Compositions for imaging or radioimmunotherapy of hematologic cancers comprising as an active ingredient a bispecific antibody specifically binding to VLA4 and CD38

Abstract

The present invention relates to a composition for imaging or radioimmunotherapy of blood cancer comprising a bispecific antibody that specifically binds to VLA4 and CD38 as an active ingredient. The bispecific antibody of the present invention can effectively treat cancer as a radioimmunotherapy agent for the treatment and suppression of recurrence of blood cancers expressing VLA4 and CD38, and can also be usefully utilized for imaging diagnosis.

Description

Technology Field

[0001] The present invention relates to a composition for imaging or radioimmunotherapy of blood cancer comprising a bispecific antibody that specifically binds to VLA4 and CD38 as an active ingredient. Background Technology

[0003] Despite recent advancements in anticancer drugs and the rise of immunotherapy technologies, treatment resistance and intolerance in cancer patients remain significant challenges that need to be addressed. In particular, since resistance to immunotherapy promotes cancer growth and metastasis and limits therapeutic efficacy, there is an urgent need to develop new treatments that can overcome the mechanisms of cancer immunotherapy resistance.

[0004] The immunosuppressive tumor microenvironment (TME) plays a significant role in cancer immunotherapy resistance and tolerance. In malignant hematological cancers, the tumor microenvironment inhibits the activity of cytotoxic T cells and natural killer (NK) cells within the tumor, thereby hindering effective immune responses and contributing to cancer growth, metastasis, and treatment resistance. Therefore, successful anticancer immunotherapy for hematological cancers requires therapeutic strategies that overcome the immunosuppressive tumor microenvironment as well as target cancer cells.

[0005] In particular, minimizing toxic side effects for patients is a critical task in cancer treatment. The development of therapeutic agents that selectively attack cancer cell surface antigens while minimizing their impact on normal cells has the potential to significantly improve patient prognosis.

[0006] CD38 is a representative target antigen expressed on the surface of hematological cancer tumor cells. CD38 regulates the interaction between cancer cells and the tumor microenvironment and mediates cancer growth and metastasis. Furthermore, it plays a role in disrupting the immune response against cancer cells by inducing resistance to anticancer treatment. For this reason, antibodies targeting CD38 are utilized in the treatment of hematological cancers. However, some hematological cancer cells within the tumor reduce CD38 expression, which causes treatment resistance to CD38-targeted antibodies, and some normal immune cells may also weakly express CD38. Additionally, since it is difficult to block the immunosuppressive tumor microenvironment with CD38-targeted antibodies, there are limitations to conquering hematological cancers with CD38 antibodies alone.

[0007] VLA4 (very late antigen-4), known as another important target, is an alpha4-beta1 integrin involved in the migration and adhesion of cancer cells and serves as a key element mediating immune response evasion within the tumor microenvironment. VLA4 weakens the attacking power of cancer cells by inhibiting the migration and activity of cytotoxic T cells and natural killer cells within tumor tissues. Therefore, targeting VLA4 can be a promising strategy for overcoming immunosuppressive microenvironments, and VLA4 expression can also be utilized as a novel imaging target to evaluate resistance to cancer immunotherapy.

[0008] Recently, bispecific antibodies (BsAbs) that bind simultaneously to two target antigens have been attracting attention in cancer treatment. Bispecific antibodies are designed to bind to either a single cancer cell surface antigen and an immune cell receptor, or to two cancer cell surface antigens, allowing for the expectation of higher anticancer effects. In particular, bispecific antibodies that simultaneously target CD38 and VLA4 have the potential to overcome the immunosuppressive microenvironment and reduce treatment resistance in refractory hematological cancers.

[0009] Although bispecific antibodies can be used as effective immunotherapies even without attached drugs, methods to maximize anticancer effects by developing them in the form of antibody-drug conjugates (ADCs) have recently emerged. For example, 177 Combining radioisotopes such as Lu allows for its use as a radioimmunotherapy and holds strong potential for the treatment of blood cancer.

[0010] Furthermore, in the development of such antibody therapies, immuno-PET (positron emission tomography) imaging technology, which can quantitatively evaluate the expression of target antigens in tumors and monitor antibody distribution, plays an important role. Immuno-PET 89 Positron-emitting radiopharmaceuticals such as Zr are utilized to visualize biological changes within tumors, thereby providing crucial information for the early diagnosis of cancer and the determination of treatment strategies. Therefore, the development of immuno-PET technology that simultaneously targets VLA4 and CD38 is expected to enable the evaluation of target expression within tumors and contribute to the advancement of bispecific antibody therapy technologies. Prior art literature

[0012] Klein C, Brinkmann U, Reichert JM, Kontermann RE. The present and future of bispecific antibodies for cancer therapy. Nat Rev Drug Disco. 2024 Apr;23(4):301-319. doi: 10.1038 / s41573-024-00896-6. The problem to be solved

[0013] As a result of diligent research efforts to develop an anticancer immunotherapeutic agent capable of simultaneously achieving early diagnosis and treatment of cancer, the inventors loaded a radioisotope onto a bispecific antibody that simultaneously targets VLA4 and CD38, confirmed its excellent binding ability to VLA4 and CD38 and its uptake effect by blood cancer cells, and completed the present invention.

[0014] Accordingly, the object of the present invention is to provide a bispecific antibody labeled with a radioisotope that specifically binds to VLA4 and CD38, and a method for producing the same.

[0015] Another objective of the present invention is to provide a pharmaceutical composition for immuno-PET imaging or radioimmunotherapy of cancer comprising, as an active ingredient, a bispecific antibody labeled with the radioisotope that specifically binds to VLA4 and CD38. means of solving the problem

[0017] According to one aspect of the present invention, the present invention provides a bispecific antibody that specifically binds to VLA4 and CD38, comprising:

[0018] (a) metal chelator;

[0019] (b) a metallic radioisotope coupled to the metal chelator; and

[0020] (c) Heterodimeric bispecific antibody targeting VLA4 and CD38.

[0022] In the present invention, "CD38" is a type II transmembrane glycoprotein called NAD+ glycosidase, and plays a role in inducing the activation, proliferation, and differentiation of mature lymphocytes and mediating apoptosis of bone marrow and lymphoid progenitor cells.

[0023] The aforementioned CD38 is a cell surface receptor equipped with multicatalytic exoenzyme functions, such as ADP-ribose hydrolase and cyclic ADP-ribose hydrolase. In cancer, CD38 participates in cancer cell adhesion, signal transduction, and calcium signaling, and is expressed at high levels in hematological cancer cells, including those of malignant lymphoma, multiple myeloma, and leukemia. Accordingly, antibodies against CD38 are being effectively utilized as markers for detecting these hematological cancers and are also attracting attention as anticancer agents.

[0024] In the present invention, the "VLA-4 (very late antigen-4)" refers to a cell surface receptor composed of an integrin dimer of CD49d (alpha 4; 155 kDa) and CD29 (beta 1; 150 kDa).

[0025] The aforementioned VLA-4 is found in hematopoietic stem cells and progenitor cells and is involved in the adhesion of various immune cells in the bone marrow where these cells are produced. Since the interaction between VLA-4 and its ligand within the tumor microenvironment of the bone marrow contributes to the immune evasion of hematological cancer cells and reduces the efficacy of anticancer drugs, blocking this interaction is considered important for the immunotherapy of hematological cancers.

[0026] As such, CD38 and VLA4 are excellent targets for the treatment of hematological cancers and are highly expressed in cancer cells within an immunosuppressive microenvironment. Although they are not expressed in most normal cells, they are exceptionally expressed relatively weakly in some normal immune cells; therefore, antibody therapies that block these molecules need to enhance their target specificity against hematological cancer cells. Accordingly, bispecific antibodies that simultaneously target CD38 and VLA4 can contribute to the selective detection of tumors expressing these molecules and the advancement of treatment technologies.

[0027] The bispecific antibody of the present invention can be used as a novel anticancer drug in itself. Furthermore, attaching various toxic agents to this antibody forms an antibody-drug conjugate, which can further enhance the anticancer effect. Using a similar strategy, radioisotopes can be attached to this antibody instead of toxic agents to be utilized for cancer diagnosis and treatment.

