Pharmaceutical compositions for treating tumors

Abstract

To provide a pharmaceutical composition for treating tumor.SOLUTION: A pharmaceutical composition comprises a molecular construct, which comprises an anti-CD38 antibody or a plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules bound to the anti-CD38 antibody. According to some embodiments of the present disclosure, the administration of the pharmaceutical composition gives rise to an effective amount of the lenalidomide molecules or the hydrolyzed lenalidomide molecules that is at least 1,000 times less than an effective amount of the lenalidomide molecule used alone or in combination with the anti-CD38 antibody for the treatment of tumor.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Cross-reference of related applications This application relates to and claims the interests of U.S. Provisional Patent Application No. 63 / 459249, filed on 13 April 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates, in general, to the field of tumor treatment. More specifically, this disclosure relates to a method of treating tumors by using an anti-CD38 antibody and a molecular construct comprising multiple lenalidomide molecules or hydrolyzed lenalidomide molecules. [Background technology]

[0003] Multiple myeloma (MM), also known as myeloma or plasma cell myeloma, is a type of bone marrow cancer in which cancerous plasma cells accumulate in the bone marrow, displacing normal blood cells and resulting in excessive monoclonal paraproteins (M proteins; abnormal antibodies), bone destruction, and replacement of other hematopoietic cell lines. Because cancerous plasma cells affect various parts of the body, the symptoms and signs of multiple myeloma vary greatly from patient to patient. Common symptoms and signs associated with multiple myeloma include bone pain, fractures, spinal cord compression, anemia, recurrent infections, hypercalcemia, abnormal bleeding, blood viscosity, fatigue, renal impairment, and neurological disorders.

[0004] The RVd regimen, namely the combination of bortezomib (VELCADE®), lenalidomide (Revlimid®), and dexamethasone, is often used as a first-line treatment for multiple myeloma. Reports indicate that over 90% of myeloma patients respond well to this treatment. However, this treatment merely reduces the number of cancerous cells in the patient's bone marrow, thereby alleviating the symptoms of multiple myeloma; it does not cure the underlying disease. Furthermore, high doses of lenalidomide or other drugs in the combination regimen usually cause serious adverse effects (e.g., bleeding, dyspnea or labored breathing, thromboembolism, neutropenia, thrombocytopenia, fever, seizures, arrhythmias, speech and motor problems, and confusion). Since most multiple myeloma patients are elderly (50-70 years old), severe side effects are a concern.

[0005] In light of the above, there is a need for novel methods for treating multiple myeloma in related technologies. [Overview of the Initiative]

[0006] The following provides a simplified overview of this disclosure to give readers a basic understanding. This overview is not a comprehensive overview of this disclosure, nor does it identify the main / essential elements of the invention or define its scope. Its sole purpose is to present some of the concepts disclosed herein in a simplified form as an introduction to the more detailed explanations that will follow.

[0007] As concretely and extensively described herein, one aspect of the present disclosure is directed to a method for treating a tumor in a subject. This method involves administering to a subject a molecular construct comprising an anti-CD38 antibody and a plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules conjugated to the anti-CD38 antibody.

[0008] According to some embodiments of the present disclosure, administration of the molecular construct achieves an effective amount of lenalidomide molecule or hydrolyzed lenalidomide molecule that is at least one thousandth of the effective amount of the lenalidomide molecule used alone or in combination with an anti-CD38 antibody for the treatment of tumors. In certain preferred embodiments, the effective amount of the lenalidomide molecule or hydrolyzed lenalidomide molecule is about one ten-thousandth of the effective amount of the lenalidomide molecule used alone or in combination with an anti-CD38 antibody for the treatment of tumors.

[0009] Preferably, the molecular construct is administered to the subject in an amount of about 0.01 to 100 mg / Kg body weight per administration; more preferably, about 0.1 to 10 mg / Kg body weight per administration. According to some preferred embodiments, the molecular construct is administered to the subject once every four weeks.

[0010] According to certain embodiments, the anti-CD38 antibody comprises a pair of CH2-CH3 segments of immunoglobulin G (IgG) and a pair of anti-CD38 single-chain variable fragments (scFv) respectively bound to the N-terminus of the pair of CH2-CH3 segments. In these embodiments, the pair of CH2-CH3 segments comprises a plurality of binding residues independently selected from the group consisting of lysine (K) residues and cysteine (C) residues, and the plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules are respectively bound to the plurality of binding residues.

[0011] According to alternative embodiments, the anti-CD38 antibody comprises a pair of CH2-CH3 segments of IgG, a pair of anti-CD38 scFv respectively bound to the N-terminus of the pair of CH2-CH3 segments, and a pair of binding peptides respectively bound to the C-terminus of the pair of CH2-CH3 segments. In these embodiments, the pair of binding peptides comprises a plurality of C residues and a plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules respectively bound to the plurality of C residues of the pair of binding peptides.

[0012] Preferably, each of the binding peptides of the anti-CD38 antibody comprises the amino acid sequence of "CGGHA" (SEQ ID NO: 1), "CPGHA" (SEQ ID NO: 2), "CGAHA" (SEQ ID NO: 3), "CPAHA" (SEQ ID NO: 4), "GCGGHA" (SEQ ID NO: 5), "ACPGHA" (SEQ ID NO: 6), or "GCPGHA" (SEQ ID NO: 7). In an exemplary embodiment, each of the binding peptides of the anti-CD38 antibody comprises the amino acid sequence of "ACPGHA" (SEQ ID NO: 6).

[0013] According to some exemplary embodiments, each of the binding peptides of the anti-CD38 antibody consists of the amino acid sequence of "CGGHA" (SEQ ID NO: 1), "CPGHA" (SEQ ID NO: 2), "CGAHA" (SEQ ID NO: 3), "CPAHA" (SEQ ID NO: 4), "GCGGHA" (SEQ ID NO: 5), "ACPGHA" (SEQ ID NO: 6), or "GCPGHA" (SEQ ID NO: 7). In one specific example, each of the binding peptides of the anti-CD38 antibody consists of the amino acid sequence of "ACPGHA" (SEQ ID NO: 6).

[0014] Optionally, the molecular construct further comprises a linker unit configured to bind a lenalidomide molecule or a hydrolyzed lenalidomide molecule and the binding peptide. The linker unit in its structure comprises a central core and a plurality of binding arms. The central core comprises 2 to 10 K residues, at least one filler independently disposed between two K residues, and a terminal spacer having two ends. One of the ends is bound to the N-terminus of the first K residue or the C-terminus of the last K residue, and the other end is bound to the C residue of the binding peptide of the anti-CD38 antibody. According to some optional embodiments of the present disclosure, one end of each binding arm is bound to one of the K residues of the central core, and the other end of each binding arm is bound to each lenalidomide molecule or hydrolyzed lenalidomide molecule.

[0015] According to various embodiments of the present disclosure, each of the filler and terminal spacer independently comprises (1) 1 to 12 non-K amino acid residues or (2) PEGylated amino acids having 1 to 12 repeats of ethylene glycol (EG) units. Preferably, the terminal spacer comprises at least 3 negatively charged amino acid residues. In some exemplary embodiments, the terminal spacer comprises the amino acid sequence "EDEDEAGG" (SEQ ID NO: 8), "EGEGEAGG" (SEQ ID NO: 9), or "EGEGE" (SEQ ID NO: 10). In one specific example, the central core comprises the amino acid sequence "EDEDEGAGGKGAGKGAGKG" (SEQ ID NO: 11).

[0016] The binding arm comprises a polyethylene glycol (PEG) chain having 2 to 12 non-K amino acid residues and 2 to 24 repeats of an EG unit, or a combination thereof. In one exemplary embodiment, the binding arm comprises a valine-alanine (Val-Ala) dipeptide and a PEG chain having 3 repeats of an EG unit.

[0017] In certain embodiments, each of the binding arms is bound to the ε-amino group of the K residue.

[0018] Tumors treatable by this method may be solid or diffuse tumors. Examples of solid tumors include, but are not limited to, melanoma, esophageal cancer, gastric cancer, brain tumor, small cell lung cancer, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, thyroid cancer, testicular cancer, head and neck squamous cell carcinoma, and combinations thereof. Exemplary diffuse tumors include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), Hodgkin lymphoma, non-Hodgkin lymphoma (e.g., lymphocytic plasmacytic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, or marginal zone lymphoma), multiple myeloma, and combinations thereof. According to one embodiment of the present disclosure, the tumor is multiple myeloma.

[0019] Many of the accompanying features and advantages described herein should be better understood by referring to the following detailed explanations, which are considered in conjunction with the attached drawings.

[0020] This description should be better understood by referring to the attached drawings and the following detailed explanation. [Brief explanation of the drawing]

