Anti-CD19 antibodies and methods of use and preparation thereof

Abstract

An isolated monoclonal antibody (mAb) or antigen-binding fragment thereof having binding specificity for human CD19 wherein the isolated mAb or antigen-binding fragment comprises an amino acid sequence having identity to a sequence selected from the group consisting of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93 wherein the identity is not less than 95%.

Description

[0001] This application is a divisional application of the Chinese invention patent application (application number: 202180005432X; invention name: anti-CD19 antibodies and their use and preparation methods).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application Serial No. 62 / 984,731, filed on March 3, 2020, the entire disclosure of which is incorporated herein by reference. Technical Field

[0004] The present disclosure relates generally to the field of biotherapeutic technology and, more particularly, to making and using multispecific antibodies. Background Art

[0005] Lymphomas account for 4.3% of all cancers diagnosed in the United States each year, and B-cell malignancies account for approximately 90% of all lymphoma diagnoses. CD19 is a B-lymphocyte-specific member of the immunoglobulin superfamily expressed by B lymphocytes at different stages of differentiation, starting with V(D)J rearrangement until B cells mature into plasma cells, at which time the surface expression of CD19 appears to be lost. Although CD19 is widely used as a pan-B cell marker, it has been found that CD19 is highly expressed in many forms of leukemia and lymphoma, with characteristics of B cell origin. For more than 30 years, CD19 has been the focus of immunotherapy development. Pharmaceutical companies are actively pursuing anti-CD19 strategies because they have the promise of directly targeting those B-cell malignancies corresponding to early B-cell differentiation stages. Targeting CD19 has been approved as an excellent immunotherapy strategy, especially when antibody treatment targeting CD22 (another pan-B cell marker expressed by B-cell malignancies) is unsuccessful.

[0006] CD19 is an important cell surface marker for normal B cells and cancers of B cell origin. Therefore, it is highly desirable to have an antibody targeting CD19 for anticancer therapy. Reports in the literature indicate that it is difficult to identify anti-CD19 antibodies that also cross-react with CD19 found in cynomolgus monkeys, a property that greatly facilitates therapeutic pharmacology and toxicology studies. The historical antibody BU12 has been shown to have high affinity for human CD19 and cross-reactivity to cynomolgus monkey CD19, however, the antibody was discovered from a mouse hybridoma and does not contain human framework sequences. Therefore, a humanized variant of BU12 is highly desirable for therapeutic use. Summary of the invention

[0007] The present application provides anti-CD19 peptides, proteins, protein complexes, antibodies, and methods for preparing and using the same.

[0008] In one aspect, the present application provides peptides with binding specificity to human CD 19. In one embodiment, the peptide has an amino acid sequence with at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93.

[0009] In one embodiment, the peptide is a scFv peptide. In one embodiment, the scFv peptide may have a binding affinity for human CD19 with a KD of no greater than 1 nM, 2 nM, 3 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 40 nM or 50 nM.

[0010] In one aspect, the present application provides antibodies or antigen-binding fragments thereof having binding specificity to human CD19. In one embodiment, the isolated antibody or antigen-binding fragment comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO.1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93. In one embodiment, the antibody comprises an isolated monoclonal antibody (mAb).

[0011] In one embodiment, the antibody is a bispecific antibody. In one embodiment, the antibody is a multispecific antibody. In one embodiment, the antibody is a trispecific antibody, a tetraspecific antibody, a pentaspecific antibody, or a hexaspecific antibody.

[0012] In one embodiment, the antibody comprises a scFv, wherein the scFv comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93.

[0013] In one embodiment, the antibody comprises a Fab, wherein the Fab comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93.

[0014] In one embodiment, the present application provides a multi-specific antibody-like protein. In one embodiment, the protein comprises a peptide having an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO.1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93. In one embodiment, the multi-specific antibody-like protein has an N-terminus and a C-terminus, and from the N-terminus to the C-terminus, it comprises a first binding domain (D1) at the N-terminus, a second binding domain (D2) comprising a light chain portion, an Fc region, a third binding domain (D3) and a fourth binding domain (D4) at the C-terminus. The light chain portion comprises a fifth binding domain (D5) covalently linked to the C-terminus, a sixth binding domain (D6) covalently linked to the N-terminus, or both, and D1, D2, D3, D4, D5 and D6 each have binding specificity for a tumor antigen, an immune signaling antigen, or a combination thereof.