[0028] to the bispecific antibody of the present invention 89 By attaching imaging radioisotopes such as Zr, immuno-PET scans can be performed to evaluate in vivo pharmacokinetics, enabling the detection of tumors and the non-invasive, quantitative assessment of CD38 and VLA-4 expression. Furthermore, by providing pharmacokinetic information to evaluate the tumor delivery of bispecific antibody drugs, this technology can be utilized to select treatment targets and predict therapeutic effects.

[0029] Also, regarding the bispecific antibody of the present invention 177 Attaching therapeutic radioisotopes such as Lu can simultaneously target CD38 and VLA-4, thereby enhancing the effect of radioimmunotherapy for intractable blood cancers.

[0030] In one embodiment of the present invention, the metal chelator is characterized as being a chelator capable of binding with a metallic radioisotope.

[0031] In this specification, the term "chelator" in the present invention refers to a molecule that forms a complex with a radioisotope, wherein the complex is stable under physiological conditions. More specifically, the chelator may be a molecule that is physiologically stable when synthesized with a radioisotope and has at least one reactive functional group for conjugation with a spacer.

[0032] In one embodiment of the present invention, the metal chelator may be selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriamine pentaacetate (DTPA), and deferoxamine (DFO), but is not limited thereto.

[0033] In the present invention, "radioisotope" or "radionuclide" refers to an atom having an unstable nucleus characterized by an excess of energy that can be utilized to impart to newly generated radioactive particles within the nucleus or through internal conversion. The radioisotope undergoes radioactive decay and emits subatomic particles such as gamma rays or alpha or beta particles. Such emission constitutes ionizing radiation. Radioisotopes may occur naturally or be artificially generated.

[0034] In the present invention, "radiation" refers to a phenomenon in which energy propagates through space or a substance that mediates such propagation, and can be emitted by various radioisotopes.

[0035] In one embodiment of the present invention, the metallic radioisotope may be a metallic radioisotope that emits radiation of alpha rays, beta rays, or gamma rays, or a combination thereof.

[0036] The above radioisotope may be, for example, an alkali metal, an alkaline earth metal, a lanthanoid, an actinoid, a transition metal, or a radioisotope of a metal other than the above metal.

[0037] When using radioactive metal-labeled antibodies for the purpose of treating a disease, α-particle emitting nuclides or β-particle emitting nuclides may be used as radioisotopes to enhance the therapeutic effect.

[0038] In the present invention, "α-particle emitting nuclide" refers to a nuclide that emits α-particles during the process of radioactive metal catastrophism, and more specifically, 212 Bi, 213 Bi, 227 Th, 225 Ac or 211 It may be at, but is not limited to.

[0039] In the present invention, "β-particle emitting nuclide" refers to a nuclide that emits β-particles during the process of radioactive metal catastrophism, and more specifically, 177 Lu, 60 Co, 59 Fe, 64 Cu, 67 Cu, 89 Sr, 90 Y, 103 Ru, 153 Sm, 165 Dy, 166 Ho, 186 Re, 188 Re, 198 Au, 203 Hg, 212 Bi, 213 Bi or 212 It may be Pb, but is not limited thereto.

[0040] In paragraph 1, the metallic radioisotope is 177 Lu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 111 In, 89 Zr, 90 Y, 203 Pb, 212 Pb, 213 Bi, 212 Bi, 227 Th 225 Ac, 105 Rh, 176 Yb, 169 Gd, 111 Ag and 166 It may be any one or more selected from the group consisting of Ho, but is not limited thereto.

[0041] In one embodiment of the present invention, the metallic radioisotope of the present invention is preferably 177 Lu or 89 It could be Zr.

[0042] In one embodiment of the present invention, the metal chelator may be conjugated to the amine group or hinge region cysteine ​​group of the bispecific antibody.

[0043] In the present invention, "conjugation" refers to a reaction in which two compounds are connected through covalent bonds or other chemical bonds. "Conjugate" refers to the combined final product formed through the above conjugation process; in the present invention, it refers to the product formed by the combination of the metal chelator and the bispecific antibody through conjugation, which can be described as a type of antibody-drug conjugate (ADC).

[0044] In the present invention, the "antibody" is a bispecific antibody against VLA4 and CD38, comprising not only the complete antibody form but also an antigen-binding fragment of the antibody molecule.

[0045] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, each light chain being connected to a heavy chain by a disulfide bond.

[0046] The "constant region (C region)" of an antibody is divided into a heavy chain constant region and a light chain constant region; the heavy chain constant region has alpha (α), gamma (γ), mu (μ), delta (δ), and epsilon (ε) types, and has subclasses alpha1 (α1), alpha2 (α2), gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), and gamma4 (γ4). The class of the antibody, such as IgA, IgM, IgD, IgG, and IgE, is determined by the type of the heavy chain constant region. It can be further divided into subclasses based on the types of the alpha (α) chain and gamma (γ) chain. The light chain constant region has kappa (κ) and lambda (λ) types.

[0047] The "variable region (V region)" refers to a domain of the heavy or light chain involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies binding to a specific antigen can be isolated using the VH or VL domain from antibodies that screen a library of VL or VH domains that are complementary to the antigen.

[0048] In the present invention, "antigen-binding fragment" refers to a fragment possessing an antigen-binding function and includes, for example, Fab, F(ab'), F(ab')2, chemically linked F(ab')2 and Fv, etc. Among the antibody fragments, Fab has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Recombinant technology for generating Fv fragments as minimal antibody fragments having only a heavy chain variable region and a light chain variable region is disclosed in PCT international published patent applications WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086, and WO 88 / 09344. Two-chain Fv has a heavy chain variable region and a light chain variable region connected by non-covalent bonds, while single-chain Fv generally has a heavy chain variable region and a single chain variable region connected by covalent bonds through a peptide linker or directly connected at the C-terminus, so they can form a dimer-like structure similar to two-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, limiting the whole antibody with papain yields Fab, and limiting it with pepsin yields F(ab')2 fragment), or they can be produced through genetic recombination technology.

[0049] In the present invention, the antibody may be in the scFv form or in the complete antibody form. Additionally, the heavy chain constant region may be selected from any one isotype among alpha (α), gamma (γ), mu (μ), delta (δ), or epsilon (ε) types, and the light chain constant region may be kappa (κ) or lambda (λ) type.

[0050] In the present invention, "heavy chain" refers to both the full-length heavy chain and its fragments, including a variable region domain VH having an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3.

[0051] In the present invention, "light chain" refers to both the full-length light chain and its fragments, which include a variable region domain VL and a constant region domain CL, comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen.

[0052] In the present invention, the "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region (HVR) of the immunoglobulin heavy chain and light chain. The heavy chains (HCDR1, HCDR2, and HCDR3) and light chains (LCDR1, LCDR2, and LCDR3) each contain three CDRs. The CDRs provide key contact residues for the binding of antibodies to antigens or epitopes.

[0053] In this invention, "Framework (FR)" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of the variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in the following order in VH (or VL):

[0054] (a) FRH1 (Framework region 1 of Heavy chain)-CDRH1 (complementarity determining region 1 of Heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4; and

[0055] (b) FRL1 (Framework region 1 of Light chain)-CDRL1 (complementarity determining region 1 of Light chain)-FRL2-CDRL2-FRL3-CDRL3-FRL4.

[0056] The antibodies of the present invention can also be generated using various phage display methods known in the art, [Brinkman et al., 1995, J. Immunol. Methods, 182:41-50]; [Ames et al., 1995, J. Immunol. Methods, 184, 177-186]; [Kettleborough et al., 1994, Eur. J. Immunol, 24, 952-958]; [Persic et al., 1997, Gene, 187, 9-18]; and [Burton et al.,1994, Adv. Immunol., 57, 191-280]; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; and WO 95 / 20401; and US 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; Includes those posted in 5,733,743; and 5,969,108.