[0021] [Figure 1A] Figure 1A is a schematic diagram showing the structure of a molecular construct 10 according to an embodiment of the present disclosure. [Figure 1B] Figure 1B is a schematic diagram showing the structure of a molecular construct 20 according to an embodiment of the present disclosure. [Figure 1C] Figure 1C is a schematic diagram showing the structure of a molecular construct 30 according to an embodiment of this disclosure. [Figure 2] Figure 2 is a schematic diagram showing the structure of a lenalidomide bundle according to Example 1 of this disclosure. [Figure 3] Figure 3 shows the reversed-phase high-performance liquid chromatography (HPLC) elution profile of the Mal-lenalidomide bundle according to Example 1 of this disclosure. [Figure 4] Figure 4 shows the results of electrospray ionization tandem mass spectrometry (ESI-MS) of the Mal-lenalidomide bundle according to Example 1 of this disclosure. [Figure 5A] Figure 5A is a schematic diagram showing the structure of the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein according to Example 2 of this disclosure. [Figure 5B] Figure 5B shows the non-reduced sodium lauryl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein according to Example 3 of this disclosure. [Figure 6A] Figure 6A is a schematic diagram showing the structure of the natural double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 4 of this disclosure. [Figure 6B]Figure 6B shows the non-reducible SDS-PAGE analysis of the recombinant double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 4 of the present invention. [Figure 7A] Figure 7A shows the results of non-reducible SDS-PAGE analysis of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 6 of the present invention. [Figure 7B] Figure 7B shows the elution profile results of size exclusion chromatography of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 6 of the present invention. [Figure 8A] Figure 8A shows the HPLC results of lenalidomide release from MM cells treated with the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 9 of this disclosure. [Figure 8B(A)] Figure 8B shows the ESI-MS results illustrating the release of lenalidomide from MM cells treated with the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 9 of this disclosure. Figure 8B(A): ESI-MS profile of lenalidomide released from MM cells. [Figure 8B(B)] Figure 8B shows the ESI-MS results demonstrating the release of lenalidomide from MM cells treated with the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 9 of this disclosure. Figure 8B(B): ESI-MS results of lenalidomide and two lenalidomide metabolites corresponding to peak (1) in the ESI-MS profile shown in Figure 8B(A). [Figure 8B(C)]Figure 8B shows the ESI-MS results demonstrating the release of lenalidomide from MM cells treated with the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 9 of this disclosure. Figure 8B(C): ESI-MS results of lenalidomide and two lenalidomide metabolites corresponding to peak (2) in the ESI-MS profile shown in Figure 8B(A). [Figure 8B(D)] Figure 8B shows the ESI-MS results demonstrating the release of lenalidomide from MM cells treated with the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 9 of this disclosure. Figure 8B(D): ESI-MS results of lenalidomide and two lenalidomide metabolites corresponding to peak (3) in the ESI-MS profile shown in Figure 8B(A). [Figure 9] Figure 9 shows the matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) results for the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle according to Example 10 of the present invention. [Figure 10] Figure 10 shows the results of an enzyme-linked immunosorbent assay (ELISA) indicating the binding affinity of the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein (unbound) and the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (stabilized complex) according to Example 13 of the present invention to human CD38-expressing cells. [Figure 11] Figure 11 shows flow cytometry results indicating the binding affinity of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (stabilized complex) according to Example 14 of this disclosure to H929 and U266-CD38+ multiple myeloma (MM) cell lines. [Figure 12A]Figure 12A shows the in vitro cytotoxicity of the designated agent according to Example 16 of this disclosure. It shows the cell survival percentage (%) of H929 cells treated for 5 hours with the stabilizing complex (i.e., the stabilizing double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle), the anti-CD38 monoclonal antibody (mAb), daratumumab, and daratumumab or lenalidomide in combination with lenalidomide. [Figure 12B] Figure 12B shows the in vitro cytotoxicity of the designated agent according to Example 16 of this disclosure. It shows the cell survival percentage (%) of H929 cells treated for 1 day with the stabilizing complex (i.e., the stabilizing double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle), the anti-CD38 monoclonal antibody (mAb), daratumumab, and daratumumab or lenalidomide in combination with lenalidomide. [Figure 12C] Figure 12C shows the in vitro cytotoxicity of the designated agent according to Example 16 of this disclosure. It shows the cell survival percentage (%) of H929 cells treated for 3 days with a stabilizing complex (i.e., a stabilizing double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle), an anti-CD38 monoclonal antibody (mAb), daratumumab, daratumumab in combination with lenalidomide, or lenalidomide. [Figure 12D] Figure 12D shows the in vitro cytotoxicity of the designated agent according to Example 16 of this disclosure. It shows the cell survival percentage (%) of H929 cells treated for 5 days with the stabilizing complex (i.e., the stabilizing double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle), the anti-CD38 monoclonal antibody (mAb), daratumumab, and daratumumab or lenalidomide in combination with lenalidomide. [Figure 12E] Figure 12E shows the in vitro cytotoxicity of the designated agent in Example 16 of this disclosure. It shows the cell survival percentage (%) of MM.1S cells treated for 5 days with a stabilizing complex, anti-CD38mAb, daratumumab, or daratumumab or lenalidomide in combination with lenalidomide. [Figure 12F]Figure 12F shows the in vitro cytotoxicity of the designated agent in Example 16 of this disclosure. It shows the cell survival percentage (%) of U266-CD38- cells treated for 5 days with the stabilizing complex, anti-CD38 mAb, daratumumab, or daratumumab or lenalidomide in combination with lenalidomide. [Figure 12G] Figure 12G shows the in vitro cytotoxicity of the designated agent in Example 16 of this disclosure. It shows the cell survival percentage (%) of Daudi cells treated for 5 days with a stabilizing complex, anti-CD38 mAb, daratumumab, or daratumumab or lenalidomide in combination with lenalidomide. [Figure 13] Figure 13 shows the in vitro stability of the specified drug in human plasma according to Example 18 of this disclosure. Daratumumab, unbound (i.e., double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein), and bound Ab (stabilized) (i.e., stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle) were dissolved in human plasma and incubated at 37°C for 7, 14, 21, and 28 days. Subsequently, stabilized ADC (total Ab): products detected by HRP-bound anti-human IgG-Fc antibody, stabilized ADC (bound Ab): products detected by anti-lenalidomide bundle antibody and anti-mouse IgG-Fc antibody were subjected to ELISA analysis. [Figure 14] Figure 14 shows the antitumor effect of the specified treatment in a xenograft tumor model according to Example 20 of this disclosure, where tumor-bearing mice (average tumor size 115 ± 15 mm3) were administered phosphate-buffered saline (PBS: serving as the control group), a stabilizing complex (i.e., a stabilized double-chain (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle), and daratumumab (Dara), respectively. [Figure 15A]Figure 15A shows the effect of the specified treatment on inhibiting tumor size in a xenograft tumor model according to Example 21 of this disclosure, where tumor-bearing mice (average tumor size 150 ± 20 mm3) were administered PBS, a stabilizing complex (i.e., a stabilizing double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle), daratumumab (Dara), daratumumab in combination with lenalidomide (Dara / Lena), and lenalidomide (Lena), respectively. ** indicates P<0.01, and *** indicates P<0.001. [Figure 15B] Figure 15B shows the effect of the specified treatment on inhibiting tumor weight in a xenograft tumor model according to Example 21 of this disclosure, where tumor-bearing mice (average tumor size 150 ± 20 mm3) were administered PBS, a stabilizing complex (i.e., a stabilizing double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle), daratumumab (Dara), daratumumab in combination with lenalidomide (Dara / Lena), and lenalidomide (Lena), respectively. ** indicates P<0.01, and *** indicates P<0.001. [Modes for carrying out the invention]

[0022] Following common practice, the various features / elements described are not drawn to scale, but rather are drawn to best illustrate the specific features / elements relevant to the invention. Furthermore, reference numerals and designations in the various drawings are used to indicate elements / parts.

[0023] The detailed description provided below in relation to the attached drawings is for illustrative purposes only and does not represent the only form in which this embodiment may be configured or used. This description explains the function of this embodiment and the sequence of steps for configuring and operating this embodiment. However, the same or equivalent functions and sequence may be achieved by different embodiments.

[0024] I. Definition For convenience, the specific terms used in this specification, the examples, and the appended claims are summarized herein. Unless otherwise specified, the scientific and technical terms used in this disclosure have meanings that are generally understood and used by those skilled in the art. Unless the context specifically requires otherwise, singular terms shall include their plural forms, and plural terms shall include their singular forms. Specifically, as used herein and in the claims, the singular forms "a" and "an" shall include plural references unless the context explicitly states otherwise. Also, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more.

[0025] While the numerical ranges and parameters describing the broad scope of the invention are approximate, the numerical values ​​described in the specific examples are reported as precisely as possible. However, all numerical values ​​inherently include a predetermined error that inevitably arises from the standard deviation observed in each test measurement. Furthermore, as used herein, the term “approximately” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “approximately” means within the mean permissible standard error as considered by those skilled in the art. Outside of the operational / executive examples, or unless expressly stated otherwise, all numerical ranges, quantities, values, and percentages disclosed herein, such as those for the quantity, duration, temperature, operating conditions, and ratios of the material, should in all cases be understood as being modified by the term “approximately.” Therefore, unless the opposite is indicated, the numerical parameters described in this disclosure and the appended claims are approximate and may vary as desired. At a minimum, each numerical parameter should be interpreted with respect to the reported number of significant figures and by applying ordinary rounding methods.

[0026] In this specification and in the claims, the term “antibody” is used in its broadest sense and includes antibody fragments that bind to an antigen, such as fully assembled antibodies, antigen-binding fragments (Fab / Fab'), F(ab')2 fragments (having two antigen-binding Fab portions linked together by disulfide bonds), variable fragments (Fv), single-stranded variable fragments (scFv), bispecific single-stranded variable fragments (bi-scFv), nanobodies (also called single-domain antibodies sdAb), unibodies, and diabodies. An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen-binding region or variable region of an intact antibody. An antibody fragment may comprise a pair of scFv fused to the N-terminus or C-terminus of a pair of CH2-CH3 segments derived from human immunoglobulin (Ig). Typically, “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Well-known immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which similarly define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. A standard immunoglobulin (antibody) structural unit is known to consist of a tetramer, comprising two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The N-terminal of each chain defines a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. According to certain embodiments of this disclosure, antibody fragments can be produced by modifying native antibodies or by de novo synthesis using recombinant DNA methods. In certain embodiments of this disclosure, antibodies may be bispecific or have various configurations. For example, a bispecific antibody may have two different antigen-binding sites (variable regions).In various embodiments, bispecific antibodies can be produced by hybridoma technology or recombinant DNA technology.

[0027] As used herein, the terms “link,” “couple,” and “conjugate” are interchangeable to refer to any means of connecting two components, whether through a direct or indirect link between them.

[0028] The terms “polypeptide” and “peptide” are used interchangeably to refer to polymers having at least two amino acid residues. Typically, polypeptides consist of amino acid residues ranging in length from 2 to approximately 200, although it also includes polymers with more than 200 amino acid residues. Where an amino acid sequence is given, L-, D-, or beta-amino acid versions of that sequence are also considered. Polypeptides also include amino acid polymers, where one or more amino acid residues are artificial chemical analogs to corresponding naturally occurring amino acids and naturally occurring amino acid polymers. Furthermore, the term also applies to amino acids joined by peptide bonds, or by other “modification bonds” where the peptide bond is substituted with, for example, α-esters, β-esters, thioamides, phosphoramides, carbomates, hydroxylates, etc.

[0029] As used herein, the term “fragment crystallizable region” or “Fc region” refers to the tail region of immunoglobulins that interacts with cell surface receptors and / or certain complement system proteins, known as Fc receptors. Structurally, the Fc region comprises, from N-terminus to C-terminus, at least a hinge region (a short sequence of the heavy chain binding the CH1 and CH2 domains), a CH2 domain (the second constant domain of the heavy chain), and a CH3 domain (the third constant domain of the heavy chain). The Fc region of an IgG1 antibody can be generated, for example, by digestion of the IgG1 antibody with papain.

[0030] The “percentage (%) sequence identity” of the amino acid sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific reference sequence, after aligning the sequences as necessary to obtain the maximum percentage of sequence identity and introducing gaps, and without considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage sequence identity can be achieved in various aspects of this art using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can identify appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. For the purposes herein, sequence comparisons between two amino acid sequences were performed using the computer program Blasp (protein-protein BLAST), provided online by the National Center for Biotechnology Information (NCBI). The percentage sequence identity of a given sequence A to a target sequence B (or, to put it another way, a given sequence A having a predetermined percentage of sequence identity with a given sequence B) is calculated using the following formula. X / Y × 100% X is the number of amino acid residues scored as identical by the sequence alignment program BLAST in the alignment of A and B in that program, and Y is the total number of amino acid residues in the target sequence B.

[0031] In certain embodiments, conservative amino acid substitutions comprising any of the sequences described herein are considered. In various embodiments, one, two, three, four, or five different residues are substituted. The term “conservative substitution” is used to refer to amino acid substitutions that do not substantially alter the activity of the molecule (e.g., biological or functional activity and / or specificity). Typically, a conservative amino acid substitution involves substituting one amino acid with another amino acid having similar chemical properties (e.g., charge or hydrophobicity). Some conservative substitutions include “analogous substitutions” in which a standard amino acid is substituted with a non-standard (e.g., rare, synthetic, etc.) amino acid that differs minimally from the parent residue. Amino acid analogs are synthetically derived from a standard amino acid without significant alteration to the parent structure, are isomers, or are metabolite precursors. In this application, the following are considered to be the four characteristic amino acids: (1) lysine, which has an amine group in its side chain; (2) cysteine, which has a thiol group in its side chain; (3) serine and threonine, which have a hydroxyl group in their side chains; and (4) aspartic acid and glutamic acid, which have a carboxyl group in their side chains. Each of these four amino acid groups contains a unique functional group in its side chain that can be applied to bind to various chemical components. Non-natural amino acids having the same functional group in their side chains may be substituted for similar purposes.

[0032] In certain embodiments, polypeptides having at least 80%, preferably at least 85% or 90%, and more preferably at least 95% or 98% sequence identity with any of the sequences described herein may also be considered.

[0033] The term "PEGylated amino acid" as used herein refers to a polyethylene glycol (PEG) chain having one amino group and one carboxyl group. According to embodiments of this disclosure, the PEGylated amino acid is NH2-(CH2CH2O) n The formula has the equation -CO2H. In this disclosure, the value of n is in the range of 1 to 20, preferably in the range of 2 to 12.