[0015] In one embodiment, the multispecific antibody-like protein is pentaspecific.In one embodiment, the antibody-like protein comprises a binding domain comprising D1, D2, D3, D4 and D6.

[0016] In one embodiment, the multispecific antibody-like protein is hexaspecific.

[0017] In one embodiment, D1 comprises a peptide comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93.

[0018] In one embodiment, D1 comprises a peptide having an amino acid sequence having 95% sequence identity to SEQ ID NO. 7 or 19.

[0019] In one embodiment, D2 comprises a peptide having an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93.

[0020] In one embodiment, D2 comprises a peptide having an amino acid sequence having 95% sequence identity with SEQ ID NO. 91 or 93.

[0021] In one embodiment, D6 comprises a peptide having an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93.

[0022] In one embodiment, D6 comprises a peptide having an amino acid sequence having 95% sequence identity to SEQ ID NO. 7 or 19.

[0023] In one embodiment, the present application provides a multispecific monoclonal antibody comprising a multispecific antibody-like protein as claimed herein.

[0024] In one embodiment, the multispecific monoclonal antibody may have a binding affinity for human CD19 with a Kd of no greater than 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, or 50 nM.

[0025] In one embodiment, the antibody is a human antibody. In one embodiment, the multispecific monoclonal antibody is an IgG.

[0026] In one embodiment, the application provides an isolated nucleic acid encoding an isolated mAb or antigen binding fragment, IgG1 heavy chain, kappa light chain, variable light chain or variable heavy chain, as disclosed herein.

[0027] In one aspect, the present application provides an isolated nucleic acid sequence encoding the amino acid sequence of the multispecific monoclonal antibodies disclosed herein.

[0028] In one embodiment, the present application provides an expression vector comprising an isolated nucleic acid sequence as disclosed herein.

[0029] In one embodiment, the present application provides a host cell comprising a nucleic acid sequence as disclosed herein. In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0030] In one aspect, the present application provides a method for producing an antibody, comprising culturing a host cell to produce the antibody.

[0031] In one aspect, the application provides an immunoconjugate. In one embodiment, the immunoconjugate comprises an isolated mAb or an antigen-binding fragment thereof and a drug unit, wherein the drug unit is connected to the isolated mAb or antigen-binding fragment via a linker, and wherein the linker comprises a covalent bond selected from an ester bond, an ether bond, an amine bond, an amide bond, a disulfide bond, an imide bond, a sulfone bond, a phosphate bond, a phosphoester bond, a peptide bond, a hydrazone bond, or a combination thereof.

[0032] In one embodiment, the drug unit comprises a cytotoxic agent, an immunomodulatory agent, an imaging agent, or a combination thereof. In one embodiment, the cytotoxic agent is selected from a growth inhibitor or a chemotherapeutic agent from a class of tubulin binding agents, DNA intercalators, DNA alkylating agents, enzyme inhibitors, immunomodulators, antimetabolites, radioisotopes, or a combination thereof. In one embodiment, the cytotoxic agent is selected from calicheamicin, camptothecin, ozogamicin, monomethyl auristatin E, emtansine, derivatives thereof, or combinations thereof.

[0033] In one embodiment, the immunomodulatory agent activates or inhibits immune cells, T cells, NK cells, B cells, macrophages, or dendritic cells. In one embodiment, the imaging agent can be a radionuclide, a fluorescent agent, a quantum dot, or a combination thereof.

[0034] In one aspect, the present application provides a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises an isolated mAb or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may also include a chemotherapeutic agent, a growth inhibitor, a cytotoxic agent from a class of calicheamicins, an antimitotic agent, a toxin, a radioisotope, a therapeutic agent, or a combination thereof.

[0035] In one aspect, the present application provides a pharmaceutical composition comprising the immunoconjugate disclosed herein and a pharmaceutically acceptable carrier.

[0036] In one aspect, the present application provides a method for treating a subject suffering from cancer. In one embodiment, the method comprises administering to the subject an effective amount of an isolated mAb or its disclosed antigen-binding fragment. In one embodiment, the method may also comprise co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. In one embodiment, the subject is a human.