[0057] In the present invention, "specifically binds" or such means that an antibody or its antigen-binding fragment, or other components such as scFvs, form a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10 -6 M or less (e.g., 9 x 10 -7 M, 8 Х 10 -7 M, 7 Х 10 -7 M, 6 X 10 -7 M, 5 X 10 -7 M, 4 Х 10 -7 M, 3 X 10 -7 M, 2 X 10 -7 M, or 1 x 10 -7 M), preferably 1 X 10 -7 M or less (e.g., 9 x 10 -8 M, 8 Х 10 -8 M, 7 Х 10 -8 M, 6 X 10 -8 M, 5 X 10 -8 M, 4 Х 10 -8 M, 3 X 10 -8 M, 2 X 10 -8 M, or 1 x 10 -8 M), more preferably 1 Х 10 -8 M or less (e.g., 9 x 10 -9 M, 8 Х 10-9 M, 7 Х 10 -9 M, 6 X 10 -9 M, 5 X 10 -9 M, 4 Х 10 -9 M, 3 X 10 -9 M, 2 X 10 -9 M, or 1 x 10 -9 The equilibrium dissociation constant of M) (e.g., K smaller than this). d It can be characterized as (indicating a tighter bond). Methods for determining whether two molecules specifically bind are well known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0058] In this specification, the term “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). Unless otherwise specified, as used herein, “binding affinity” refers to the intrinsic binding affinity reflecting the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity between molecule Y and its partner Y is generally given by the dissociation constant (K d It can be expressed as ). Affinity can be measured by conventional methods known in the art, including those described herein.

[0059] In one embodiment of the present invention, the heterodimer bispecific antibody targeting VLA4 and CD38 of the present invention is a bivalent antibody having two antigen recognition sites, and each antigen recognition site targets VLA4 and CD38, respectively.

[0060] In this specification, the term “human antibody” has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody excludes humanized antibodies containing non-human antigen-binding residues.

[0061] In this specification, the term “chimeric” antibody means an antibody in which part of the heavy chain and / or light chain is derived from a specific source or species, and the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0062] In this specification, the term “humanized antibody” refers to a chimeric immunoglobulin containing a minimal sequence derived from a non-human (e.g., mouse) antibody, its immunoglobulin chain, or fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody). In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the recipient’s complementarity-determining region (CDR) are replaced by residues of the CDR of a non-human species (donor antibody), e.g., mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some cases, residues of the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, the humanized antibody may contain residues not found in the recipient antibody or in the imported CDR or framework sequence. Such modifications are made to further improve and optimize antibody performance. Generally, the humanized antibody will comprise at least one, and typically two, substantially all variable domains, wherein all or substantially all of the CDR region in the domain corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR region has the sequence of the FR region of a human immunoglobulin. The humanized antibody comprises at least a portion of the immunoglobulin constant region (Fc region) or a substantial sequence of the human immunoglobulin constant region (Fc region).

[0063] In one embodiment of the present invention, the antibody applied to the present invention may be selected from the group consisting of IgG, IgA, IgM, IgE, and IgD, but is not limited thereto, and preferably may be an IgG antibody.

[0064] In one embodiment of the present invention, the antibody of the present invention may be selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, and preferably may be IgG1 or IgG2.

[0065] In the present invention, the bispecific antibody of the present invention refers to an antibody that specifically binds simultaneously to two antigenic sites of CD38 and VLA-4.

[0066] In one embodiment of the present invention, the heterodimer bispecific antibody targeting VLA4 and CD38 of the present invention comprises a heavy-light chain pair targeting VLA4 and a heavy-light chain pair targeting CD38.

[0067] In a specific embodiment of the present invention, the heterodimer bispecific antibody targeting VLA4 and CD38 of the present invention comprises a heavy-light chain pair of a humanized antibody targeting VLA4 and a heavy-light chain pair derived from a mouse antibody targeting CD38.

[0068] In a specific embodiment of the present invention, the amino acid sequences of the heavy chain (4ky1_H) and light chain (4ky1_L) of a humanized HP1 / 2 FAB clone for human VLA4 (anti-human ITGA4) are as follows ( http: / / www.abysis.org / abysis / searches / search / search_form.cgi?qs=humanized%20HP1 / 2 ):

[0069] 4ky1_H: VQLVQSGAEVKKPGATVKISCKSSGYNIKDVYMHWVQQAPGKGLEWMGRIDPASGDTKYDPKFQVRVTITADTSDTAYMELSSLRSEDTAVYYCATG MWVSTGYALDFWGQGTLVTVSS

[0070] 4ky1_L: DIVMTQSPDSLAVSLGERATINCKASQSVTNDVAWYQQKPGQPPKLLIYYASNRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQDYSSPYTFGQGTKVEIKRTV

[0071] In the present invention, the anti-human CD38 antibody followed the gene sequence for clone OKT10.

[0072] In the present invention, anti-CD38 VHH corresponds to a single domain variable region of the heavy chain and follows the gene sequence for Ab02729 (Catalogue Sequence - Clone C2) of Absolute Antibody.

[0073] In another embodiment of the present invention, the heterodimer bispecific antibody targeting VLA4 and CD38 of the present invention is formed such that a heavy chain-light chain pair targeting VLA4 and a heavy chain-light chain pair targeting CD38 are combined in a knobs-in-hole manner, but is not limited thereto.

[0074] The above-mentioned knobs-in-hole is a technique for designing and producing bispecific antibodies, and is a structural design method that induces heterodimerization of the heavy chain. This method is designed to efficiently generate antibodies capable of binding to two different antigens. Specifically, it involves creating large protrusions ("knobs") through amino acid mutations on one side of the heavy chain (the Fc region) and creating small depressions ("holes") at corresponding locations on the other heavy chain. The knobs and holes are then designed to interlock precisely, thereby inducing a preference for the formation of heavy-chain heterodimers. This method is known to prevent the formation of homodimers and enable the efficient production of bispecific antibodies.

[0075] Methods for producing the heterodimer-type bispecific antibody of the present invention include, but are not limited to, CorssMab, Dual-Variable Domain Antibody (DVD-Ig), Quadroma (Hybrid Hybridoma), Tandem scFv, etc.

[0077] According to another aspect of the present invention, the present invention provides a method for producing a radioisotope-labeled bispecific antibody that specifically binds to VLA4 and CD38, comprising the following steps:

[0078] (a) a step of conjugating a metal chelator to a bispecific antibody that specifically binds to VLA4 and CD38; and

[0079] (b) A step of loading a radioactive isotope by reacting the bispecific antibody conjugated with the metal chelator with a metallic radioactive isotope.

[0081] The present invention is described below in each step.

[0083] Step (a): A step of conjugating a metal chelator to a bispecific antibody

[0084] This step involves conjugating a metal chelator capable of forming a complex with a radioisotope for radioisotope labeling to the bispecific antibody of the present invention.

[0085] In one embodiment of the present invention, the molar ratio of the metal chelator and the bispecific antibody is 150:1 to 5:1, 150:1 to 10:1, 150:1 to 20:1, 150:1 to 50:1, 150:1 to 100:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 50:1, 80:1 to 5:1, 80:1 to 10:1, 80:1 to 20:1, 80:1 to 50:1, 60:1 to 5:1, 60:1 to 10:1, 60:1 to 20:1, 50:1 to 5:1, 50:1 to 10:1, 50:1 to 20:1, 30:1 to 5:1, 30:1 to 10:1, or 30:1 to 20:1, but not limited thereto.

[0086] In one embodiment of the present invention, the metal chelator may be deferoxamine-SCN or DOTA-SCN, but is not limited thereto.

[0087] If the metal chelator is deferoxamine-SCN or DOTA-SCN, the metal chelator is conjugated to the amine group of the antibody.

[0088] In one embodiment of the present invention, the bispecific antibody that specifically binds to VLA4 and CD38 of the present invention may first be reduced for conjugation with a metal chelator.

[0089] The reduction of the above bispecific antibody is to reduce the disulfide bond chain of the hinge region of the antibody.

[0090] In one embodiment of the present invention, the reduction is achieved by reacting a bispecific antibody with a compound selected from the group consisting of TCEP (tris(2-carboxyethyl)phosphine, 2-Mercaptoethylamine·HCl (2-MEA), dithiotreitol, and mercaptoethanol, but is not limited thereto.

[0091] In a specific embodiment of the present invention, the reduction is achieved by reacting with TCEP (tris(2-carboxyethyl)phosphine).

[0092] In one embodiment of the present invention, the metal chelator may be deferoxamine-maleimide or maleimide-DOTA (Maleimido-mono-amide-DOTA, MMA-DOTA), but is not limited thereto.

[0093] When the metal chelator is deferoxamine-maleimide or maleimide-DOTA (Maleimido-mono-amide-DOTA, MMA-DOTA), the metal chelator is conjugated to the cysteine ​​group of the hinge region of the antibody.