[0034] As used herein, the term “terminus” in relation to polypeptides refers to an amino acid residue at the N-terminus or C-terminus of a polypeptide. In relation to polymers, the term “terminus” refers to a constituent unit of the polymer (e.g., polyethylene glycol in this disclosure) located at the end of the polymer backbone. In this specification and the claims, the term “free end” is used to mean that a terminal amino acid residue or constituent unit is not chemically bonded to any other molecule.

[0035] The terms “application” and “administration” are used interchangeably here to mean the application of the molecular construct of the present invention to a subject requiring such treatment.

[0036] The terms “to treat / process,” “treating / processing,” or “treatment / treatment / processing” as used herein include prophylactic (e.g., preventive), curative, or palliative treatments / treatments / processing. In particular, the terms “to treat / process,” “treating / processing,” or “treatment / treatment / processing” as used herein mean the application or administration of the molecular construct to a subject with a medical condition (e.g., cancer), symptoms associated with a medical condition, a secondary disease or disorder to a medical condition, or a predisposition to a medical condition, for the purpose of partially or completely alleviating, improving, relieving, delaying the onset, inhibiting the progression, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of the particular disease, disorder, and / or condition described herein. Treatments / treatments / processing may be applied to subjects who show no signs of disease, disorder, and / or condition, or subjects who show only early signs of disease, disorder, and / or condition, for the purpose of reducing the risk of progressive pathology associated with the disease, disorder, and / or condition. A treatment / therapy / process is generally considered “effective” if it reduces one or more symptoms or clinical markers as defined herein. Alternatively, a treatment / therapy / process is considered “effective” if it alleviates or cessates symptoms, impairment, or medical condition.

[0037] The term "effective dose" as used herein refers to a sufficient amount of the molecular construct to obtain the desired therapeutic response. An effective dose of a drug is not necessary to cure the disease or condition, but provides treatment to the disease or condition in such a way that the onset of the disease or condition is delayed, inhibited, or prevented, or the disease or condition is improved. The effective dose may be divided into one, two, or more doses in an appropriate form administered once, twice, or more times over the entire specified period. The specific effective or sufficient dose varies depending on factors such as the specific condition being treated, the patient's physical condition (e.g., weight, age, or sex), the type of subject being treated, the duration of treatment, the nature of any concomitant therapy, and the specific formulation and structure of the compound or its derivative employed. The effective dose may be expressed, for example, as the total amount of the active ingredient (e.g., grams, milligrams, or micrograms) or as the ratio of the mass of the active ingredient to body weight, e.g., milligrams per kilogram (mg / kg) or nanomoles per kilogram (nmol / kg). Those skilled in the art can calculate the human equivalent dose (HED) for a drug (such as this molecular construct) based on doses determined from animal models. For example, when estimating the maximum safe starting dose for use in human subjects, one can follow the industrial guidance issued by the U.S. Food and Drug Administration (FDA) entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers."

[0038] As used herein, the term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. In this specification and in the claims, the term "tumor" includes solid tumors and diffuse tumors.

[0039] The term "solid tumor" as used herein refers to an abnormal mass of tissue that does not typically contain cysts or fluid areas. Various types of solid tumors are named according to the type of cells that form them. Examples of solid tumors include, but are not limited to, sarcomas and carcinomas. Generally, a "sarcoma" is a cancer that arises from connective or supporting tissue, such as bone or muscle. A "carcinoma" is a cancer that arises from glandular and epithelial cells that cover body tissues.

[0040] As used herein, the term "diffuse tumor" refers to leukemia and / or hematological malignancies that originate from hematopoietic (blood-forming) cells and affect the blood, bone marrow, or lymph nodes. Examples of diffuse tumors include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), Hodgkin lymphoma, non-Hodgkin lymphoma, and myeloma.

[0041] As used herein, the term “subject” refers to an animal, including human species, that can be treated by the molecular constructs and / or methods of the present invention. Unless otherwise specifically indicated, the term “subject” refers to both male and female sexes. Thus, the term “subject” includes any mammal that could benefit from the treatment methods of this disclosure. Examples of “subjects” include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cattle, birds, and poultry. In exemplary embodiments, the subject is human.

[0042] II. Description of the Invention In conventional antibody-drug conjugate (ADC) constructs, the antibody is a very large polymer with a molecular weight of approximately 150,000 daltons, while the drug molecule (payload) is a small compound with a molecular weight in the range of several hundred daltons. Standard doses of ADCs range from 100 mg to several hundred mg. Therefore, for therapeutic agents applicable for use in combination with antibodies against ADC constructs that mediate cytotoxicity, immunomodulatory activity, or other types of effects on tumors, the efficacy of the drug is in the sub-nanomolelic range. 50It must have and be very high (see, for example, David Dahlgren et al., Antibody-Drug Conjugates and Targeted Treatment Strategies for Hepatocellular Carcinoma: A Drug-Delivery Perspective; Molecules (2020), 25, 2861; or Alain Beck et al., Strategies and challenges for the next generation of antibody-drug conjugates; Nature Reviews Drug Discovery (2017), 16:315-337). For example, microtubule disruptors (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine (DM-1), and eribulin), topoisomerase I inhibitors (e.g., camptothecin, exatecan, and SN38), and DNA synthesis inhibitors (e.g., pyrrolobenzodiazepines (PBD) and their derivatives) are effective in the sub-nanomole range, i.e., in the sub-nanomole range of EC 50 It has a lethal effect on target cells. Lenalidomide, a thalidomide analog, is a clinically used drug for multiple disease indications, most notably in multiple myeloma, usually in combination with other drugs. It is taken orally at a dose of 20-40 mg per day for most days of treatment. Various studies have shown that lenalidomide is effective in EC levels ranging from 3 μM to 30 μM. 50 It is known to possess (see, for example, A. Lopez-Girona et al., Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide; Leukemia (2012), 26:2326-2335). Therefore, it is well known that lenalidomide is not suitable for use as a therapeutic agent in ADC constructs.

[0043] This disclosure is based, at least to some extent, on the finding that ADC constructs employing lenalidomide as a conjugate agent exhibit therapeutic efficacy in reducing or eliminating tumor growth. According to embodiments of this disclosure, the conjugation of an anti-CD38 antibody to a lenalidomide molecule significantly enhances the therapeutic effect of the lenalidomide molecule on tumors (e.g., multiple myeloma) compared to a standard lenalidomide treatment schedule with a 28-day cycle. During the 28-day cycle, the lenalidomide molecule is administered daily until either disease progression or unacceptable toxicity occurs, and the single-dose combination treatment (i.e., a bioconjugate of anti-CD38 antibody and lenalidomide molecule) provides a satisfactory effect in suppressing tumor growth in the subject, thereby avoiding repeated administrations of the lenalidomide molecule and significantly reducing its adverse effects.

[0044] Accordingly, this disclosure provides a method for treating a tumor in a subject. The method involves administering to the subject a molecular construct comprising an anti-CD38 antibody and a plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules conjugated to the anti-CD38 antibody.

[0045] According to some embodiments of the present disclosure, the molecular construct comprises a plurality of lenalidomide molecules conjugated to an anti-CD38 antibody. In these embodiments, administration of the molecular construct achieves an effective amount of lenalidomide molecules at least 1 / 1000th of an effective amount of lenalidomide molecules used for tumor treatment, either alone or in combination with an anti-CD38 antibody (i.e., in a combination treatment in which the lenalidomide molecules and the anti-CD38 antibody are administered separately to the subject). For example, effective doses of lenalidomide molecules used alone or in combination with anti-CD38 antibodies to achieve therapeutic objectives are 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, and 510. 0, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, The effective amount may be 1 / 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000, 11000, 12000 or more. According to some preferred embodiments, the effective amount of lenalidomide molecules is about 1 / 10000 of the effective amount of lenalidomide molecules used alone or in combination with an anti-CD38 antibody. In one exemplary embodiment, the effective amount of lenalidomide molecules is about 1 / 10640 of the effective amount of lenalidomide molecules used alone or in combination with an anti-CD38 antibody.

[0046] According to some embodiments of this disclosure, the molecular construct comprises a plurality of hydrolyzed lenalidomide molecules conjugated to an anti-CD38 antibody. In these embodiments, administration of the molecular construct achieves an effective amount of hydrolyzed lenalidomide molecules that is at least one-thousandth of an effective amount of lenalidomide molecules used alone or in combination with an anti-CD38 antibody for the treatment of tumors. For example, the effective doses of hydrolyzed lenalidomide molecules used alone or in combination with anti-CD38 antibodies to achieve therapeutic objectives are 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5 100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800 The effective amount may be 1 / 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000, 11000, 12000 or more. According to some preferred embodiments, the effective amount of hydrolyzed lenalidomide molecules is about 1 / 10000 of the effective amount of lenalidomide molecules used alone or in combination with an anti-CD38 antibody. In one exemplary embodiment, the effective amount of hydrolyzed lenalidomide molecules is about 1 / 10640 of the effective amount of lenalidomide molecules used alone or in combination with an anti-CD38 antibody.

[0047] According to some embodiments, the lenalidomide molecule is [ka] The hydrolyzed lenalidomide molecule has the following structure: [ka] It has the structure of [the object].

[0048] According to certain embodiments, the subject is a mouse. In these embodiments, the molecular construct is administered in amounts of approximately 1 nmol / kg to 1000 nmol / kg (approximately 0.12 mg / kg to 120 mg / kg) per body weight per dose. For example, to achieve a therapeutic objective, the molecular construct is administered in amounts of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 7 10, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 or 1000 nmol / kg (or approximately 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18) It may be administered in amounts of 0.19, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, or 120 mg / kg). Preferably, the molecular construct is administered in an amount of approximately 1 nmol / kg to 100 nmol / kg (approximately 0.12 mg / kg to 12 mg / kg) per dose. More preferably, the molecular construct is administered in an amount of approximately 10 nmol / kg to 50 nmol / kg (approximately 1.2 mg / kg to 6 mg / kg) per body weight per dose.In some exemplary embodiments, a dose of approximately 20 nmol / kg (approximately 2.3 mg / kg to 2.4 mg / kg) of this molecular construct is sufficient to inhibit tumor growth in the subject.

[0049] An expert can easily determine the human equivalent dose (HED) of the molecular construct based on the doses determined from animal studies given in the effective examples of this application.Therefore, the effective dose of this molecular construct suitable for use in human subjects is in the range of approximately 0.08 nmol / kg to 100 nmol / kg (approximately 0.01 mg / kg to 12 mg / kg) per dose per body weight, for example, approximately 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nmol / kg (or approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.0 8, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6 It can be 0.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9 or 12 mg / kg).Preferably, the molecular construct is administered at a dose of approximately 0.8 nmol / kg to 83 nmol / kg (approximately 0.1 mg / kg to 10 mg / kg) per body weight. More preferably, the molecular construct is administered at a dose of approximately 0.8 nmol / kg to 33 nmol / kg (approximately 0.1 mg / kg to 4 mg / kg) per body weight. The dose may be administered as a single aliquot or as two or more aliquots. An experienced practitioner or clinician may adjust the dosage or regimen according to the patient's physical condition or the severity of the disease.

[0050] Preferably, the molecular construct is administered to the subject once every four weeks. In certain cases, the molecular construct is administered to the subject multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times) at four-week intervals until the desired therapeutic effect is achieved. As can be understood, a skilled person may adjust the dosage or regime depending on the patient's physical condition or the severity of the disease.