[0037] In another aspect, the present application provides a solution comprising an effective concentration of a multispecific monoclonal antibody as disclosed herein. In one embodiment, the solution is plasma from a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The foregoing and other features of the present disclosure will become more apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the present disclosure and are therefore not to be considered limiting of its scope, the present disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:

[0039] Figure 1The increase in humanness scores (Z-scores) from the mouse sequence (grey line) to the humanized framework (black line) in the variable regions of humanized BU12, H4 (Vk of kappa light chain in 1A, VH of heavy chain in 1B), and H5 (Vk of kappa light chain in 1C, VH of heavy chain in 1D) are shown;

[0040] FIG2 shows a sequence alignment of the variable regions (VL of the light chain in 2A and VH of the heavy chain in 2B) of humanized mouse BU12 (H1-H6 and H7) and a human antibody (21D4);

[0041] Figure 3 DLS thermal stability of SI-63C1 (BU12-chimeric), SI-63C2 (humanized BU12, H1), and SI-34C1 (human antibody, 21D4) are shown;

[0042] FIG4 shows histograms depicting the cross-reactivity of the SI-63C2 antibody to human, cynomolgus and rhesus CD20+ B cells (4A) and CD20- lymphocytes (4B);

[0043] Figure 5 shows dose-response curves of SI-63C2 antibody binding to human (5A), cynomolgus monkey (5B), and rhesus monkey (5C) CD20+ lymphocytes compared to its parental control (SI-63C1) and mouse anti-human CD19 antibody controls (SJ25C, LT19, HIB19, and 4G7);

[0044] Figure 6 Analytical SEC profiles showing DLS thermal stability of SI-63R1 (H1), protein A purified recombinant anti-CD19 scFv-HIS protein (6A), and SI-63R1 (H1) with an unfolding temperature of approximately 58.8°C (6B);

[0045] Figure 7 Analytical SEC profiles of protein A purified recombinant anti-CD19 scFv-single Fc proteins (H1 to H6) with 90% of the protein of interest (POI) are shown;

[0046] Figure 8 Schematic diagram depicting the six binding domains (D1-D6) in the hexaspecific GNC antibody comprising the core Fab (D2) and Fc region, as well as additional D1, D3 and D4 on the heavy chain (HC) and D5 and D6 on the light chain;

[0047] Fig. 9 The expression and purification of three hexaspecific GNC antibodies, SI-77H3, SI-77H6, and SI-55H11, whose humanized anti-CD19 domains are H4 at D1, H7 (Fab) at D2, and H4 at D6, respectively, are shown;

[0048] Figure 10 shows the dose-response curves of direct cytotoxicity (ADCC) of antibodies (SI-38E17, SI-55H11, SI-77H3 and SI-77H6, respectively) against human (10A) or cynomolgus monkey (10B) PBMCs; and

[0049] Figure 11 shows that the humanized CD19 binding domains of hexaspecific antibodies such as SI-77H, SI-77H6 and SI-55H11 mediate cytolysis of Raji lymphoma cells that express only CD19 but not other tumor antigens (11A), with an effective dose-response curve comparable to that of SI-38E17 (human anti-CD19 antibody (21D4) (11B)). DETAILED DESCRIPTION

[0050] In the following detailed description, reference is made to the accompanying drawings which form a part thereof. In the drawings, similar symbols generally identify similar components unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure as generally described herein and shown in the accompanying drawings may be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0051] The present disclosure provides, among other things, isolated antibodies; methods for preparing such antibodies; monoclonal and / or recombinant monospecific antibodies, multispecific antibodies, antibody-drug conjugates and / or immunoconjugates composed of such antibodies or antigen-binding fragments, pharmaceutical compositions containing the antibodies, monoclonal and / or recombinant monospecific antibodies, multispecific antibodies, antibody-drug conjugates and / or immunoconjugates, methods for preparing antibodies and compositions; and methods for treating cancer using the antibodies and compositions disclosed herein. Specifically, the present disclosure provides isolated monoclonal antibodies (mAbs) or antigen-binding fragments thereof having binding specificity to human CD19 (Table 1), wherein the isolated mAb or antigen-binding fragment comprises an amino acid sequence having the same identity as a sequence selected from SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93.

[0052] As used herein, the terms "a," "an," and "the" are defined to mean "one or more" and include the plural unless the context is inappropriate.