[0095] Step (b): A step of loading a metallic radioisotope onto a bispecific antibody conjugated with a metal chelator.

[0096] This step is to prepare a radioactive isotope-labeled bispecific antibody by loading a metallic radioisotope onto the metal chelator-conjugated bispecific antibody prepared in step (a) above.

[0097] In one embodiment of the present invention, the loading of the metallic radioisotope can be carried out under appropriate solvent, pH conditions, temperature, and reaction time conditions.

[0098] In one embodiment of the present invention, the solvent may be a suitable buffer solution to increase the binding efficiency between the metal chelator and the isotope. In a specific embodiment of the present invention, the buffer solution may be a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, but is not limited thereto.

[0099] In one embodiment of the present invention, the pH conditions for loading the metallic radioisotope may generally be at pH 4 to pH 7.5, but are not limited thereto.

[0100] In one embodiment of the present invention, the temperature conditions for loading the metallic radioactive isotope may be carried out at a temperature of 20-50°C, 20-40°C, 20-37°C, 20-35°C, or 20-30°C to strengthen the binding of the radioactive isotope.

[0101] In one embodiment of the present invention, the reaction time condition for loading the metallic radioactive isotope is between 30 minutes and 2 hours, and this may vary depending on the characteristics of the isotope and the chelator.

[0102] In one embodiment of the present invention, the bispecific antibody after the loading reaction is completed may undergo a neutralization and / or chromatographic purification process to remove unnecessary radioactive residues.

[0103] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for radioimmunotherapy of cancer comprising a bispecific antibody as an active ingredient.

[0104] In the present invention, "radioimmunotherapy" refers to a treatment in which a radioisotope is labeled on an antibody, antibody fragment, or immunological binding molecule that specifically binds to a tumor-associated antigen, and the tumor cells are selectively killed through radiation emitted from the radioisotope.

[0105] In one embodiment of the present invention, the radioimmunotherapy comprises a radioisotope to a bispecific antibody that simultaneously binds to VLA-4 and CD38 expressed in tumor cells or the tumor microenvironment. 89 It refers to a treatment method in which Zr is labeled, the antibody selectively accumulates at the tumor site, and then tumor cells are killed through radiation emitted from a radioisotope.

[0106] Unlike conventional radioimmunodiagnostic technology that is limited to imaging tumors using a single target antibody, the present invention can selectively deliver radiation to the tumor site and induce apoptosis of tumor cells by labeling a radioisotope on a bispecific antibody that simultaneously targets VLA-4 and CD38.

[0107] In the present invention, "treatment" includes the suppression of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement of the disease state, remission, alleviation, or improved prognosis, etc., by the administration of the pharmaceutical composition according to the present invention.

[0108] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations and include, but are not limited to, ascorbic acid, sodium ascorbate, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, antioxidants, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0109] The pharmaceutical composition of the present invention may be administered orally or parenterally, for example by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0110] Suitable dosages of the pharmaceutical composition of the present invention vary depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, rate of excretion, and responsiveness, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. As specified herein, the term "pharmaceutical effective dose" means an amount sufficient to prevent or treat the aforementioned disease.

[0111] The pharmaceutical composition of the present invention can be additionally administered in combination with other anticancer agents, and thereby effectively inhibit general cancer cell proliferation and cancer metastasis, etc., and can be used for the treatment or prevention of cancer.

[0112] In the present invention, the anticancer agents include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, cemasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, pertuzumab, trastuzumab-emtansine, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscolumabum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tussetane, heptaplatin, methylaminolevulinic acid, amsacrin, alemtuzumab, procarbazine, alprostadil, and holmium nitrate. Chitosan, Gemcitabine, Doxyfluridine, Pemetrexed, Tegafur, Capecitabine, Gimeracin, Oteracil, Azacitidine, Methotrexate, Uracil, Cytarabine, Fluorouracil, Fludabin, Enositabine, Flutamide, Capecitabine, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Carmoper, Raltitrexed, Docetaxel, Paclitaxel, Irinotecan, Belotecan, Topotecan, Vinorelbine, Etoposide, Vinblastine, Idarubicin, Mitomycin, Bleromycin, Dactinomycin, Pirarubicin, Aclarubicin, Pepromycin, Temsirolimus, Temozolomide, Busulfan, Ifosfamide, Cyclophosphamide, Melphalan, Altretmine, One or more selected from the group consisting of dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutesimide, anagrelide, olaparib, nabelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, lomustine, 5-FU, vorinostat, entinostet, and carmustine may be used, but are not limited thereto.

[0113] In another embodiment of the present invention, the anticancer agent that can be co-administered may be an anti-PD-1 antibody or an anti-PD-L1 antibody. The anti-PD-1 antibody or anti-PD-L1 antibody may be, for example, one or more selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, atezolizumab, durvalumab, and avelumab, but is not limited thereto.

[0114] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0115] In one embodiment of the present invention, the cancer is blood cancer.

[0116] In a specific embodiment of the present invention, the blood cancer is selected from the group consisting of leukemia, multiple myeloma, and lymphoma, but is not limited thereto.

[0118] According to one aspect of the present invention, the present invention provides a pharmaceutical composition for imaging or diagnosing cancer, comprising the aforementioned bispecific antibody as an active ingredient.

[0119] In the present invention, "cancer imaging" refers to the process of diagnosing cancer, establishing a treatment plan, and monitoring follow-up using various imaging technologies such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET).

[0120] In one embodiment of the present invention, the imaging technology of the present invention may be PET (positron emission tomography), but is not limited thereto.

[0121] In a specific embodiment of the present invention, the image of the present invention may be characterized as being obtained by immuno-PET, but is not limited thereto.

[0122] In one embodiment of the present invention, cancer imaging of the present invention refers to a process of visually confirming a cancer lesion using a signal emitted from a radioisotope labeled with a bispecific antibody.

[0123] In one embodiment of the present invention, the immuno-PET refers to a technique in which a radioisotope is labeled on an antibody that specifically binds to a tumor-associated antigen, and then the distribution of the antibody in the body and the pattern of tumor accumulation are visualized through positron emission tomography.

[0124] In the present invention, "cancer diagnosis" means evaluating the presence, extent, or target expression pattern of cancer based on the imaging results.

[0125] The cancer imaging or diagnosis of the present invention may be used for at least one of initial diagnosis, staging, establishment of a treatment plan, evaluation of treatment response, or confirmation of recurrence, but is not limited thereto.

[0126] A method for producing a bispecific antibody according to one embodiment of the present invention, a pharmaceutical composition for radioimmunotherapy comprising the same as an active ingredient, and a pharmaceutical composition for imaging or diagnosing cancer are either methods for producing the bispecific antibody of the present invention as described above or inventions comprising the same as they are; therefore, common components between the two inventions can be applied identically. Furthermore, to prevent complexity in this specification, descriptions of overlapping components are omitted. Effects of the invention

[0128] The bispecific antibody of the present invention can effectively treat cancer as a radioimmunotherapy agent for the treatment and suppression of recurrence of blood cancers expressing VLA4 and CD38, and can also be usefully utilized for imaging diagnosis. Brief explanation of the drawing