[0051] According to certain embodiments of the present disclosure, the molecular construct is in the form of an antibody-drug conjugate (ADC), which, as is well known in the art, consists of a monoclonal antibody (mAb) covalently attached to a therapeutic agent (e.g., a cytotoxic agent). Specifically, in the embodiments, the anti-CD38 antibody of the molecular construct has a conventional structure, namely comprising a pair of heavy chains and a pair of light chains, each heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable (HV) domain, a CH1 domain (first constant domain of the heavy chain), a hinge domain, a CH2 domain (second constant domain of the heavy chain), and a CH3 domain (third constant domain of the heavy chain), and each light chain comprising, from N-terminus to C-terminus, a light chain variable (VL) domain and a CL domain (constant domain of the light chain). The anti-CD38 antibody has multiple binding residues in its constant domain, preferably in the CH2 and CH3 domains (i.e., the CH2-CH3 segment), and a lenalidomide molecule or a hydrolyzed lenalidomide molecule is bound to the binding residues of the anti-CD38 antibody, respectively. The binding residues are independently selected from the group consisting of K and C residues. According to one embodiment, the anti-CD38 antibody has multiple K residues in its CH2-CH3 segment. In this case, the lenalidomide molecule or a hydrolyzed lenalidomide molecule is bound to the K residues by forming an amide bond with them, respectively. According to another embodiment, the anti-CD38 antibody has multiple C residues in its CH2-CH3 segment. In this case, the lenalidomide molecule or a hydrolyzed lenalidomide molecule is bound to the C residues, respectively, by reacting with the sulfhydryl (SH) group of the C residue.

[0052] Herein, we refer to Figure 1A, a schematic diagram showing a molecular construct 10 according to a part of the present disclosure. As shown in the figure, the anti-CD38 antibody of molecular construct 10 comprises, as described above, a pair of heavy chains 110a, 110b (each heavy chain 110a, 110b comprising a VH domain, a CH1 domain, a CH2 domain, and a CH3 domain) and a pair of light chains 120a, 120b (each light chain 120a, 120b comprising a VL domain and a CL domain), with four binding residues (starred in Figure 1A 130a, 130b, 130c, 130d) positioned in the CH2 and CH3 domains, respectively. Thus, four therapeutic agents T (i.e., four lenalidomide molecules or four hydrolyzed lenalidomide molecules) are conjugated to the anti-CD38 antibody via the binding residues 130a, 130b, 130c, 130d, respectively.

[0053] Depending on the intended purpose, each of the lenalidomide molecule and the hydrolyzed lenalidomide molecule may be bound to the binding residue in the presence or absence of a linker. Preferably, each of the lenalidomide molecule and the hydrolyzed lenalidomide molecule is bound to the binding residue via a linker, such as a cleavable linker or a non-cleavable linker. Exemplary cleavable linkers include, but are not limited to, protease-sensitive linkers (e.g., valine-citrulline dipeptide, valine-alanine dipeptide, valine-lysine dipeptide, valine-arginine dipeptide, and glutamic acid-valine-citrulline tripeptide), pH-sensitive linkers (e.g., hydrazone linkers, ester linkers, and amide linkers), and glutathione-sensitive linkers (e.g., N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP)). Non-limiting examples of non-cleavable linkers include maleimidocaproyl (MC), maleimidomethylcyclohexane-1-carboxylate (MCC), and succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). Alternatively, the linker may be a linker known in the art to link two functional motifs in an immune complex (e.g., linking an antibody and an ADC payload). An expert may select an appropriate linker to produce the molecular construct depending on the intended purpose. According to some exemplary embodiments, the linker linking the anti-CD38 antibody and the lenalidomide molecule / hydrolyzed lenalidomide molecule comprises a polyethylene glycol (PEG) chain and a protease-sensitive linker attached to the PEG chain. Preferably, the PEG chain has 1 to 10 repeats of EG units. In one specific embodiment, the linker comprises a PEG chain and a valine-alanine dipeptide attached to the PEG chain, the PEG chain having 3 repeats of EG units.

[0054] According to some embodiments of this disclosure, the molecular construct is in the form of an scFv-Fc fusion protein, which comprises an scFv fused to the Fc region of an immunoglobulin (e.g., IgG). In these embodiments, the anti-CD38 antibody of the molecular construct comprises a pair of CH2-CH3 segments of the immunoglobulin and a pair of anti-CD38scFvs, each bound to the N-terminus of the pair of CH2-CH3 segments via hinge domains. The anti-CD38 antibody comprises a plurality of binding residues in the CH2-CH3 segments, and a lenalidomide molecule or a hydrolyzed lenalidomide molecule is bound to the binding residues of the anti-CD38 antibody, respectively. The binding residues are independently selected from the group consisting of K and C residues. According to one embodiment, the anti-CD38 antibody comprises a plurality of K residues in its CH2-CH3 segment. In this case, a lenalidomide molecule or a hydrolyzed lenalidomide molecule is bound to the K residues, respectively, by forming an amide bond with it. According to another embodiment, the anti-CD38 antibody comprises a plurality of C residues in its CH2-CH3 segment. In this case, the lenalidomide molecule or the hydrolyzed lenalidomide molecule is bound to the C residue by reacting with the SH group of the C residue. As described above, each lenalidomide molecule / hydrolyzed lenalidomide molecule is preferably bound to the binding residue via a linker, for example, a cleavable linker or a non-cleavable linker.

[0055] Figure 1B gives a schematic diagram of a molecular construct 20 according to a partial embodiment of the present disclosure. In the structure, the anti-CD38 antibody of molecular construct 20 comprises a pair of CH2-CH3 domains 220a, 220b of an immunoglobulin (e.g., IgG), a pair of anti-CD38scFv 210a, 210b conjugated to the N-terminuses of the pair of CH2-CH3 domains 220a, 220b, respectively, with six binding residues (starred in Figure 1B: 230a, 230b, 230c, 230d, 230e, 230f) positioned on the CH2 and CH3 domains 220a, 220b, respectively. Thus, six therapeutic agents T (i.e., six lenalidomide molecules or six hydrolyzed lenalidomide molecules) are conjugated to the anti-CD38 antibody via the binding residues 230a, 230b, 230c, 230d, 230e, 230f, respectively.

[0056] To understand this, the number of lenalidomide molecules or hydrolyzed lenalidomide molecules supported on this molecular construct (in the form of an ADC or scFv-Fc fusion protein) depends on the number of binding residues consisting of the CH2-CH3 segment of the anti-CD38 antibody. Therefore, a skilled person may adjust the number of binding residues as needed to optimize therapeutic efficacy.

[0057] According to alternative embodiments of the present disclosure, the molecular construct is in the form of a drug bundle. In these embodiments, the molecular construct comprises an anti-CD38 antibody and at least one linker unit conjugated to the anti-CD38 antibody, each linker unit carrying a plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules. Specifically, the anti-CD38 antibody comprises a pair of CH2-CH3 segments of immunoglobulin (e.g., IgG), a pair of anti-CD38scFv conjugated to the N-terminuses of the pair of CH2-CH3 segments via hinge domains, and a pair of binding peptides conjugated to the C-terminuses of the pair of CH2-CH3 segments. Each pair of binding peptides comprises a plurality of C residues, and at least one linker unit is conjugated to the anti-CD38 antibody via the C residues of the binding peptides. According to some preferred embodiments, each of the pair of conjugated peptides comprises the amino acid sequence of "CGGHA" ​​(SEQ ID NO: 1), "CPGHA" (SEQ ID NO: 2), "CGAHA" (SEQ ID NO: 3), "CPAHA" (SEQ ID NO: 4), "GCGGHA" ​​(SEQ ID NO: 5), "ACPGHA" (SEQ ID NO: 6), or "GCPGHA" (SEQ ID NO: 7). In a specific embodiment, each of the conjugated peptides comprises the amino acid sequence of "ACPGHA" (SEQ ID NO: 6). According to a particular exemplary embodiment, each of the pair of conjugated peptides comprises the amino acid sequence of "CGGHA" ​​(SEQ ID NO: 1), "CPGHA" (SEQ ID NO: 2), "CGAHA" (SEQ ID NO: 3), "CPAHA" (SEQ ID NO: 4), "GCGGHA" ​​(SEQ ID NO: 5), "ACPGHA" (SEQ ID NO: 6), or "GCPGHA" (SEQ ID NO: 7). In an exemplary embodiment, each of the conjugated peptides comprises the amino acid sequence of "ACPGHA" (SEQ ID NO: 6).

[0058] Herein, we refer to Figure 1C, which shows a schematic diagram of a molecular construct 30 according to a particular embodiment of the present disclosure. As shown in the figure, the anti-CD38 antibody of molecular construct 30 comprises a pair of CH2-CH3 domains 320a, 320b of an immunoglobulin (e.g., IgG), a pair of anti-CD38scFv 310a, 310b conjugated to the N-terminuses of the pair of CH2-CH3 domains 320a, 320b, respectively, and a pair of conjugating peptides 330a, 330b conjugated to the C-terminuses of the pair of CH2-CH3 domains 320a, 320b, respectively, each of the conjugating peptides 330a, 330b comprising a C residue. In this case, two linker units 340a, 340b are conjugated to the C residues of the conjugating peptides 330a, 330b of the anti-CD38 antibody, respectively.

[0059] To understand this, the number of linker units bound to the anti-CD38 antibody depends on the number of C residues in the binding peptide. For example, if each binding peptide has two C residues, four linker units may be bound to the anti-CD38 antibody. Experienced engineers may adjust the number of C residues in the binding peptide depending on the actual application.

[0060] According to certain embodiments of the present disclosure, a linker unit comprises a central core and a plurality of binding arms bonded to the central core. The central core is a linear polypeptide comprising 2 to 10 K residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 K residues) and terminal spacers. The binding arms are each bonded to a K residue of the central core, and lenalidomide molecules or hydrolyzed lenalidomide molecules are each bonded to the free ends of the binding arms (i.e., ends that are not chemically bonded to any molecule).

[0061] According to some exemplary embodiments, a molecular construct designated as “Natural double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle” is a pair of binding peptides of an anti-CD38 antibody (each binding peptide is the amino acid sequence of “ACPGHA” (SEQ ID NO: 6, Zn 2+-A molecular construct comprising two linker units each bound to a binding motif (which is designated in this disclosure as "BM"), each linker unit carrying three lenalidomide molecules via binding arms, thereby forming a drug bundle at the C-terminus of the anti-CD38 antibody. According to some exemplary embodiments, a molecular construct designated as "stabilized double-strand (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle" comprises two linker units each bound to a pair of binding peptides of the anti-CD38 antibody (each binding peptide having the amino acid sequence "ACPGHA" (SEQ ID NO: 6)), each linker unit carrying three hydrolyzed lenalidomide molecules via binding arms, thereby forming a drug bundle at the C-terminus of the anti-CD38 antibody. In one embodiment, the hydrolyzed lenalidomide molecules are [ka] It has the structure. In other embodiments, the hydrolyzed lenalidomide molecule is [ka] It has the structure of [the object].

[0062] The linker unit is bound to the C residue of the anti-CD38 antibody binding peptide via a terminal spacer. According to various embodiments of this disclosure, the terminal spacer may be an N-terminal spacer or a C-terminal spacer. In some embodiments, the terminal spacer is an N-terminal spacer having two ends (i.e., a first end and a second end), one of which is bound to the N-terminus of the first K residue of the central core (starting from the N-terminus of the central core), and the other end of which is bound to the C residue of the anti-CD38 antibody binding peptide. According to some exemplary embodiments, the N-terminal spacer has a CO2H group at the first end and an SH reactive group (e.g., a maleimide group, a sulfone group, a haloacetyl group, or a pyridyl disulfide group) at the second end. In this case, the first end of the terminal spacer is bound to the N-terminus of the first K residue by forming an amide bond between the CO2H group of the N-terminal spacer and the NH2 group of the first K residue, and the second end of the terminal spacer is bound to the C residue of the anti-CD38 antibody binding peptide by a thiol-maleimide reaction that occurs between the SH reactive group of the C-terminal spacer and the SH group of the C residue.