[0053] As used herein, the terms "polypeptide," "peptide," and "protein" are interchangeable and are defined to refer to biological molecules composed of amino acids linked by peptide bonds.

[0054] The term "antigen" refers to an entity or fragment thereof that can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions thereof that are responsible for antigenicity or antigenic determinants.

[0055] The terms "antigen or epitope binding portion or fragment", "variable region", "variable region sequence" or "binding domain" refer to antibody fragments that are capable of binding to an antigen (such as CD19 in the present application). These fragments may have the antigen binding function and other functions of the intact antibody. Examples of binding fragments include, but are not limited to, single-chain Fv fragments (scFv), which consist of the variable light chain (VL) and variable heavy chain (VH) domains of a single arm of an antibody connected in a single polypeptide chain by a synthetic linker; or Fab fragments, which are monovalent fragments consisting of VL, constant light chain (CL), VH and constant heavy chain 1 (CH1) domains. Antibody fragments can be even smaller subfragments and can consist of domains as small as a single CDR domain, particularly CDR3 regions from VL and / or VH domains (see, e.g., Beiboer et al., J. Mol. Biol. 296: 833-49 (2000)). Antibody fragments are produced using conventional methods known to those skilled in the art. The utility of antibody fragments can be screened using the same techniques used for intact antibodies.

[0056] "Antigen or epitope binding portions or fragments," "variable regions," "variable region sequences," or "binding domains" can be derived from the antibodies of the present disclosure by a number of techniques known in the art. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Appropriate fractions containing the Fab fragments are then collected and concentrated by membrane filtration, etc. For further description of general techniques for isolating antibody active fragments, see, for example, Khaw, BA et al., J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al., Methods Enzymology, 121:663-69, Academic Press, 1986.

[0057] The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal antibodies and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions with multiple epitope specificities, and antibody fragments such as Fab, F(ab')2, and Fv, as long as they exhibit the desired biological activity. In some embodiments, the antibody can be a monoclonal, polyclonal, chimeric, single-chain, multispecific or multi-effective, human and humanized antibody, and active fragments thereof. Examples of active fragment molecules that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, including products of Fab immunoglobulin expression libraries and epitope binding fragments of any of the above antibodies and fragments.

[0058] The term "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain and a light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable region interact to define the antigen binding site on the surface of the VH-VL dimer. These six CDRs together give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0059] In some embodiments, antibodies may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain binding sites and immunospecifically bind to antigens. A typical antibody refers to a heterotetrameric protein that generally comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain consists of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The light chains of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains. The VH and VL regions can be further subdivided into domains of hypervariable complementary determining regions (CDRs) and more conservative regions called framework regions (FRs). Each variable domain (VH or VL) generally consists of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. Within the variable regions of the light and heavy chains are binding regions that interact with antigens.

[0060] Immunoglobulins can be divided into different classes according to the amino acid sequence of their heavy chain constant domain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of the different types of immunoglobulins are well known.

[0061] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" represents the characteristics of antibodies obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can be prepared by the hybridoma method first described by Kohler & Milstein, Nature, 256: 495 (1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Recombination" refers to the use of recombinant nucleic acid technology to produce antibodies in exogenous host cells.

[0062] Monoclonal antibodies can be produced using a variety of methods, including but not limited to mouse hybridomas, phage display, recombinant DNA, molecular cloning of antibodies directly from primary B cells, and antibody discovery methods (see Siegel, Transfus. Clin. Biol. 2002; Tiller, New Biotechnol. 2011; Seeber et al., PLOS One. 2014). Monoclonal antibodies may include "chimeric" antibodies (immunoglobulins), in which a portion of the heavy chain and / or light chain portion is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (as long as they exhibit the desired biological activity) (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855

[1984] ).

[0063] As used herein, the term "multispecific" antibody refers to an antibody having at least two binding sites, each binding site having binding affinity for an epitope of an antigen. As used herein, the term "bispecific, trispecific, tetraspecific, pentaspecific or hexaspecific" antibody refers to an antibody having 2, 3, 4, 5 or 6 antigen binding sites. For example, an antibody disclosed herein having 5 binding sites is pentaspecific, and an antibody having 6 binding sites is hexaspecific.