[0130] Figure 1 is a schematic diagram illustrating the concept of a process in which a bispecific antibody specifically binding to VLA4 and CD38 and a bispecific antibody loaded with a payload such as a radioisotope binds to VLA4 and CD38 on the surface of a cancer cell, is ingested by the cancer cell, and then releases the payload. FIG. 2a shows a schematic diagram of a crossmab knobs-in-hole construct of a bispecific antibody that specifically binds to VLA4 and CD38 of the present invention. Figure 2b shows the results of confirming the size of the bispecific antibody that specifically binds to VLA4 and CD38, prepared by the method of Figure 2a, using SDS-PAGE and SEC-HPLC. FIG. 3a shows a schematic diagram of a bispecific antibody that specifically binds to VLA4 and CD38 of the present invention, constructed using a Fab / VHH heterodimeric knobs-in-hole construct. Figure 3b shows the results of confirming the size of the bispecific antibodies that specifically bind to VLA4 and CD38, prepared by the method of Figure 3a, using SDS PAGE and SEC-HPLC. Figure 4a shows the results of quantitatively analyzing antigen-antibody binding kinetics using the inverse ELISA technique to evaluate the target-specific binding ability of bispecific antibodies that specifically bind to VLA4 and CD38 constructed with a crossmab knobs-in-hole construct. Figure 4b shows the results of quantitatively analyzing antigen-antibody binding kinetics using the inverse ELISA technique to evaluate the target-specific binding ability of bispecific antibodies that specifically bind to VLA4 and CD38 constructed from the Fab / VHH heterodimeric knobs-in-hole construct. Figure 5a illustrates a process in which the hinge-region cysteine ​​residue of a bispecific antibody that specifically binds to VLA4 and CD38 is reduced to TCEP, thereby enabling the position-specific loading of a payload. Figure 5b shows the results of confirming the change in molecular size by SDS PAGE after TCEP reduction or DFO-SCN conjugation during the process of loading a payload, such as a radioisotope, onto hinge-region cysteine ​​of a bispecific antibody that specifically binds to VLA4 and CD38. Figure 6a shows the amine groups of a bispecific antibody that specifically binds to VLA4 and CD38. 89 It shows a form in which the Zr radioisotope is loaded via DFO chelator conjugation. Figure 6b shows the amine groups of bispecific antibodies that specifically bind to VLA4 and CD38 constructed with a crossmab knobs-in-hole construct. 89 This shows the profile of radioactivity measured by a gamma counter and the labeling efficiency results when Zr was labeled, purified by passing through a PD-10 column, and collected in 0.5 ml fractions. Fig. 6c shows the hinge-region cysteine ​​residue of a bispecific antibody specifically binding to VLA4 and CD38 constructed with the Fab / VHH heterodimeric knobs-in-hole construct. 89 This shows the radioactivity profile and labeling efficiency of the fraction when purified by passing it through a PD-10 column after specifically labeling with Zr. Fig. 6d shows the hinge-region cysteine ​​residue of a bispecific antibody specifically binding to VLA4 and CD38 constructed with the Fab / VHH heterodimeric knobs-in-hole construct. 89 This shows the results of autoradiography analysis of the fractions after purification by passing them through a PD-10 column following specific labeling with Zr. Figure 7a shows the amine groups of bispecific antibodies that specifically bind to VLA4 and CD38 constructed with a crossmab knobs-in-hole construct. 89 This shows the results of evaluating in vitro stability by analyzing with radio-iTLC after labeling with Zr and incubating in PBS (left) or FBS (right) for several days. Figure 7b shows the hinge-region cysteine ​​residue of a bispecific antibody specifically binding to VLA4 and CD38 constructed with the Fab / VHH heterodimeric knobs-in-hole construct. 89 This shows the results of evaluating in vitro stability by radio-iTLC analysis after specifically labeling with Zr and incubating in PBS (left) or FBS (right) for several days. Figure 8 shows the results of confirming the expression levels of VLA4 and CD38 antigens in RPMI8266, MM1S, and U266 human multiple myeloma cancer cells, and HL60 and MOLT4 human acute leukemia cancer cells by western blotting. Figure 9a shows the amine groups of bispecific antibodies that specifically bind to VLA4 and CD38 constructed with a crossmab knobs-in-hole construct. 89 This is the result of comparing the degree of binding when human MOLT4 T cell leukemia cancer cells were reacted with Zr-labeled cells (‡P <0.001). FIG. 9b is of FIG. 9a above.89 The degree of binding when bispecific antibodies specifically binding to Zr-labeled VLA4 and CD38 are reacted with RPMI multiple myeloma cancer cells (left), and the binding specificity inhibited when 0.5 μM of VLA4 antibody or both antibodies are administered simultaneously (right) (**P <0.01; ‡P <0.001). Fig. 10a shows the hinge-region cysteine ​​residue of a bispecific antibody specifically binding to VLA4 and CD38 constructed with the Fab / VHH heterodimeric knobs-in-hole construct. 89 This indicates the degree of binding when MOLT4 human T cell leukemia cancer cells are reacted after being specifically labeled with Zr. FIG. 10b is of FIG. 10a above. 89 This shows the effect of temperature difference on cell binding and the degree of binding when blocking with 0.5 μM cold unlabeled antibody when Zr-labeled bispecific antibodies were reacted with MOLT4 cancer cells. FIG. 10c is of FIG. 10a above. 89 When Zr-labeled bispecific antibodies were reacted with human MM.S1 and U266 human multiple myeloma cancer cells, they exhibited binding specificity that was inhibited by co-administration of CD38 antibody, VLA4 antibody, or a combination thereof (*P <0.05; †P <0.005; ‡P <0.001). Fig. 9d 89 This shows the effect of Zr-labeled bispecific antibodies on the degree of binding when retinoic acid is treated to HL60 human acute leukemia cancer cells with low CD38 expression. (*P <0.05; †P <0.005; ‡P <0.001). Figure 11 shows the amine groups of bispecific antibodies that specifically bind to VLA4 and CD38 constructed with a crossmab knobs-in-hole construct. 89These are the results of confirming the body distribution and tumor uptake rate 7 days after administration when Zr-labeled and intravenously injected into a mouse model transplanted with MOLT4 human T lymphoma. (†P <0.005). Fig. 12a shows the hinge-region cysteine ​​residue of a bispecific antibody specifically binding to VLA4 and CD38 constructed with the Fab / VHH heterodimeric knobs-in-hole construct. 89 This shows the results of confirming the distribution in the body and lymphoma uptake rate 2, 4, or 7 days after administration when Zr was specifically labeled and intravenously injected into a mouse model transplanted with SUDHL1 human lymphoma (‡P <0.001). FIG. 12b is of FIG. 12a above. 89 The results of confirming the distribution and tumor uptake rate in the body on day 7 after intravenous injection of a Zr-labeled bispecific antibody into a MOLT4 lymphoma mouse model (‡P <0.001). FIG. 13 is of FIG. 12a above. 89 This shows a positron emission tomography (PET) image acquired on day 7 after transplantation in a mouse model in which a MOLT4 tumor injected with a Zr-labeled bispecific antibody was transplanted. Figure 14 shows hinge-region cysteine ​​residues in a bispecific antibody specifically binding to VLA4 and CD38 constructed with a Fab / VHH heterodimeric knobs-in-hole construct. 177 This shows the profile and labeling efficiency results obtained by collecting 0.5 ml fractions and measuring the radioactivity with a gamma counter when purifying by passing through a PD-10 column after specifically labeling Lu (left), and the in vitro labeling stability when incubated in FBS (right). Specific details for implementing the invention

[0131] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0133] Examples

[0135] Throughout this specification, “%” used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0137] Example 1: Preparation of a bispecific antibody specifically binding to VLA4 and CD38

[0138] The inventors intended to develop a VLA4 and CD38 bispecific antibody that simultaneously targets VLA4 and CD38 antigens. The schematic form thereof and the concept of the process in which the bispecific antibody, which specifically binds to VLA4 and CD38 carrying a payload, binds to VLA4 and CD38 on the surface of cancer cells and is then ingested by the cancer cells to release the payload are shown in Fig. 1.

[0140] Example 1-1: Preparation of a bispecific antibody specifically binding to VLA4 and CD38 in the form of crossmab knobs-in-holes

[0141] The method for preparing bispecific antibodies against VLA4 and CD38 utilized recombinant IgG synthesis technology widely known in the art.

[0142] The crossmab knobs-in-hole form corresponds to a general bispecific antibody production technique, and a bispecific antibody that specifically binds to VLA4 and CD38 was produced using the above method, and a schematic diagram of the antibody is shown in Fig. 2.

[0143] The Fc backbone of the antibody was based on the mouse IgG2a sequence. For the antibody portion against VLA4, the gene sequences of the heavy chain (4ky1_H) and light chain (4ky1_L) of the humanized HP1 / 2 FAB clone against anti-human ITGA4 were utilized. For the antibody portion against CD38, the gene sequence of the widely known clone OKT10 was used.

[0145] Examples 1-2: Preparation of bispecific antibodies specifically binding to Fab / VHH heterodimeric knobs-in-hole forms of VLA4 and CD38

[0146] A bispecific antibody that specifically binds to VLA4 and CD38 in the form of Fab / VHH heterodimeric knobs-in-hole was also prepared using recombinant IgG synthesis technology widely known in the art, and a schematic diagram of the antibody is shown in Fig. 3.