[0063] In some embodiments, the terminal spacer is a C-terminal spacer having two ends (i.e., a first end and a second end), one of which is bound to the C-terminus of the last K residue of the central core (starting from the N-terminus of the central core), and the other end of which is bound to the C residue of the anti-CD38 antibody-binding peptide. According to some exemplary embodiments, the C-terminal spacer has an NH2 group at the first end and an SH reactive group (e.g., a maleimide group, a sulfone group, a haloacetyl group, or a pyridyl disulfide group) at the second end. In this case, the first end of the terminal spacer is bound to the C-terminus of the last K residue by forming an amide bond between the NH2 group of the C-terminal spacer and the CO2H group of the last K residue, and the second end of the terminal spacer is bound to the C residue of the anti-CD38 antibody-binding peptide by a thiol-maleimide reaction occurring between the SH reactive group of the N-terminal spacer and the SH group of the C residue.

[0064] According to certain embodiments of the present disclosure, the central core has a length of 3 to 120 amino acid residues and contains at least two lysine (K) residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more K residues) in its amino acid sequence, where any two K residues are adjacent to each other or separated by fillers.

[0065] Each filler and terminal spacer independently comprises a PEGylated amino acid having (1) 1 to 12 non-K amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 non-K amino acid residues) or (2) 1 to 12 repeats of an ethylene glycol (EG) unit (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 repeats of an EG unit). Generally, each non-K amino acid residue is independently selected from the group consisting of glycine (G), aspartic acid (D), glutamic acid (E), serine (S), arginine (R), histidine (H), threonine (T), asparagine (N), glutamine (Q), proline (P), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), and tryptophan (W) residues. Preferably, the terminal spacer comprises at least three negatively charged amino acid residues at pH=7, such as aspartic acid (D) and / or glutamic acid (E) residues. According to some exemplary embodiments, the terminal spacer comprises the amino acid sequence "EDEDEAGG" (SEQ ID NO: 8), "EGEGEAGG" (SEQ ID NO: 9), or "EGEGE" (SEQ ID NO: 10). In one specific example, the central core has the amino acid sequence "EDEDEGAGGKGAGKGAGKG" (SEQ ID NO: 11).

[0066] According to certain embodiments of this disclosure, the binding arm comprises a PEG chain having 2 to 12 non-K amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 non-K amino acid residues), 2 to 24 repeats of EG units (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 EG units), or a combination thereof. In one specific embodiment, the binding arm comprises a PEG chain and a valine-alanine (Val-Ala) dipeptide attached to the PEG chain, the PEG chain having 3 repeats of EG units. Optionally, the peptide or PEG chain of the binding arm may be replaced with a polymer of substantially the same length. Polymers comprising carbohydrates or other hydrophilic building blocks are suitable for use as binding arms.

[0067] In the structure, each binding arm has two ends (i.e., a first end and a second end), the first end being bound to one of the K residues of the central core, and the second end being bound to a lenalidomide molecule or a hydrolyzed lenalidomide molecule. According to some embodiments, the first end of the binding arm is bound to the ε-amino group of the K residue by forming an amide bond with it, and the second end of the binding arm is bound to a lenalidomide molecule or a hydrolyzed lenalidomide molecule via a para-aminobenzylcarbamate (PABC) reagent.

[0068] Alternatively, the second end of the binding arm has a functional group for binding a lenalidomide molecule or a hydrolyzed lenalidomide molecule. Depending on the intended purpose, the functional group may be an NH2 group, a CO2H group, an N-hydroxysuccinimidyl (NHS) group, an azide group, an alkyne group, a cyclooctin group, a tetrazine group, or a cyclooctene group. The lenalidomide molecule / hydrolyzed lenalidomide molecule is bound to the second end of the binding arm by one of the following chemical reactions. (1) To form an amide bond between them. In this case, the functional group is an NH2 group, a CO2H group, or an NHS group, and the lenalidomide molecule / hydrolyzed lenalidomide molecule has an NH2 group or a CO2H group (i.e., the lenalidomide molecule / hydrolyzed lenalidomide molecule is modified with an NH2 group or a CO2H group). (2) Copper(I) catalyzed alkyne-azide cycloaddition reaction (CuAAC reaction). In this reaction, one of the functional group and the lenalidomide molecule / hydrolyzed lenalidomide molecule has an azide group or a picolyl azide group, and the other has an alkyne group. (3) Reverse electron-required Diels-Alder (iEDDA) reaction. In this reaction, one of the functional group and the lenalidomide molecule / hydrolyzed lenalidomide molecule has a tetrazine group, and the other has a cyclooctene group (e.g., a TCO group or a norbornene group). (4) Strain-accelerated azide-alkyn click chemistry (SPAAC) reaction. In this reaction, one of the functional group and the lenalidomide molecule / hydrolyzed lenalidomide molecule has an azide group, and the other has a cyclooctin group.

[0069] According to various embodiments of the present disclosure, the tetrazine group is 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, 1,2,4,5-tetrazine, or a derivative thereof; the cyclooctene group is norbornene or trans-cyclooctene (TCO); and the cyclooctin group is selected from the group consisting of dibenzocyclooctin (DIBO), difluorinated cyclooctin (DIFO), bicyclononine (BCN), and dibenzoazacyclooctin (DIBAC or DBCO). According to one embodiment of the present disclosure, the tetrazine group is 6-methyltetrazine.

[0070] To make it clear, the number of lenalidomide molecules or hydrolyzed lenalidomide molecules supported on this linker unit depends on the number of K residues in the central core (and therefore the number of binding arms). For example, if the central core has three K residues, three binding arms are bound to the K residues of the central core, and three lenalidomide molecules / hydrolyzed lenalidomide molecules are bound to the free ends of the binding arms, respectively. Alternatively, if the central core has ten K residues, ten binding arms are bound to the K residues of the central core, and ten lenalidomide molecules / hydrolyzed lenalidomide molecules are bound to the free ends of the binding arms, respectively. Therefore, those skilled in the art can adjust the number of lenalidomide molecules or hydrolyzed lenalidomide molecules supported on the linker unit by changing the number of K residues in the central core (and therefore the number of binding arms) according to the desired purpose.

[0071] According to alternative embodiments of the present disclosure, a lenalidomide molecule or a hydrolyzed lenalidomide molecule is conjugated to an anti-CD38 antibody, respectively, by reacting with the SH group of the C residue of the conjugated peptide via a linker that may be a cleavable or non-cleavable linker, as described above.

[0072] Tumors treatable by this method may be solid tumors or diffuse tumors. Examples of solid tumors include, but are not limited to, melanoma, esophageal cancer, gastric cancer, brain tumors, small cell lung cancer, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, thyroid cancer, testicular cancer, head and neck squamous cell carcinoma, and combinations thereof. Exemplary diffuse tumors include, but are not limited to, ALL, CLL, AML, CML, Hodgkin lymphoma, non-Hodgkin lymphoma (e.g., lymphocytic plasmacytic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, or marginal zone lymphoma), multiple myeloma, and combinations thereof. According to one embodiment of this disclosure, the tumor is multiple myeloma.

[0073] Subjects that can be treated with this method include mammals such as humans, mice, rats, guinea pigs, hamsters, monkeys, pigs, dogs, cats, horses, sheep, goats, cattle, and rabbits. Preferably, the subject is a human.

[0074] The molecular constructs of this disclosure may be administered to subjects by appropriate routes such as oral, enteral, nasal, topical, transmucosal, or parenteral administration. Depending on the intended purpose, parenteral administration may be intratumoral, intramuscular, intravenous, intraperitoneal, or intra-arterial injection.

[0075] Pharmaceutical compositions for treating tumors (e.g., solid tumors or diffuse tumors) are also disclosed herein. According to some embodiments of this disclosure, the pharmaceutical composition comprises the molecular construct comprising an anti-CD38 antibody and a plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules conjugated to the anti-CD38 antibody, as described above. In these embodiments, administration of the pharmaceutical composition delivers an effective amount of lenalidomide molecules or hydrolyzed lenalidomide molecules at least 1 / 1000th of an effective amount of lenalidomide molecules used alone or in combination with the anti-CD38 antibody for the treatment of tumors.

[0076] According to some preferred embodiments, the pharmaceutical composition is administered in an amount of about 0.1 to 10 mg / kg. Preferably, the pharmaceutical composition is administered once every four weeks.

[0077] The following embodiments are provided to illustrate specific aspects of the present invention and to assist those skilled in the art in carrying it out. These embodiments should not be considered to limit the scope of the present invention in any way. Without further detail, those skilled in the art will be able to make the most use of the present invention based on the description herein. All publications cited herein are incorporated herein by reference in their entirety. [Examples]

[0078] Example 1: Synthesis of Mal-Lenalidomide Bundle A drug bundle containing three lenalidomide molecules was synthesized in this example. As shown in the structure in Figure 2, the central core had the sequence "EDEDEGAGGKGAGKGAGKG" (from N-terminus to C-terminus, SEQ ID NO: 11). A maleimide-ethyl-CO2H group (acting as a conjugate group) was attached to the N-terminus of the central core by forming an amide bond between the NH2 group of the first amino acid residue (i.e., the first "E" residue in SEQ ID NO: 11) and the CO2H group of the conjugate group. Three binding arms were attached to the ε-amino groups of the lysine (K) residues of the central core. Each binding arm consisted of three repeats of an ethylene glycol (EG) unit, a Val-Ala dipeptide recognized and cleaved by cathepsin B, and a para-aminobenzylcarbamate (PABC) for binding the lenalidomide molecule.

[0079] The maleimide-containing lenalidomide bundle (hereinafter referred to as "Mal-lenalidomide bundle") was synthesized using a combined method in which a standard Fmoc-based solid-phase synthesis was performed to synthesize the central core and assemble the drug bundle, and a liquid-phase synthesis was performed to synthesize the building blocks. Manufacturing was outsourced to WuXi STA Co., Ltd. (Shanghai, China). The synthesis of this Mal-lenalidomide bundle consisted of three steps: (i) building block synthesis, (ii) central core synthesis, and (iii) drug bundle assembly. Briefly, a Boc-protected Val-Ala dipeptide (compound 1) was sequentially bonded in solution to (4-aminophenyl)methanol (compound 2) and bis(4-nitrophenyl)carbonate to obtain compound 3, which was then similarly bonded to lenalidomide to obtain compound 4. After removing the Boc protecting group, the resulting compound 5 was bonded to (EG)3 by solid-phase peptide synthesis (SPPS) to produce compound 6, which was then bonded to the ε-amino group of Lys in the Fmoc-protected Gly-Lys-Gly tripeptide via SPPS to form building block 7. The central core 8 was synthesized by standard Fmoc-based SPPS and subsequently bonded to 2,3,4,5,6-pentafluorophenol at the C-terminus in solution to obtain compound 9. Using building block 7, compound 9, Fmoc-Ala-OH, and N-succinimidyl 3-maleimide propionate, the lenalidomide bundle 10 was assembled by SPPS.

[0080] The purified sample of the resulting Mal-lenalidomide bundle was analyzed by reverse-phase high-performance liquid chromatography (HPLC). Figure 3 shows the reverse-phase HPLC profile of the Mal-lenalidomide bundle, which indicates that the peak of the Mal-lenalidomide bundle had a residence time of 13.777 minutes.

[0081] The Mal-lenalidomide bundle was identified by mass spectrometry ESI-MS. Figure 4 shows the results of mass spectrometry ESI-MS, and the molecular construct was identified as [M+3H] 3+At 1379.235 corresponding thereto, it had a strong molecular ion. This indicates that the actual molecular weight (M.W.) of the Mal-lenalidomide bundle was 1379.235 × 3 - 3 = 4134.705 daltons, which matched the calculated 4135.1474.