[0064] The term "guidance and navigation control (GNC)" protein refers to a multispecific protein that can bind to at least one effector cell (such as an immune cell) antigen and at least one target cell (such as a tumor cell, an immune cell, or a microbial cell) antigen. The GNC protein can adopt an antibody core structure including a Fab region and an Fc region, wherein various binding domains are attached to the antibody core, in which case the GNC protein is also referred to as a GNC antibody. The GNC protein can adopt an antibody-like structure, in which case the Fv fragment can be replaced with a non-antibody-based binding domain such as NKG2D, 4-1BBL (4-1BB receptor ligand), 4-1BBL trimer or receptor of 4-1BB.

[0065] The term "GNC antibody" refers to a GNC protein having an antibody structure capable of simultaneously binding to at least one effector cell (e.g., an immune cell) and at least one target cell (e.g., a tumor cell, an immune cell, or a microbial cell). As used herein, the term "bispecific GNC, trispecific GNC, tetraspecific GNC, pentaspecific GNC, or hexaspecific GNC" antibody refers to a GNC antibody having 2, 3, 4, 5, or 6 antigen binding sites, wherein at least one antigen binding site has binding affinity for immune cells and at least one antigen binding site has binding affinity for tumor cells. In one embodiment, the GNC antibodies disclosed herein have 4-6 binding sites (or binding domains), and are tetraspecific GNC, pentaspecific GNC, and hexaspecific GNC antibodies, respectively. In some embodiments, the GNC antibody includes an antibody binding domain (e.g., Fab and scFv) without the need for additional protein engineering in the Fc region. In one embodiment, the GNC antibody also has the advantage of retaining the divalence of each target antigen. In addition, in one embodiment, the GNC antibody has the advantage of an avidity effect that results in a higher affinity and a lower dissociation rate for the antigen. This bivalency for each antigen is in contrast to many multispecific platforms, which are monovalent for each target antigen and thus often lose the beneficial avidity effects that allow the antibodies to bind so strongly.

[0066] The term "humanized antibody" refers to an engineered antibody type having CDRs derived from non-human donor immunoglobulins, with the remaining immunoglobulin-derived portions of the molecule derived from one (or more) human immunoglobulins. In addition, framework support residues may be altered to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art (see, e.g., Queen et al., Proc. Natl Acad Sci USA, 86: 10029-10032 (1989); Hodgson et al., Bio / Technology, 9: 421 (1991)).

[0067] The terms "isolated" or "purified" refer to a biomolecule that is free from at least some of its naturally occurring components. When used to describe the various polypeptides disclosed herein, "isolated" or "purified" refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Generally, a purified polypeptide will be prepared by at least one purification step. An "isolated" or "purified" antibody refers to an antibody that is substantially free of other antibodies with different antigen-binding specificities.

[0068] The term "immunogenic" refers to a substance that elicits or enhances antibodies, T cells, or other reactive immune cells to an immunogenic agent in a human or animal, or an immune response in an animal. An immune response occurs when an individual produces enough antibodies, T cells, and other reactive immune cells to alleviate or alleviate the condition to be treated in response to an administered immunogenic composition of the present disclosure. Although an immunogenic response typically includes both the cellular (T cell) and humoral (antibody) arms of an immune response, antibodies to a therapeutic protein (anti-drug antibodies, ADA) may be composed of IgM, IgG, IgE, and / or IgA isotypes.

[0069] The term "specific binding" or "specifically binds to" or "is specific for a particular antigen or an epitope" means that the binding is clearly distinguishable from non-specific interactions. For example, specific binding can be measured by comparing the binding of the test molecule to the binding of a control molecule, which is typically a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0070] Specific binding to a particular antigen or epitope can be determined, for example, by a KD for the antigen or epitope of at least about 10 -4 M, at least about 10 -5M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 , or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 The multispecific antibodies are displayed as antibodies with a KD of M or greater, where KD refers to the dissociation rate of a specific antibody-antigen interaction. Typically, the multispecific antibodies that specifically bind to an antigen have a KD relative to the antigen or epitope that is 20, 50, 100, 500, 1000, 5,000, 10,000 or more times that of a control molecule.

[0071] Furthermore, for example, specific binding for a particular antigen or epitope can be demonstrated by an antibody having a KA or Ka for the antigen or epitope that is at least 20, 50, 100, 500, 1000, 5,000, 10,000 or more times greater than that for a control epitope, where KA or Ka refers to the association rate of the specific antibody-antigen interaction.