[0147] The antibody portion against VLA4 utilized the gene sequences of the heavy chain (4ky1_H) and light chain (4ky1_L) of the humanized HP1 / 2 FAB clone against anti-human ITGA4. The antibody portion against CD38 utilized the VHH form corresponding to the single domain variable region of the heavy chain, which utilized the gene sequence of Absolute Antibody's Ab02729 (catalogue Sequence - clone C2).

[0149] Examples 1-3: ELISA analysis of VLA4 and CD38 antigen binding of bispecific antibodies specifically binding to VLA4 and CD38

[0150] To evaluate the target-specific binding ability of the bispecific antibodies that specifically bind to VLA4 and CD38 prepared in Examples 1-1 and 1-2, the inventors coated an Immuno plate with integrin α4β1 corresponding to purified antigens CD38 and VLA4 at a concentration of 1 μg / mL and then performed an inverse ELISA analysis.

[0151] Specifically, 100 μL of antigen at a concentration of 1 μg / mL was dispensed onto the plate at a rate of 1 μg / mL per well, and the plate was coated by reacting at 4°C for 19 hours. After coating, the solution was removed from the plate, and washing was performed three times for 5 minutes each with 200 μL of washing buffer (0.1% Tween 20 in PBS).

[0152] Next, 200 μL of Blocking Buffer (1% BSA in PBS) was added, and the mixture was incubated at room temperature for 2 hours. After 2 hours, the solution was removed from the plates. Then, 100 μL of the first-stage VLA4 and CD38 co-targeting bispecific antibody, diluted half-to-half in dilution buffer, was added, and the mixture was incubated at room temperature for 1 hour. Upon completion of the reaction, the solution was removed from the plates, and washing was performed three times with washing buffer for 5 minutes each. Subsequently, 100 μL of the second-stage anti-mouse-HRP conjugated antibody, diluted in PBS, was added, and the mixture was incubated at room temperature for 1 hour.

[0153] After removing the solution from the plate by repeating the washing buffer three times for 5 minutes, 100 μL of Ultra-TMB solution (Thermo Scientific, #34028) was added and reacted for 5 to 10 minutes to induce color development. Then, 100 μL of stop solution (Sigma, #S5814) was added to terminate the reaction, and the absorbance was measured at a wavelength of 450 nm using a spectrophotometer. The measured values ​​were analyzed for the equilibrium dissociation constant (Kd) using Graphpad Prism 8.0 software.

[0154] The experimental results are shown in Figures 4a and 4b.

[0155] As shown in Figure 4a, the ELISA analysis results showed that the isotype control antibody exhibited only very low levels of non-specific binding. The bispecific antibody, which specifically binds to VLA4 and CD38 in the form of crossmab knobs-in-holes, was found to bind to the CD38 antigen coated on the plate with a high affinity of 1.713 and an equilibrium dissociation constant (Kd) of 9.175 nM. Additionally, it was found to bind to the coated VLA4 antigen with a high affinity of 1.898 and an equilibrium dissociation constant (Kd) of 14.8 nM.

[0156] In addition, as shown in Figure 4b, the binding affinity of a bispecific antibody that specifically binds to Fab / VHH heterodimeric knobs-in-hole VLA4 and CD38 was analyzed, and it was confirmed that the antibody bound to the CD38 antigen coated on the plate with a high affinity of a B max value of 2.21 and an equilibrium dissociation constant (Kd) of 0.5559 nM. Furthermore, it was confirmed that the antibody bound to the VLA4 antigen coated on the plate with a high affinity of a B max value of 1.93 and an equilibrium dissociation constant (Kd) of 4.325 nM.

[0158] Therefore, these results show that both the bispecific antibody specifically binding to VLA4 and CD38 constructed with the crossmab knobs-in-hole construct and the bispecific antibody specifically binding to VLA4 and CD38 constructed with the Fab / VHH heterodimeric knobs-in-hole construct bound to purified human VLA4 and CD38 with high affinity, thus demonstrating that they are successful antibodies with bispecificity.

[0160] Example 2. 89 Synthesis of a bispecific antibody specifically binding to VLA4 and CD38 loaded with Zr radioisotope

[0161] It is possible to conjugate a payload that aids in imaging or treatment to the amine group or the hinge region cysteine ​​group of the bispecific antibody of the present invention. For example, by loading a radioisotope, it is possible to detect the antibody or induce radioactive exposure in cells that have ingested the antibody.

[0162] In this embodiment, for video 89 It was confirmed that by loading the Zr radioisotope, it specifically binds to blood cancer cells that highly express VLA4, CD38, or a combination thereof, and excellent and specific images of blood cancer tumors can be obtained when scanned by immuno-PET.

[0163] In the following, imaging for the bispecific antibody that simultaneously targets VLA4 and CD38 according to the present invention 89 Zr and therapeutic 177 The method for loading Lu radioisotopes, binding to cells, and analyzing tumor uptake in a mouse model was described in detail.

[0165] Example 2-1. Deferoxamine-NHS conjugation to the amine groups of a bispecific antibody simultaneously targeting VLA4 and CD38

[0166] The inventors conjugated deferoxamine[DFO]-SCN to an amine group to load a Zr-89 radioisotope onto a bispecific antibody that simultaneously targets VLA4 and CD38 in the form of cross-mab knobs-in-holes.

[0167] First, 1 mg of the above-mentioned bispecific antibody was reacted with 2 mg / ml DFO-Bz-SCN stock (1:10 molar ratio) at 37°C for 1 hour after replacing the buffer with 0.1 M NaHCO3 solution using a spin column to attach a chelator to the amine group of the antibody.

[0168] Subsequently, the reaction product was eluted using a PD-10 column with a 0.25 M sodium acetate (pH 5.5) solution containing 0.5% gentisic acid to obtain a fraction of DFO-conjugated bispecific antibodies. At this time, the molar ratio of DFO-SCN to antibodies was 10:1. The obtained fraction of DFO-conjugated bispecific antibodies was concentrated using a 100K amicon filter and then used for loading the Zr-89 radioisotope.

[0170] In addition, the inventors conjugated DOTA-SCN to an amine group to load the Lu-177 radioisotope onto a bispecific antibody that simultaneously targets VLA4 and CD38 in the form of cross mab knobs-in-holes.

[0171] Specifically, 1 mg of the bispecific antibody was reacted with 100 mM p-SCN-Bn-DOTA stock (1:100 molar ratio) overnight at room temperature using a spin column after exchanging the buffer with 0.1 M Na2CO3 solution to attach a chelator to the amine group of the antibody. Subsequently, the reaction product was eluted with 1X PBS using a PD-10 column to obtain a fraction of the DOTA-conjugated bispecific antibody. At this time, the molar ratio of DOTA-SCN to antibody was 100:1. The obtained DOTA-conjugated bispecific antibody fraction was concentrated using a 100K amicon filter and subsequently used for loading the Lu-177 radioisotope.

[0173] Example 2-2. Reduction of VLA4xCD38 BsAb and Cysteine-Specific Deferoxamine-Maleimide Conjugation

[0174] The inventors conjugated deferoxamine[DFO]-maleimide site-specifically to sulfohydryl residues within cysteine ​​residues to load the Zr-89 radioisotope onto a bispecific antibody targeting VLA4 and CD38 in the form of Fab / VHH heterodimeric knobs-in-holes.

[0175] To this end, a bispecific antibody simultaneously targeting VLA4 and CD38 is reduced to TCEP at a molar concentration of 70 to 130 times or more and then reacted with a deferoxamine-maleimide chelator, thereby [attaching] the disulfide binding site of the antibody hinge region 89 It was possible to specifically bind Zr atoms, and the specific method is as follows.

[0177] First, 2 mg of the above bispecific antibody was reduced by reacting it with 100 mM TCEP (1:100 molar ratio) at room temperature for 20 minutes. Subsequently, the reduced bispecific antibody was diluted in a 0.1 M sodium phosphate solution containing 150 mM NaCl and 1 mM EDTA (ethylene diamine tetraacetic acid). 56.4 μL of 2 mM deferoxamine-maleimide (DFO-Mal) chelator was added to this solution and reacted at room temperature for 1 hour to attach to the sulfohydryl residues of the cysteine ​​residues in the hinge region of the bispecific antibody.

[0178] The reaction product was eluted with a PD-10 column to concentrate the fraction of DFO-conjugated bispecific antibody. DFO-conjugated IgG was obtained from the fraction. At this time, the molar ratio of deferoxamine-maleimide to antibody was 60:1.