[0082] Example 2 Construction of Recombinant Double-Stranded (Anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM Fusion Protein The V L and V H of the scFv specific for human CD38 were derived from daratumumab. The gene sequence encoding the anti-CD38scFv-Fc fusion protein was constructed by fusing it upstream of the gene sequence encoding the Fc region of human IgG1 (hIgG.Fc having a flexible hinge region, CH2 domain and CH3 domain). The gene sequence encoding the Zn 2+ binding motif ACPGHA (SEQ ID NO: 6, serving as a binding peptide and designated as "BM" in this study) was fused downstream of the gene sequence encoding the CH3 domain of hIgG1.Fc (lacking the C-terminal Lys) and a short (Gly)3 linker. The resulting gene sequence construct was inserted into the FREEDOM (registered trademark) pCHO1.0 expression cassette. The resulting fusion protein was in the form of a dimer of (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM, which had the amino acid sequence of SEQ ID NO: 12, and the anti-CD38scFv had V L -linker-V H and had V L and V H connected by the hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 13).

[0083] The composition of the prepared double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein is given in FIG. 5A.

[0084] Example 3: Expression and Purification of Recombinant Double-Stranded (Anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM Fusion Protein To ensure stable expression of the fusion protein, the expression plasmid was linearized by SspI digestion and then transfected into CHO-S® cells. The transfected cells were incubated at 37°C for 40–48 hours in an orbital shaker (150 rpm), followed by incubation in a selective medium containing 10 μg / mL puromycin and 200 nM methotrexate (MTX) for stable pool selection. The selective medium was changed twice weekly, and the expression of the recombinant double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein was confirmed weekly. After 2–3 weeks, cells stably expressing the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein were cryopreserved. The fusion protein was recovered from the supernatant of stable CHO-S® cells and purified using protein A chromatography. The buffer solution was replaced with phosphate-buffered saline (PBS), and the concentration of the fusion protein was identified and analyzed using SDS-PAGE. The data in Figure 5B show the analysis results, and the fusion protein was identified as the main band at approximately 110 kDa (lane 1 in Figure 5B), which is consistent with the predicted size. M is an abbreviation for protein marker. The antibody was dissolved in PBS with 50% glycerol and stored at approximately -20°C for further analysis.

[0085] Example 4 Synthesis of a molecular construct containing a recombinant double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein and two lenalidomide bundles In this example, a molecular construct was prepared having two lenalidomide bundles, each bound to two cysteine ​​residues of the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein (i.e., ACPGHA, SEQ ID NO: 6). A schematic diagram of the structure of this molecular construct is shown in Figure 6A. The purified double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein from Example 3 was prepared in conjugation buffer (10 mM sodium succinate, 30 mM sucrose, pH 6.0). The final concentration was adjusted to 15 μM, and then reduced by incubation with 6 equivalents of tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 minutes to form Zn molecules that can form unnecessary disulfide bonds with Cys or glutathione in the culture medium. 2+ The bound Cys was released. The reduced protein was dialyzed in a conjugation buffer containing 60 μM ZnCl2 using a slide-A-lyzer dialysis cassette at 25°C for 2 hours to (i) reconstruct the hinged disulfide bond, and (ii) Zn 2+ Bonding motif is Zn 2+ The fusion protein was combined with the fused protein. Three equivalents of the Mal-lenalidomide bundle from Example 1 (10 mM sodium succinate buffer, 30 mM sucrose, 60 μM ZnCl2, final concentration of 45 μM at pH 6.0) were added and incubated with the fusion protein in a round-bottom flask at 25°C for 10 minutes with stirring (600 rpm). To solubilize the product, an equal volume of 100% (w / v) sucrose was added to the resulting solution and stirred at 25°C for 16-18 hours. The fused product was analyzed by SDS-PAGE.

[0086] As shown in the data in Figure 6B, the molecular construct with two lenalidomide bundles had a MW of approximately 120 kDa (see the protein band labeled "#1" in lane 2), which was somewhat larger than expected. The unbound molecular construct is in lane 1 in Figure 6B and is labeled #2. M stands for protein marker. As shown in Figure 6B, the yield of the complex of the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein (hereinafter referred to as "natural double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle") with two lenalidomide bundles was approximately 85%.

[0087] Example 5: Stabilization of the native double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle in basic solution. To prevent the β-elimination reaction of the thioether bond, hydrolysis buffer (100 mM Tris, 100 mM NaCl, 100 mM L-arginine, and 50% sucrose (w / v), pH 9.0) at 1 / 10 volume was added to the double-chain (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle of Example 4 to hydrolyze the thiol-maleimide complex and induce ring-opening of the maleimide ring. The resulting solution was then stirred at 25°C for 2 days to hydrolyze the maleimide ring.

[0088] The reaction products of the molecular constructs obtained in Example 4 were exchanged using a column to obtain Tris buffer (100 mM Tris, 100 mM L-arginine, and 100 mM sodium chloride at pH 9.0). The resulting solution was then heated at 37°C for 5 hours. The solution was cooled, and the buffer was exchanged by centrifugation to obtain 50 mM Bis-Tris buffer at pH 5.5. The final sample was concentrated to approximately 1-3 mg / mL of protein. The resulting product was designated as "stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle".

[0089] Example 6 Purification of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle Prior to purification, the stabilization product from Example 5 was replaced with Buffer A (50 mM Na2HPO4, 1 M NaCl, pH 7.0). To remove unbound molecules or complexes with only one drug bundle, the mixture was applied to a pre-equilibrated hydrophobic interaction column (HIC), followed by three washing steps with 0, 50, and 65% Buffer B (50 mM sodium phosphate, pH 7.0) for 10, 20, and 15 column volumes, respectively. Complexes with two drug bundles were eluted with 100% Buffer B for 20 column volumes at a flow rate of 1.0 ml / min. The recovered samples from the preceding HIC purification were applied to a size exclusion chromatography (SEC) column to separate molecular constructs from protein aggregates. After HIC and SEC purification, the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle was obtained.

[0090] Figure 7A shows the results of SDS-PAGE analysis of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, where the lanes labeled 1 and 2 correspond to the unbound molecular construct and the molecular construct bound to the two lenalidomide bundles, respectively. The elution profile from size exclusion chromatography showed that the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle had a purity of over 95% (Figure 7B).

[0091] Example 7 Purification of the natural double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle To obtain the natural double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, the product from Example 4 was replaced with Buffer A (50 mM Na2HPO4, 1 M NaCl, pH 6.0) before purification. Briefly, the reaction mixture was applied to a pre-equilibrated hydrophobic interaction column (HIC), followed by three washing steps with 0, 50, and 65% Buffer B (50 mM sodium phosphate, pH 6.0) for 10, 20, and 15 column volumes, respectively. The complex with the two lenalidomide bundles was eluted with 100% Buffer B for 20 column volumes at a flow rate of 1.0 ml / min. The recovered samples from the preceding HIC purification were applied to a size exclusion chromatography column to separate the molecular constructs from protein aggregates. After HIC and SEC purification, a highly purified natural double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle was obtained.

[0092] Example 8 Establishment of an HPLC-based lenalidomide release assay A drug release assay was established to evaluate the release of free lenalidomide from the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle after internalization. In short, lenalidomide (1 μM) dissolved in PBS or RPMI medium, the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (1 μM) dissolved in RPMI medium, H929 cells cultured in RPMI medium, and 1 × 10⁶ cells in RPMI medium. 6Stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundles (1 μM) co-cultured with H929 cells were incubated at 37°C for 5 days. All supernatants were collected under the above conditions and centrifuged to remove cell debris. Molecules with MW less than 3 kDa were collected by column. The prepared samples were injected into a C8 column for HPLC analysis. The HPLC system was operated in gradient mode at a flow rate of 1 mL / min. Solvent A consisted of water with 0.1% trifluoroacetic acid (TFA), and solvent B consisted of acetonitrile with 0.1% TFA. The HPLC program was maintained for 8 minutes starting with 0% solvent B, then a linear gradient was started with 0% solvent B and increased to 100% solvent B within 20 minutes. After maintaining pure solvent B for 10 minutes, the column was regenerated with 100% solvent A within 7 minutes. The injection volume was 100 mL, and the column oven temperature was set to 25°C. The total operating time was 45 minutes.

[0093] Example 9: Structural analysis of lenalidomide molecules released from a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle. In the study analyzing the structure of the released lenalidomide molecule, lenalidomide incubated in PBS for 5 days was used as a control group. After long-term incubation, it showed a peak corresponding to the lenalidomide molecule (referred to as p3) and two further peaks (p2 and p1) corresponding to its two hydrolysis forms, respectively (Figure 8A, Group (1): Lenalidomide). The three peaks in the HPLC profile were confirmed by mass spectrometry. As shown in the analysis results in Figure 8B, the three peaks (referred to as (1), (2), and (3), respectively) were detected after replacing the lenalidomide in PBS with RPMI medium, and peak (1) was... [ka] This corresponds to hydrolyzed lenalidomide having the structure (Panels (A) and (B) in Figure 8B), and peak (2) is [ka] This corresponds to hydrolyzed lenalidomide having the structure (Panels (A) and (C) in Figure 8B), and peak (3) is [ka] This corresponds to lenalidomide having the structure shown (Panels (A) and (D) in Figure 8B). The two hydrolysis forms of lenalidomide (p1 and p2) could be detected in the culture supernatant of H929 cells incubated for 5 days with a stabilized double-chain (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, while the original lenalidomide (p3) was not detected (Figure 8A, Group (6): H929 cells + complex). The p1, p2, or p3 peaks were not observed in RPMI medium alone (Figure 8A, Group (2): RPMI), in the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle incubated in RPMI medium for 5 days (Figure 8A, Group (4): RPMI+ complex), or in the supernatant of H929 cells cultured in RPMI medium for 5 days (Figure 8A, Group (5): H929 cells).

[0094] Therefore, these results indicate that the lenalidomide molecule supported on the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein is released in hydrolytic forms (p1 and p2).

[0095] Example 10: MALDI-TOF analysis of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle for DAR identification. The stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle was analyzed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF). Saturated sinapic acid (2 mg / mL in 0.1% TFA with acetonitrile:water 30:70 (v / v)) was directly spotted onto a MALDI target plate as the desorption matrix. Each spot was analyzed using a MALDI TOF / TOF equipped with a 200 Hz laser. Data acquisition and processing were performed using software. The analysis results showed that the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle had a m / z (z=1):[M+H] + and m / z(z=2):[M+2H] 2+ The corresponding proteins have MWs of 58382 and 116705 Daltons (lower panel of Figure 9), while the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein (untreated, serving as a control) has MWs of 54300 and 108537 Daltons, and their m / z (z=1):[M+H] + and m / z(z=2):[M+2H] 2+ This corresponds to (upper panel in Figure 9).

[0096] In MALDI-TOF analysis, the confirmed m / z values ​​for the single-charged (Z=1) and double-charged (Z=2) stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundles were 116820 and 58501 Da, respectively, while the corresponding m / z values ​​for the drug-free double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein were 108534 and 54311 Da, respectively (data not shown). Since the lenalidomide bundle has a MW of 4205 Da, the increase in MW of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle by 8286 Da compared to the unbound double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein against Z=1 represents the MW of the two lenalidomide bundles. Therefore, the drug-antibody ratio (DAR) of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle molecule is 6.