[0072] The present disclosure may be understood more readily by reference to the following detailed description of specific embodiments and examples included herein.While the present disclosure has been described with reference to specific details of certain embodiments of the present disclosure, such details are not intended to be construed as limitations on the scope of the present disclosure.

[0073] Example

[0074] Example 1. Design of humanized anti-CD19 sequences.

[0075] All calculation steps are performed in the Discovery Studio software package (Dassault Systemes). First, mouse BU12 sequence is used to generate a structural model (McDonagh et al., 2009). Hidden Markov model (HMM) is used to identify the antibody framework region in the input sequence and compare it with the database of antibody variable domains, and MODELLER software is used to compare this for building and scoring models. CDR ring modeling is carried out by CDRL1, CDRL2, CDRL3, CDRH1 and CDRH2 districts and the structural mapping of known normative classes, and a ring model is built similarly to the framework.

[0076] The framework regions of the mouse BU12 antibody were aligned and matched to the closest human germline sequence, and the CDR regions were copied into the human sequence, except for important structural residues (Vernier residues [Almagro and Fransson, 2008]). Mutations predicted to stabilize the previously constructed structural model were evaluated by 1000 steps of steepest descent, with an RMS gradient tolerance of 3, followed by conjugate gradient minimization, and stabilizing mutations matching common human residues were selected based on individual and combined ΔΔG comparisons to the initial model. Mutation stabilization energy analysis was performed on the discovery studio with sequence H1 as a reference. The H2, H3 and H4 forms are mutant variants with negative mutation energy values ​​(ΔΔG of -0.8, -1.5 and -1.1 kCal, respectively), and are assumed to be more stable than the H1 form. The humanization of the resulting humanized sequences (H1, SEQ ID NO.1 and 13) was tested using the Abysis web server based on the method of Abhinandan and Martin (2007). As Figure 1 As shown in A and 1B, using H4 as an example for its light chain (Vk) and heavy chain (VH), the humanized sequence showed a higher humanization score than the corresponding mouse sequence (BU12) (SEQ ID NOs. 25 and 27).

[0077] In addition, a direct CDR transplantation method was used to generate a humanized version of H5. The reference antibody framework was mutated to similar human germline residues, and the CDRs were directly transplanted into the mutated framework to generate H5. Using the resulting humanized sequence H5 as a building sequence, more mutations were made on the H5 framework to improve the framework stability of the generated humanized sequence (H6). Based on the method of Abhinandan and Martin (2007), the humanization of the humanized sequence H5 (SEQ ID NO.9 and 21) was tested using the Abysis web server. The humanized sequence showed a higher humanization score ( ) than the mouse sequence (BU12) (SEQ ID NO.25 and 27). Figure 1 C and 1D).

[0078] H1 is the first humanized form of the variable domain derived directly from the Fab sequence of BU12, with the characteristic amino acid sequence LEIK at the C-terminus. As shown in Figure 2, the last three residues (EIK) are mainly present in the naturally occurring variable kappa chain and provide stability when specifically located in the Fab domain. In this case, H4, which ends with VTVL in the Fab position, may not be ideal for the stability of the antibody. The H7 form is a modified H4 that is hypothesized to improve protein stability when located in the Fab domain ( https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5058631 / ). H7 was generated by reducing EIK and introducing a disulfide staple between H7VL and H7VH by Q to C and G to C mutations, respectively.

[0079] As shown in Figure 2, in order to compare and distinguish the priority of these designed sequences, all humanized variable regions (H1, H2, H3, H4, H5, H6 and H7) are compared with the sequence from human anti-CD19 antibody 21D4 (Rao-Naik et al., 2009). The percentage identity with H1 is 98.1% VL and 99.1-100% VH for H2, H3, H4 and H7, 85% VL and 86% VH for H5 and H6, and 70% VL and 51% VH for 21D4. These findings suggest significant flexibility in the primary sequence of anti-CD19 binding domains.

[0080] To predict the immunogenicity of humanized anti-CD19 sequences, the MixMHC2pred algorithm (Gfeller Lab, https: / / github.com / GfellerLab / MixMHC2pred ) is used to predict the degree of major histocompatibility complex-II (MHC-II) binding of peptides in mouse and humanized (VH / VL) sequences. The algorithm detects the number of "core" peptides in a given amino acid sequence, which will bind to MHCII with sufficient affinity to form T cell epitopes. The higher the number of MHCII binding peptides identified in the sequence, the more potential T cell epitopes the sequence contains. A large number of core peptides increases the possibility of containing some immunogenic original peptides. Therefore, reducing the number of core peptides in the antibody variable region can help reduce ADA by eliminating potential T cell epitopes.