[0180] Next, the reduction status of the obtained DFO-conjugated bispecific antibody was analyzed using the SDS-PAGE (Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis) method.

[0181] Specifically, 2 μg of DFO-conjugated bispecific antibody was diluted in water, mixed with 5x non-reducing sample buffer with dithiothreitol removed, boiled at 95°C for 10 minutes, and then electrophoresis was performed on an 8% SDS PAGE gel and stained with 0.5% Coomassie blue.

[0182] The result is shown in Fig. 5b.

[0183] As shown in Figure 5b, it was confirmed that there was no difference in the change in molecular size after TCEP reduction and after DFO-SCN conjugation.

[0185] Example 2-3. To deferoxamine-conjugated VLA4xCD38 BsAb 89 Zr equipped

[0186] The dual-specific antibodies that specifically bind to DFO-conjugated VLA4 and CD38 prepared in Examples 2-1 and 2-2 above 89 Reacted with Zr-oxalate at room temperature, as shown in Fig. 6a 89 We synthesized a bispecific antibody that specifically binds to Zr-loaded VLA4 and CD38.

[0187] first, 89 Zr-oxalate (50 μL; Korea Atomic Energy Research Institute) was neutralized with 25 μL of 2 M Na2CO3. Then, the DFO-conjugated bispecific antibody was diluted in 75 μL of 0.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5), and then89 The reaction was labeled by reacting with Zr-oxalate and incubating at room temperature for 1 hour. Using a 0.25 M sodium acetate solution containing 0.5% gentisic acid as the elution buffer, the reaction product was purified by size-exclusion chromatography with a PD-10 column as shown in Figures 6b and 6c. Finally, 89 The fraction in which Zr-labeled BsAb eluted was separated and purified. As a result of gamma counting the eluted fractions, in the crossmab knobs-in-hole and Fab / VHH heterodimeric knobs-in-hole bispecific antibodies appearing at the first radioactivity peak 89 The Zr labeling efficiencies were 60.8% and 66.8%, respectively.

[0189] The above-mentioned obtained 89 For the Zr-loaded bispecific antibody, we confirmed whether the antibody was properly labeled through autoradiography. For autoradiography, first 89 Zr-bispecific antibodies were separated using 8% native PAGE and then exposed to a photosensitive plate.

[0190] The results of the autoradiography experiment are shown in Fig. d.

[0191] As shown in Fig. 5d, a person or mouse 89 A radioactive IgG band was detected at the expected 170kD position for Zr-VLA4xCD38BsAb.

[0193] Example 3. 89 In vitro labeling stability of Zr-loaded bispecific antibodies specifically binding to VLA4 and CD38

[0194] The final obtained in Examples 2-3 above 89When the bispecific antibodies that specifically bind to Zr-labeled VLA4 and CD38 were analyzed by radio-iTLC (radio-instant thin layer chromatography), the radiochemical purity was over 98%.

[0195] 89 When Zr-VLA4xCD38 BsAb was added to PBS or FBS and reacted at 37°C for 5 to 7 days, and then in vitro label stability was analyzed by radio-iTLC using 50 mM ethylene diamine tetraacetic acid (EDTA, pH 5.5) as a solvent, the intact (Intact) 89 While Zr-VLA4xCD38BsAb remains at the basal position, the free 89 Zr4+ ions and 89 Zr-EDTA moved along the solvent front and was separated.

[0196] As a result, as shown in Figures 7a and 7b, on VLA4xCD38 bispecific antibodies in the crossmab knobs-in-hole and Fab / VHH heterodimeric knobs-in-hole forms in PBS and FBS 89 Zr labeling was stable and exceeded 70% in PBS and FBS up to day 7 (n = 2).

[0198] Example 4. In blood cancer cells 89 Uptake kinetics of Zr-VLA4xCD38 BsAb

[0199] Example 4-1. Confirmation of VLA4 and CD38 expression levels by cancer cell type and establishment of a blood cancer cell model

[0200] The inventors intended to confirm the expression of VLA4 and CD38 markers on the surface of various types of human blood cancer cell lines using western blotting with anti-VLA4 IgG and anti-CD38 IgG.

[0201] Specifically, the above-mentioned cancer cells were lysed to isolate proteins, and the proteins were quantified. 15 μg of each protein was subjected to electrophoresis on a 10% gel, transferred to a membrane, and reacted overnight at 4°C with VLA4 IgG and CD38 IgG primary antibodies (1:1000 dilution). Subsequently, the membrane was washed three times for 10 minutes each with TBST buffer, and then reacted with HRP-conjugated anti-rabbit secondary antibody (1:2000 dilution) at room temperature for 1 hour. After washing three times again for 10 minutes each with TBST buffer, the membrane was reacted with an enhanced chemiluminescence substrate, and the film was exposed to detect and quantify the protein band intensities.

[0202] The result is shown in Fig. 8.

[0203] As shown in Figure 8, very high VLA4 expression was confirmed in MM1S and U266 human multiple myeloma cancer cell lines and HL60 and MOLT4 human acute leukemia cancer cells. Meanwhile, very high CD38 expression was confirmed in RPMI8266 and U266 human multiple myeloma cancer cell lines and MOLT4 human acute leukemia cancer cells, and these were used as cell models.

[0205] Example 4-2. In a blood cancer cell model 89 Cell uptake kinetics of Zr-VLA4xCD38 BsAb

[0206] The inventors used MOLT4 cancer cells with high VLA4 and CD38 expression in vitro 89 The binding specificity of Zr-VLA4xCD38 BsAb was analyzed.

[0207] First, after culturing the above cells, the present invention 89After reacting with Zr-VLA4xCD38 BsAb, the cells were washed, and the uptake rate was measured in %ID units using a gamma counter in the conventional manner. In some cells, binding was inhibited with large amounts of cold anti-VLA4 and anti-CD38 antibodies, or both antibodies (binding inhibition experiment), and binding specificity was quantified as % blocking.

[0208] The results are shown in FIGS. 9a and 9b, and FIGS. 10a to 10d.

[0209] As shown in Figures 9a and 10a, in MOLT4 cancer cells with very high VLA4 expression, both the cross mab knobs-in-hole bispecific antibody and the Fab / VHH heterodimeric knobs-in-hole bispecific antibody showed very high VLA4 target-specific binding, with uptake by the VLA4 antibody severely inhibited to less than 20% of the control group.

[0210] Furthermore, cell binding was further inhibited when VLA4 and CD38 antibodies were present simultaneously, and this target-specific cell uptake was not significantly affected by the reaction temperature with the cells.

[0211] In addition, as shown in Figures 9b and 10b, in the case of RPMI8266 cancer cells with very low VLA4 expression, the uptake of the cross-mab knobs-in-hole bispecific antibody was lower than 20% of that of MOLT4 cells. While the inhibition of uptake by the VLA4 antibody was weak, uptake was more strongly inhibited when the CD38 antibody or both antibodies were present.

[0212] In addition, as shown in Fig. 10c, MM1S and U266 human multiple myeloma cancer cells, which have high VLA4 and CD38 expression similar to MOLT4 cancer cells, also showed results of uptake inhibition in the form of Fab / VHH heterodimeric knobs-in-hole in a pattern similar to that of MOLT4 cancer cells.

[0213] In the case of HL-60 cancer cells with low CD38 expression at baseline, when treated with retinoic acid (ATRA) which increases CD38 expression, the uptake of the VLA4xCD38 bispecific antibody significantly increased as CD38 expression increased compared to the cell group not treated with ATRA (Fig. 9d).

[0215] Example 5. In a mouse blood cancer model 89 Pharmacokinetics and In vivo distribution of Zr-VLA4xCD38 BsAb

[0216] To create a mouse tumor model, MOLT4 human T leukemia cancer cells were cultured, and 1 x 10 of the cells were placed on the shoulder site of immunodeficient balb / C nude mice. 7 The dogs were injected subcutaneously. A 1 cm tumor formed approximately 21 days after transplantation. In the tail veins of these mice (n = 6) in which the tumors formed 89 After 5 days of injecting Zr-VLA4xCD38 BsAb, the animals were sacrificed by oral dislocation, and tumors, blood, and major organs were extracted to measure radioactivity levels.

[0217] The experimental results are shown in Figure 11.

