615 620 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 625 630 635 640 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 645 650 655 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 660 665 670 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 675 680 685 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 690 695 700Gly Lys 705

Claims (21)

HCDR1 region having an amino acid sequence represented by the following general formula (1); HCDR2 region having the amino acid sequence of SEQ ID NO: 1; And an antibody or antigen-binding fragment thereof that specifically binds to CD55, comprising a heavy chain variable region comprising an HCDR3 region having an amino acid sequence represented by the following general formula 2:
general formula 1
DRGM- X ₁ ;
general formula 2
NA- X ₂ -AGGWHAAYIDA,
In Formula 1, X ₁ is Ala or Val, and in Formula 2, X ₂ is selected from the group consisting of Val, Ala, Ile, Leu, Phe and Met.
The antibody or antigen-binding fragment thereof according to claim 1, wherein X ₁ is Ala.
The antibody or antigen-binding fragment thereof according to claim 1, wherein X ₂ is Val.
The method according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises an LCDR1 region having the amino acid sequence of SEQ ID NO: 2; LCDR2 region having an amino acid sequence represented by the following general formula 3; And an antibody or antigen-binding fragment thereof, characterized in that it further comprises a light chain variable region comprising an LCDR3 region having the amino acid sequence of SEQ ID NO: 3:
general formula 3
WN- X ₃ -KRPS,
In Formula 3, X ₃ is Asn or Asp.
The antibody or antigen-binding fragment thereof according to claim 4, wherein X ₃ is Asp.
An antibody or antigen-binding fragment thereof that specifically binds to CD55, comprising the following light chain variable region and heavy chain variable region:
a) the light chain variable region of SEQ ID NO: 4; Or, in the light chain variable region of SEQ ID NO: 4, the 32 amino acid sequence Y (Tyr) is substituted with F (Phe) and the 48th amino acid sequence D (Asp) is selected from the group consisting of N (Asn) substitution A light chain variable region comprising one or more substitutions, and
b) the heavy chain variable region of SEQ ID NO: 5; Or, in the heavy chain variable region of SEQ ID NO: 5, the 5th amino acid sequence V(Val) is substituted with A(Ala), the 23rd amino acid sequence A(Ala) is substituted with V(Val), the 25th amino acid sequence S(Ser) is substituted with R(Arg), 35th amino acid sequence A(Ala) is substituted with V(Val), 40th amino acid sequence A(Ala) is substituted with S(Ser), 68th amino acid The sequence A(Ala) is substituted with T(Thr), the 85th amino acid sequence S(Ser) is substituted with N(Asn), and the 101st amino acid sequence G(Gly) is V(Val), L(Leu) , I(Ile), F(Phe), M(Met), or a heavy chain variable region comprising one or more substitutions selected from the group consisting of substitutions with A(Ala).
The antibody or antigen-binding fragment thereof according to claim 1 or 6, wherein the antigen-binding fragment is an scFv, Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment.
A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.
A pharmaceutical composition for the prevention or treatment of cancer comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 as an active ingredient.
The composition according to claim 9, wherein the composition further comprises one or more pharmacological components selected from the group consisting of an anti-CD20 antibody and a tyrosine kinase inhibitor.
11. The composition of claim 10, wherein the anti-CD20 antibody is Rituximab.
11. The composition of claim 10, wherein the tyrosine kinase inhibitor is at least one inhibitor selected from the group consisting of imatinib and gefitinib.
10. The composition of claim 9, wherein the cancer is a CD55 positive cancer.
A composition for diagnosis of cancer comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 as an active ingredient.
15. The composition of claim 14, wherein the cancer is a CD55 positive cancer.
7. A bispecific antibody or functional fragment thereof comprising an antigen-binding fragment of an antibody that specifically binds to CD55 according to any one of claims 1 to 6 and an antigen-binding fragment of an antibody that specifically binds to CD20.
The bispecific antibody or functional fragment thereof according to claim 16, wherein the antibody specifically binding to CD20 is Rituximab.
The bispecific antibody or functional fragment thereof according to claim 16, wherein the antigen-binding fragment of the antibody that specifically binds to CD55 comprises an scFv.
The bispecific antibody or functional fragment thereof according to claim 16, wherein the antigen-binding fragment of the antibody that specifically binds to CD55 is in the form of VL-linker-VH-CH2-CH3.
The bispecific antibody or functional fragment thereof according to claim 16, wherein the antigen-binding fragment of the antibody that specifically binds to CD20 comprises Fab.
The bispecific antibody or functional fragment thereof according to claim 16, wherein the antigen-binding fragment of the antibody specifically binding to CD20 is in the form of VL-CK-linker-VH-CH1-CH2-CH3.

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