[0081] Anti-CD19 variable sequences are run as scFv (VH-(G4S)4-VL) by the MixMHC2pred algorithm. The algorithm includes the option of scoring multiple alleles. In this case, "the score from each peptide is considered to be its best percentile rank among all alleles". This scoring strategy allows the sequence to be checked to find the strongest ligand for any allele of MHCII. For sequence analysis of antibody variable domains, the number of core peptides is calculated according to the number of peptides that can be bound to any MHCII allele in the sequence, where the score is in the first 0.2% of the interaction. As shown in Table 1, most humanized sequences have lower scores than their parental mouse sequences, indicating weaker MHCII binding peptides and lower immunogenicity risks. In this article, the total score of core peptides in the variable region predicted to be strongly bound to MHCII is reduced from 9 in the mouse sequence to 5 in the humanized sequence (H1 to H4 and H7, H5 and H6 are unchanged). The humanized scores of light and heavy chains are calculated using the humanized Z score analysis algorithm (Abhinandan & Andrew, 2007). For VH sequences, H1-H4 forms have similar humanization to 21D4, while H5 and H6 have higher humanization and H7 has lower humanization. For VK sequences, H1-H7 all have similar humanization, slightly lower than 21D4. It is worth noting that all humanized sequences (H1-H7, VH and Vk) have significantly higher humanization scores than the original mouse sequences (Table 1). Considering the humanization and MHC-II peptide binding scores, H1-H4 and H7 are candidates for generating humanized anti-CD19 antibodies.

[0082] Example 2. Expression of humanized anti-CD19 monoclonal antibody

[0083] To characterize the humanized light and heavy chain CDRs and framework regions, DNA sequences encoding H1 and other peptides were synthesized as overlapping fragments and cloned into linearized pTT5 vectors (NE Builder) containing C-terminal human κ sequences or human IgG CH1 and Fc regions, respectively, to produce mAb forms (SEQ ID NOs. 37 and 39). DNA sequences of 21D4 and mouse (BU12) variable regions were also synthesized and cloned into linearized pTT5 vectors containing C-terminal human κ sequences or IgG CH1 and Fc regions, respectively, to produce chimeric mAb forms (SEQ ID NOs. 33, 35, 83, and 85). Plasmid DNA containing antibody sequences was expressed using the ExpiCHO expression system (ThermoFisher). Three recombinant antibodies SI-63C1 (with BU12 mouse parent anti-CD19 variable sequence), SI-63C2 (with H1 humanized anti-CD19 variable sequence, also referred to as SI-huCD19) and SI-34C1 (with 21D4 human anti-CD19 variable sequence) were purified from culture supernatant using a protein A affinity chromatography column (mabSelect Resin, Ge healthcare) washed with PBS (5X Cv) followed by elution with 20mM glycine pH 3.5. The resulting protein was neutralized with 100X Tris pH 8.5 and dialyzed overnight in PDB buffer. To check stability and monodispersity, the purified antibodies were concentrated to 1 mg / ml and injected onto analytical HPLC (waters, column waters BEH200A 300mm column). The purified anti-CD19 antibodies showed sharp monodisperse peaks of the correct size with 1.8-2.5% aggregates (Table 2).

[0084] Example 3. Characterization of SI-63C2

[0085] The binding affinity of purified SI-63C1, SI-63C2 and SI-34C1 antibodies was tested using biofilm interferometry (ForteBio OctetRED 384). The antibodies were bound to an anti-human Fc biosensor and human CD19 protein (R&D Biosystems Cat#9269-CD-050) was used as the analyte in a 4-point series of 2-fold dilutions with a maximum concentration of 200 nM. The results of the Octet analysis showed that SI-63C3 (also known as SI-huCD19) had a binding affinity of 3.8 nM and 3.6 nM for human CD19 and cynomolgus monkey CD19, respectively, which was comparable to the human anti-CD19 antibody (21D4) of 2.1 nM and 3.8 nM. In addition, the humanized anti-CD19 variable sequence of SI-63C2 not only retained the binding specificity to human and cynomolgus CD19, but also exhibited binding affinity (KD) comparable to that of SI-63C1 (with BU12 variable sequence) and SI-34C1 (with 21D4 variable sequence) (Table 2).