[0218] As shown in FIG. 11, in a mouse model implanted with the MOLT4 tumor, 89When Zr-loaded cross-mab knobs-in-hole bispecific antibodies were intravenously administered and the subjects were sacrificed 7 days later to measure uptake by organ, the antibody uptake in the tumor reached 3.03 ± 0.33 %ID / g. This corresponded to a high uptake, 5.03 times the amount of radioactivity remaining in the blood, which was 0.60 ± 0.13 %ID / g. Spleen uptake was slightly higher than that of the tumor, and liver uptake was similar to that of the tumor, but uptake in the heart, lungs, stomach, kidneys, and bones was lower than that of the tumor, and muscle uptake was significantly lower at 0.19 ± 0.03 %ID / g.

[0219] Furthermore, after first injecting 0.8 mg of unlabeled VLA4 antibody into the mouse model, 89 When Zr-VLA4xCD38 BsAb was injected, BsAb uptake in MOLT4 tumors was significantly reduced to 1.87 ± 0.25 %ID / g, which corresponded to inhibited uptake of 61.7% (n = 3, P <0.005, see Fig. 11a).

[0220] These results 89 This means that Zr-VLA4xCD38BsAb has high binding specificity.

[0221] In contrast, in blood and other organs, due to unlabeled antibodies 89 There was no change in Zr-VLA4xCD38 BsAb intake.

[0222] Accordingly, the present invention 89 It was confirmed that Zr-VLA4xCD38BsAb exhibits excellent tumor-specific targeting and binding effects in vivo.

[0224] Next, 89A Zr-loaded Fab / VHH heterodimeric knobs-in-hole bispecific antibody was intravenously injected into a mouse model implanted with a SUDHL1 tumor having low expression of the target antigen, and changes in its distribution in the body were investigated on a daily basis after 2, 4, and 7 days, and the results are shown in Figure 12a.

[0225] As shown in Figure 12a, the amount of radioactivity in the blood gradually decreased day by day, and at the same time, tumor uptake gradually increased, so that on the 7th day, tumor uptake was more than twice as high as that of the blood. Liver uptake was similar to tumor uptake, but on the 7th day, uptake of the heart, lungs, stomach, kidneys, etc., was lower than tumor uptake, and muscle uptake was significantly lower.

[0226] Next, the above 89 We wanted to determine the distribution and lymphoma uptake rate of Zr-VLA4xCD38 BsAb when injected into a mouse model implanted with a MOLT4 tumor, and the results are shown in Figure 12b.

[0227] As shown in Figure 12b, the antibody uptake of the tumor reached 5.35 ± 1.67 %ID / g, which is 3.69 times the amount of radioactivity remaining in the blood, 1.45 ± 0.33 %ID / g. At this time, the uptake of all organs, including the spleen and liver, was lower than that of the tumor.

[0228] Furthermore, after first injecting 0.8 mg of unlabeled VLA4 antibody into the above mouse model, the present invention 89 When Zr-VLA4xCD38 BsAb was injected, BsAb uptake in MOLT4 tumors was 1.21 ± 0.21 %ID / g, which was significantly reduced to 77.4% of the control group (n = 5, ‡P <0.001).

[0229] These results 89 This means that Zr-VLA4xCD38BsAb has high binding specificity.

[0231] Example 6. In a mouse blood cancer model 89 Immuno-PET / CT imaging of Zr-VLA4xCD38 BsAb

[0232] In the mouse model implanted with the above MOLT4 tumor 89 PET / CT images were acquired using an Inveon micro PET / CT scanner for small animals 7 days after intravenously injecting a cross-mab knobs-in-hole bispecific antibody loaded with Zr.

[0233] At this time, by pre-injecting some mice with an unlabeled VLA antibody or CD38 antibody at a 5:1 molar ratio 1 hour prior, the present invention 89 Target-specific tumor uptake of Zr-VLA4xCD38 BsAb was confirmed.

[0234] The results of the micro-PET / CT imaging are shown in Figure 13.

[0235] As shown in Fig. 13, high with excellent contrast in the tumor site 89 Zr-VLA4xCD38BsAb uptake was demonstrated. In addition, 0.8 mg of cold unlabeled VLA antibody was first injected into the same mouse model, and then 89 When Zr-VLA4xCD38BsAb was injected, tumor uptake was significantly reduced, and contrast was observed to decrease.

[0237] Example 7. DOTA-bonded VLA4xCD38 BsAb 177 Lu equipped

[0238] The DFO-conjugated VLA4xCD38 BsAb prepared in Examples 2-1 and 2-2 above 177 Reacted with Lu-oxalate at room temperature, as shown in Fig. 13 177 VLA4xCD38 BsAb loaded with Lu was synthesized.

[0240] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A bispecific antibody specifically binding to VLA4 and CD38 comprising: (a) a metal chelator; (b) a metallic radioisotope bound to said metal chelator; and (c) a heterodimer bispecific antibody targeting VLA4 and CD38. Claim 2 A bispecific antibody according to claim 1, characterized in that the metal chelator is a chelator capable of binding to a metallic radioisotope. Claim 3 A bispecific antibody according to claim 1, wherein the metal chelator is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriamine pentaacetate (DTPA), and deferoxamine (DFO). Claim 4 In paragraph 1, the metallic radioisotope is 177 Lu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 111 In, 89 Zr, 90 Y, 203 Pb, 212 Pb, 213 Bi, 212 Bi, 227 Th 225 Ac, 105 Rh, 176 Yb, 169 Gd, 111 Ag and 166 A bispecific antibody characterized by being one or more selected from the group consisting of Ho. Claim 5 A bispecific antibody according to claim 1, wherein the metal chelator is conjugated to the amine group or hinge region cysteine ​​group of the bispecific antibody. Claim 6 In claim 1, the heterodimer bispecific antibody targeting VLA4 and CD38 is a bivalent antibody having two antigen recognition sites, wherein each antigen recognition site targets CD38 and VLA4, respectively. Claim 7 The bispecific antibody according to claim 1, wherein the heterodimer type bispecific antibody targeting VLA4 and CD38 comprises a heavy-light chain pair targeting CD38 and a heavy-light chain pair targeting VLA4. Claim 8 In claim 1, the heterodimer bispecific antibody targeting VLA4 and CD38 is a bispecific antibody in which a heavy chain-light chain pair targeting CD38 and a heavy chain-light chain pair targeting VLA4 are combined in a knobs-in-hole manner. Claim 9 A method for preparing a bispecific antibody labeled with a radioisotope that specifically binds to VLA4 and CD38, comprising the following steps: (a) conjugating a metal chelator to a bispecific antibody that specifically binds to VLA4 and CD38; and (b) reacting the bispecific antibody conjugated with the metal chelator with a metallic radioisotope to load the radioisotope. Claim 10 A method of preparation according to claim 9, wherein the metal chelator is deferoxamine-SCN or DOTA-SCN, and the metal chelator is conjugated to the amine group of the antibody. Claim 11 A method of preparation according to claim 9, wherein the bispecific antibody specifically binding to VLA4 and CD38 is in a reduced state, the metal chelator is deferoxamine-maleimide or maleimide-DOTA (Maleimido-mono-amide-DOTA, MMA-DOTA), and the metal chelator is conjugated to the cysteine ​​group of the hinge region of the antibody. Claim 12 A method of preparation according to claim 11, wherein the reduction is to reduce the disulfide bond chain of the hinge region of the antibody. Claim 13 A method of preparation according to claim 11, wherein the reduction is achieved by reacting a bispecific antibody with a compound selected from the group consisting of TCEP (tris(2-carboxyethyl)phosphine, 2-Mercaptoethylamine·HCl (2-MEA), dithiotreitol, and mercaptoethanol. Claim 14 A pharmaceutical composition for radioimmunotherapy of cancer comprising, as an active ingredient, a bispecific antibody of any one of claims 1 to 8. Claim 15 A pharmaceutical composition for radioimmunotherapy of cancer, wherein the cancer is blood cancer, in accordance with claim 14. Claim 16 A pharmaceutical composition for radioimmunotherapy of cancer according to claim 15, wherein the blood cancer is selected from the group consisting of leukemia, multiple myeloma, and lymphoma. Claim 17 A pharmaceutical composition for imaging or diagnosing cancer, comprising as an active ingredient a bispecific antibody according to any one of claims 1 to 8.

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