[0097] Example 11: Analysis of drug bundle binding sites for stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle. To identify cysteine ​​residues in the stabilized double-stranded (anti-CD38scFv)-Fc-BM-lenalidomide bundle, samples were digested and analyzed using LC-MS. Briefly, to reduce disulfide bonds, stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (2 μg) was dissolved in PBS at pH 7.4 and incubated with 18 mM TCEP in 17 mM triethylammonium bicarbonate (TEAB) buffer at 55°C for 10 minutes in the dark. After alkylation with iodoacetamide (IAA) at a final concentration of 15.6 mM for 30 minutes at room temperature in the dark, each sample was incubated overnight with trypsin (0.2 μg) at 37°C. Samples were detected by liquid chromatography (LC) ESI-MS using a mass spectrometer. The digestion solution (5 μl) was injected into a capillary column (C18, 0.075 mm × 150 mm, ID 3 μm) at a flow rate of 1 μl / min. The following gradient program was used for chromatographic separation. First, 100% mobile phase A (0.1% formic acid in water) was used at a flow rate of 300 nL / min, which was reduced to 98% over 2 minutes with 2% mobile phase B (0.1% formic acid in 80% acetonitrile), and further reduced to 60% over 40 minutes with 40% mobile phase B. Full scan mass spectrometry was recorded, and the target m / z was isolated for collision-induced dissociation using NCE35 and a maximum injection time of 100 ms. The LC-MS spectrum was searched for the expected molecular weight of the peptide (1366 daltons) by additional MW of carbamide methyl group (58 daltons) or lenalidomide bundle (4205 daltons). The cysteine-containing peptide sequence was identified using the Mascot search engine.

[0098] Mass spectrometry analysis revealed that the m / z value of the fragment in the MS spectrum corresponds to 5571.29 daltons (data not shown), which is consistent with the molecular weight of the fragment containing the amino acid sequence "SLSLSPGGGGACPGHA" (sequence number 14, amino acid residues 472-487 of sequence number 12) (1366 daltons) and one lenalidomide bundle (4205 daltons).

[0099] Example 12: Generation of stable human CD38 overexpressing HEK293T cells Full-length human CD38 cDNA was cloned into a pCDH-CMV-MCS-Ef1α-Puro expression vector to generate the lentivirus-based CD38 expression plasmid pCDH-CMV-CD38. The lentivirus was packaged by co-transfecting HEK293T cells with pCDH-CMV-CD38, pCMV-VSV-G, and pCMVδR8.91. Human CD38-overexpressing HEK293T cells were transduced by lentivirus infection and cultured with 1 μg / mL puromycin 3 days after infection. Human CD38 expression was confirmed by flow cytometry.

[0100] Example 13: Binding activity of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle to human CD38-overexpressing HEK293T cells. To confirm that the two lenalidomide bundles bound at the antibody C-terminus did not alter the antibody's targeting ability, the binding activity of the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein and the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle to human CD38 was identified by cell-based ELISA using human CD38-overexpressing 293T cells. The stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (labeled as "stabilized complex") and the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein (labeled as "unbound") showed similar binding activity to human CD38 (Figure 10). The results showed that the EC of the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle and the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein 50The values ​​were 0.65 and 0.84 nM, respectively. Therefore, the binding of two lenalidomide bundles to the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein did not impair its binding affinity to human CD38.

[0101] Example 14: Binding activity of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle to human multiple myeloma H929 cell line. Surviving human CD38 + Multiple myeloma H929 cells (H929 and U266-CD38 + The binding affinity of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle to cells (including cells) was verified in this example.

[0102] In short, approximately 2 x 10 5 Individual H929 cells or U266-CD38 + Cells were washed three times with flow cytometry (FACS) buffer (PBS containing 1% fetal bovine serum) and incubated for 20 minutes with stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundles and daratumumab at different concentrations (1 μM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, and 1 pM). After washing with FACS buffer, cells were labeled with phycoerythrin (PE)-conjugated anti-human IgG-Fc antibody, and the levels of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundles and daratumumab on the cell surface were detected. Unstained cells and cells treated with secondary antibodies only were used as negative controls. Cell-associated fluorescence was identified using flow cytometry and analyzed by software.

[0103] Similar to the "parent" daratumumab (labeled as "dara"), the stabilized double-strand (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (labeled as "stabilized complex") specifically and dose-dependently controls H929 and U266-CD38 + It showed binding activity to cells (Figure 11). However, this was not observed with H929 cells (4.06 nM) and U266-CD38. + EC for the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle against cells (1.66 nM) 50 The values ​​were higher than those for the "parent" daratumumab (1.02 and 0.13 nM, respectively), indicating that the binding activity of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle to human CD38 was lower than that of daratumumab.

[0104] Example 15: Intracellular relocation of a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle. An internalization assay was performed in the examples to verify the degree of internalization of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle into H929 cells.

[0105] In short, 2 x 10 5 H929 cells per well were incubated on ice for 20 minutes with 1 μM stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (in FACS buffer). After washing and resuspending with fresh medium, the cells were incubated at 37°C for 0.5, 1, 2, or 3 hours. The cells were washed and incubated on ice for 20 minutes with PE-conjugated goat anti-human IgG-Fc antibody. After washing, cell-associated fluorescence was identified by flow cytometry and analyzed by software. The internalization percentage was calculated from dM, the average fluorescence intensity at a given time t relative to the average background fluorescence intensity, according to equation (1). %Internalization=[dM(t=0)-dM(t=x)]×100 / dM(t=0) (1)

[0106] The signal intensity of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle in viable H929 cells decreased by approximately 15% after 30 minutes of incubation and by a further 60% after 3 hours (Table 1, viable cells), indicating that fewer molecules of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle remained bound to the cell surface. In contrast, no loss of signal intensity of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle was detected in fixed cells after 3 hours of incubation (Table 1, fixed cells), indicating that the decrease in signal intensity in viable cells was not due to dissociation of the molecular construct during incubation. Table 1. Dynamics of internalization of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle in H929 cells [Table 1]

[0107] Example 16: In vitro cytotoxic activity of a stabilized double-chain (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle via hydrolysis of lenalidomide molecule release. To evaluate the in vitro cytotoxic activity of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, the alamarBlue® cell viability assay was performed. In short, H929, U266-CD38 -MM.1S and Daudi cells were seeded at 5000 cells / well in 96-well plates and co-cultured at 37°C for 5 hours, 1 day, 3 days, or 5 days with fresh medium containing the indicated concentrations of the test substance. Cell viability was determined using alamarBlue® cell viability reagent. Approximately 10 μl of alamarBlue® cell viability reagent was added to the cells at a final concentration of 10% v / v, followed by incubation at 37°C for 1.5 hours. Fluorescence was measured in any fluorescence unit using a microplate reader, following excitation at 560 nm and emission at 590 nm.

[0108] The in vitro tumor cell-killing effect of a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle against a panel of multiple myeloma (MM) cells was compared to (1) daratumumab, (2) daratumumab co-administered with lenalidomide in a molar ratio of 1:6 similar to the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, and (3) three other anti-MM agents containing lenalidomide (equal to the amount of lenalidomide used in the daratumumab / lenalidomide combination). Different MM cells were incubated with a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (labeled as "stabilization complex"), a double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein (unbound, labeled as "α-CD38mAb"), daratumumab, a combination of daratumumab and lenalidomide (labeled as "daratumumab / lenalidomide"), and serial dilutions of lenalidomide. H929 cells were incubated for 5 hours, 1 day, 3 days, or 5 days (Figures 12A-12D) to obtain MM.1S, U266-CD38 - The Daudi cells were incubated for 5 days (Figures 12E-12G). Cell viability was then determined.

[0109] Treatment with the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (i.e., the "stabilizing complex") resulted in approximately 0.45 μM EC2 for both H929 and MM.1S cells. 50 Dose-dependent cytotoxic effects were observed (Figures 12B-12E). In contrast, because daratumumab-mediated cell death depends on the presence of other immune cells such as natural killer cells, the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein (unbound, i.e., "anti-CD38mAb") and parental daratumumab did not show a cell death effect on the same cells, even at doses greater than 20 μM (Figures 12B-12E). Lenalidomide or daratumumab / lenalidomide combination at doses greater than 10 μM showed only weak killing activity on H929 and MM.1S cells (Figures 12B-12E), indicating that only a small amount of lenalidomide entered the target MM cells, and once inside, it showed a cytotoxic effect on the MM cells. The hydrolyzed lenalidomide molecule (not lenalidomide) was released from the stabilized double-chain (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (i.e., the "stabilizing complex"), and the results in Figures 12D and 12E showed that the stabilized double-chain (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle was more than 100 times more effective than lenalidomide alone or in combination with daratumumab. U266-CD38 - Regarding cells, the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (i.e., the "stabilizing complex"), the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM (unbound, i.e., "anti-CD38mAb"), and daratumumab did not have a killing effect. However, lenalidomide and its combination with daratumumab showed a killing effect at high concentrations, possibly due to the effect of lenalidomide (Figure 12F). Daudi B lymphoma cells expressed CD38, but none of the five treatments showed a killing effect, likely because these cells were resistant to lenalidomide (Figure 12G).

[0110] Furthermore, the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle exhibited the desired toxicity to tumor cells by tightly binding to CD38 on the surface of MM cells to form a CD38 complex. This allowed for efficient internalization of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle via endocytosis, followed by cleavage by lysosomal cathepsin B, releasing cytotoxic lenalidomide and leading to cell death. Lenalidomide, daratumumab, or their combination did not possess this mechanism of action. Lenalidomide unbound to daratumumab could not easily enter cells, but daratumumab required Fc-dependent endo-effector function to kill CD38-expressing tumor cells. Therefore, the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle exhibited more effective cytotoxic effects in in vitro assays than lenalidomide, daratumumab, or combinations thereof.

[0111] Example 17: In vitro cytotoxic activity of a natural double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle via release of a natural lenalidomide molecule. The in vitro cytotoxic activity assay of the native double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle from Example 4 against human multiple myeloma H929 and MM.1S cells was performed as described in the above example.

[0112] The stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (i.e., the "stabilized complex") was used as a positive control. The results in Table 2 show that the native double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (i.e., the "native complex") yielded 0.722 μM EC for 2 hours of treatment and 0.207 μM EC for 5 days of treatment in H929 cells. 50 The values ​​demonstrated effective cytotoxicity. Table 2 In vivo cytotoxicity of specified treatments in H929 cells [Table 2]

[0113] Furthermore, the results in Table 3 show that the natural double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (i.e., the "natural complex") yielded 0.661 μM EC for MM.1S cells after a 2-hour treatment and 0.190 μM EC for a 5-day treatment. 50 The values ​​demonstrated effective cytotoxicity. Table 3 In vivo cytotoxicity of specified treatments in MM.1S cells [Table 3]

[0114] Example 18 Plasma stability of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle To evaluate the plasma stability of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, the required amount of double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM (unbound antibody), stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, or daratumumab was dissolved in 90% human plasma at a final concentration of 2 mg / mL and incubated at 37°C for 28 days. Sample aliquots were collected on days 0, 3, 7, 10, 14, 21, and 28 and analyzed by ELISA. Recombinant human CD38 protein was coated as an antigen to capture daratumumab, double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM (unbound antibody), and stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundles. Three CD38-binding antibodies were detected using an HRP-conjugated anti-human IgG-Fc antibody as a secondary antibody. To determine whether the two lenalidomide bundles remained bound to the antibody on the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, an anti-lenalidomide bundle scFv-mouse IgG-Fc antibody, which is further detectable by an HRP-conjugated anti-mouse IgG-Fc antibody, was used. 96-well ELISA plates were coated with recombinant human CD38 protein (0.1 μg / 100 μL / well) at 4°C for 16 hours. After washing with PBST buffer (PBS containing 0.1% TWEEN® 20), each well was blocked with blocking buffer (PBS containing 1% fetal bovine serum) for 30 minutes. Standards were prepared by dissolving a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle with human plasma and diluting it in blocking buffer. All samples were prepared by diluting them in blocking buffer to match the linearity of the calculated curve. Standards and diluted samples were added to each well and incubated at room temperature for 1.5 hours. After washing three times with PBST, secondary antibodies of appropriate dilution in blocking buffer were added to the corresponding wells.To detect the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, the anti-lenalidomide bundle scFv mouse IgG-Fc antibody was washed away, and then the HRP-conjugated anti-mouse IgG-Fc antibody was added. Finally, the concentration of each antibody was quantified using tetramethylbenzidine reagent and a microplate reader. Half-life (T.) 1 / 2 The formula was calculated using software. The data is shown as mean ± standard deviation.

[0115] The results shown in Figure 13 indicate that daratumumab, the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein (labeled as "unbound"), and the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle had equivalent half-lives of approximately 6-7 days in human plasma (T 1 / 2 The study demonstrated that the two lenalidomide bundles were shared for 28 days. The secondary antibody, i.e., the HRP-conjugated anti-human IgG-Fc antibody, could not distinguish between the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle and the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein bound to a lenalidomide bundle of less than 1. Therefore, the anti-lenalidomide bundle scFv-mouse IgG-Fc antibody detected by the secondary antibody, i.e., the HRP-conjugated anti-mouse IgG-Fc antibody, was used to determine whether the two lenalidomide bundles remained bound to the anti-CD38 antibody. The concentrations of the lenalidomide bundle-binding antibody at various time points were used to determine its T 1 / 2 (7.7 days) (Labeled as "Stabilized ADC (Conjugated Ab)," i.e., the double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle detected by anti-lenalidomide bundle antibody and HRP-conjugated anti-mouse IgG-Fc antibody) is the total stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle detected by HRP-conjugated anti-human IgG-Fc antibody. 1 / 2(7.5 days) (labeled as "stabilized ADC (total Ab)" containing both a double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle and a double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM fusion protein bound to a lenalidomide bundle of 1 or less) showed to be nearly identical (Figure 13). This suggests that the two lenalidomide bundles were stably bound to the anti-CD38 antibody in human plasma. Furthermore, despite the decrease in the scFv of the anti-CD38 antibody, its T 1 / 2 This is the T of daratumumab. 1 / 2 It did not decrease compared to [the previous case].

[0116] Example 19: Establishment of an in vivo tumor model of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle. To generate subcutaneous tumors, 2 × 10 in PBS 7 H929 cells were mixed with 50% (v / v) extracellular matrix gel and subcutaneously injected into both flanks of NOD-SCID mice. Tumor size and body weight were recorded every 2-3 days. The volume of the in vivo subcutaneous xenograft tumors was determined using an external caliper, and the tumor volume was calculated using the modified ellipsoid formula, 1 / 2 × (length × width × width). The average tumor size was 115 ± 15 mm. 3 (7 days after transplantation) or 150±20mm 3If the period was within the range of 14 days post-transplant, stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM (unbound antibody), and daratumumab were prepared in PBS and administered intraperitoneally (ip) to mice. In all experiments, a single dose (20 nmol / kg, approximately 2.3 mg / kg) of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle or daratumumab was administered. At the end of the experiment, tumors were collected and weighed. Data were analyzed by software and expressed as mean ± SD or mean ± SEM as shown in the figure. Statistical analysis was performed using Student's t-test, and statistical differences between groups were analyzed using Bonferroni's multiple comparison test followed by one-way ANOVA.

[0117] Example 20: Antitumor efficacy of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle and daratumumab in a mouse xenograft tumor model. In a pilot study using H929 cells, tumors were 115±15mm in size. 3 Once the tumors reached their average size, a single dose (20 nmol / kg) of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle or daratumumab was administered intravenously at 7 days post-transplant. Changes in tumor size, measured every 2-3 days over 21 days, demonstrated that the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle effectively reduced tumor size, while daratumumab (labeled "Dara") only slowed tumor growth compared to PBS. Tumors were excised and weighed from all mice 21 days post-drug administration. The results showed that the stabilized double-strand (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (labeled as the "stabilized complex") exhibited significantly better growth inhibitory activity against H929 tumors compared to daratumumab.

[0118] Example 21: Antitumor efficacy of stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle, daratumumab, lenalidomide, and daratumumab / lenalidomide combination in a mouse xenograft tumor model. Furthermore, the ability of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle to inhibit tumor growth when the tumor was allowed to grow larger for several more days was further investigated. On day 14 after transplantation of H929 cells, the tumor was approximately 150 ± 20 mm. 3 Once the average volume was reached, the mice were administered (1) a single dose of daratumumab (20 nmol / kg), (2) daily intravenous injection of lenalidomide (46 mmol / kg / day), (3) a combination of daratumumab and lenalidomide (single dose of 20 nmol / kg of daratumumab + 46 mmol / kg / day of lenalidomide), and (4) a single dose of a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (labeled as "stabilized complex") (20 nmol / kg, approximately 2.3 mg / kg), respectively. The results in Figure 15A demonstrate that treatment with daratumumab (Dara) or lenalidomide (Lena) inhibited tumor growth compared to PBS treatment, and that the combination of daratumumab / lenalidomide (Dara / Lena) treatment showed a greater inhibitory effect on tumor growth. Tumors were excised and weighed from all mice 28 days after drug administration (Figure 15B). Surprisingly, treatment with the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle (stabilized complex) completely suppressed tumor growth in mice, leaving only a micronodule in one mouse (Figures 15A and 15B). The results showed that the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle exhibited a significantly better antitumor effect against H929 tumors compared to treatment with daratumumab, lenalidomide, or daratumumab / lenalidomide combination therapy.

[0119] In particular, the total amount of lenalidomide administered at a dose of 46 mmol / kg / day over a 28-day period was 10,640 times the amount of lenalidomide given by a single dose (20 nmol / kg) of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle.

[0120] Example 22: Antitumor efficacy of a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle and daratumumab / lenalidomide combination using patient-derived multiple myeloma cells. To evaluate the efficacy of a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle in patient-derived multiple myeloma (MM) cells, MM cells isolated from the bone marrow of five newly diagnosed MM patients (No. 1-5) were treated for 5 days at 37°C with various concentrations (0.00064, 0.0032, 0.016, 0.08, 0.4, 2, 10 μM) of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle or in combination with daratumumab and lenalidomide (i.e., daratumumab / lenalidomide, which served as the control group). Cell viability was determined using the alamarBlue® cell viability reagent.

[0121] The results of cell viability studies on MM cells treated with a stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle revealed a significant decrease in viability. The semi-maximal inhibitory concentration (IC) of the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle was also observed. 50 The values ​​are in the range of 0.07 to 0.27 μM, and the IC for each combined treatment group 50 The values ​​exceeded 100 μM (Table 4). Table 4 Designated Treatment IC 50 value [Table 4]

[0122] Furthermore, IC 50 The analysis of values ​​showed that IC was obtained by the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle compared to free lenalidomide in daratumumab / lenalidomide combination therapy. 50 A reduction of more than 100 times was demonstrated. This result indicates that lenalidomide supported in the stabilizing complex is more effective in killing MM cells, and that its effectiveness is further enhanced when it is internalized by MM cells.

[0123] The results suggest that the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle exhibits effective antitumor efficacy in patient-derived MM cells. IC compared with no lenalidomide. 50 The significant decrease in values ​​highlights the enhanced effectiveness of lenalidomide bundle binding.

[0124] In summary, the stabilized double-stranded (anti-CD38scFv)-hIgG1.Fc-(Gly)3-BM-lenalidomide bundle showed more effective cytotoxicity in in vivo assays than lenalidomide, daratumumab, or combinations thereof.

[0125] It should be understood that the above description of embodiments is given for illustrative purposes only and that various modifications can be made by those skilled in the art. The above specifications, examples and data provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various embodiments of the present invention have been described above with some degree of specificity or by reference to one or more individual embodiments, those skilled in the art can make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the present invention.

Claims

1. A pharmaceutical composition for treating a tumor, comprising a molecular construct comprising an anti-CD38 antibody, a plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules bound to the anti-CD38 antibody, and a linker unit, The aforementioned anti-CD38 antibody is A pair of CH2-CH3 segments of IgG, A pair of anti-CD38scFv is bonded to the N-terminuses of the pair of CH2-CH3 segments, A pair of binding peptides each attached to the C-terminus of the pair of CH2-CH3 segments, comprising a pair of binding peptides having a plurality of C residues, Equipped with, Each of the pair of binding peptides comprises the amino acid sequence "CGGHA" ​​(SEQ ID NO: 1), "CPGHA" (SEQ ID NO: 2), "CGAHA" (SEQ ID NO: 3), "CPAHA" (SEQ ID NO: 4), "GCGGHA" ​​(SEQ ID NO: 5), "ACPGHA" (SEQ ID NO: 6), or "GCPGHA" (SEQ ID NO: 7). Each of the plurality of lenalidomide molecules or hydrolyzed lenalidomide molecules is bound to the plurality of C residues of the pair of binding peptides. The aforementioned linker unit is A central core that is linear, 2 to 10 K residues, At least one filler independently positioned between two K residues, A terminal spacer having two ends, wherein one end is bound to the N-terminus of the first K residue or the C-terminus of the last K residue, and the other end is bound to the C residue of the binding peptide of the anti-CD38 antibody, Equipped with, Each of the filler and the terminal spacer independently comprises a central core having (1) 1 to 12 non-K amino acid residues or (2) PEGylated amino acids having 1 to 12 repeats of ethylene glycol (EG) units, A binding arm comprising 2 to 10 binding arms, wherein one end of each binding arm is bound to one of the K residues of the central core, and the other end of each binding arm is bound to each lenalidomide molecule or hydrolyzed lenalidomide molecule, Equipped with, The pharmaceutical composition is administered in an amount of about 0.1 to 10 mg / kg, wherein the administration of the pharmaceutical composition provides an effective amount of the lenalidomide molecule or the hydrolyzed lenalidomide molecule that is at least 1 / 1000th of the effective amount of the lenalidomide molecule used alone or in combination with the anti-CD38 antibody for the treatment of the tumor.

2. The pharmaceutical composition according to claim 1, wherein the effective amount of the lenalidomide molecule or the hydrolyzed lenalidomide molecule is about 1 / 10,000th of the effective amount of the lenalidomide molecule used alone or in combination with the anti-CD38 antibody for the treatment of the tumor.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered once every four weeks.

4. The pharmaceutical composition according to claim 1, wherein each of the pair of binding peptides comprises the amino acid sequence "ACPGHA" (SEQ ID NO: 6).

5. The pharmaceutical composition according to claim 1, wherein the terminal spacer comprises at least three negatively charged amino acid residues.

6. The pharmaceutical composition according to claim 5, wherein the terminal spacer comprises the amino acid sequence "EDEDEAGG" (SEQ ID NO: 8), "EGEGEAGG" (SEQ ID NO: 9), or "EGEG" (SEQ ID NO: 10).

7. The pharmaceutical composition according to claim 6, wherein the central core comprises the amino acid sequence "EDEDEGAGGGKGAGKGAGKG" (SEQ ID NO: 11).

8. The pharmaceutical composition according to claim 1, wherein each of the binding arms comprises a polyethylene glycol (PEG) chain having 2 to 12 non-K amino acid residues, 2 to 24 repeats of an EG unit, or a combination thereof.

9. The pharmaceutical composition according to claim 1, wherein each of the binding arms is bonded to the ε-amino group of the K residue.

10. The pharmaceutical composition according to claim 1, wherein the tumor is a solid tumor or a diffuse tumor.

11. The pharmaceutical composition according to claim 10, wherein the solid tumor is melanoma, esophageal cancer, gastric cancer, brain tumor, small cell lung cancer, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, thyroid cancer, testicular cancer, or head and neck squamous cell carcinoma.

12. The pharmaceutical composition according to claim 10, wherein the diffuse tumor is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

Images