[0086] To test the thermal stability of SI-63C2, dynamic light scattering was used while the temperature was increased from 25°C to 75°C at 0.5°C / min and the radius of the protein (1 mg / ml) was monitored by using a Wyatt DynaPro microplate reader III. Figure 3 As shown in Table 2, the results indicate that SI-63C2 and SI-63C1 exhibit similar unfolding temperatures, as measured by DLS Tm, which are higher than that of SI-34C1.

[0087] Example 4. Binding specificity of SI-63C2

[0088] Due to the similarity of non-human primates (NHPs) such as cynomolgus or rhesus macaque to humans, predictable metabolic stability and historically established toxicity characteristics, they are currently needed to provide risk assessment data for antibody drug development. In order to minimize the use of NHPs and improve efficiency, antibody drug candidates should have high target specificity and cross-reactivity. In this case, CD19 is a pan-B cell marker and is expressed by most malignant B cells. CD19 covers B cell development and differentiation more widely than CD20, which is another pan-B cell marker from lymphocytes of humans and NHPs such as cynomolgus and rhesus macaques. Among many mouse anti-human CD19 antibodies, BU12 can cross-react with B lymphocytes derived from cynomolgus macaques with lower binding affinity (Liu et al., 2016).

[0089] In order to determine whether humanization changes cross-reactivity, flow cytometry was performed. SI-63C2 antibody is used to bind peripheral blood mononuclear cells from people, cynomolgus monkeys and rhesus monkeys, respectively. Lymphocytes were gated based on forward scatter and side scatter, and then single cells were gated based on the ratio of forward scatter signal height and area. Based on the exclusion of membrane permeability amine-active dyes and the binding level of CD20 antibodies, surviving CD20+B cells and CD20-lymphocytes were gated (clone 2H7, Biolegend). The combination of labeled antibodies was determined as the geometric mean fluorescence intensity (gMFI) of the cell population of the fluorescent conjugate emission channel. As shown in the histogram analysis of Figure 4, SI-63C2 antibody binds to CD20+B cells from people, cynomolgus monkeys and rhesus monkeys (4A), but does not bind to their CD20-lymphocytes (4B). When a panel of anti-CD19 antibodies (i.e., SJ25C, LT19, HIB19, and 4G7) was used for comparison, only SI-63C2 and its parent antibody SI-63C1 showed significant binding affinity to CD20+B cells from humans, cynomolgus monkeys, and rhesus monkeys (Figure 5). These data confirm that the binding specificity of SI-63C2 to human, cynomolgus monkey, and rhesus monkey B cells is retained, however, as measured by EC50, its cross-reactivity to cynomolgus monkeys is still lower than its response to human CD19 (Table 3).

[0090] Example 5. His-tagged humanized anti-CD19 scFv protein

[0091] To characterize the humanized anti-CD19 binding domain as a scFv unit, the DNA sequence encoding the humanized anti-CD19 variable region (H1) was cloned into a His-tagged scFv expression vector containing residues GSHHHHHH (SEQ ID NO. 41) at the C-terminus of the scFv. The humanized anti-CD19 scFv-His-tagged protein was expressed using the ExpiCHO expression system, purified by protein L affinity chromatography, and named SI-63R1. Data from analytical SEC indicated that SI-63R1 had 70% of the target protein, and the DLS thermal stability test measured the unfolding temperature of SI-63R1 at 58.8°C ( Figure 6 ).

[0092] To evaluate the binding affinity of SI-63R1, Octet binding assay was used. SI-63R1 protein was loaded onto the AR2G sensor at 10 μg / ml by covalent coupling and combined with a serial dilution of His-tagged human CD19 (1:2.5 dilution starting from the highest concentration of 200 nM). The results showed that SI-63R1 had a low nanomolar binding affinity for human CD19 (Table 2).

[0093] Example 6. Humanized anti-CD19 scFv monomer Fc fusion protein.

[0094] In order to further screen and compare all humanized peptides, the DNA sequences encoding humanized CD19 binding variants (H1, H2, H3, H4, H5 and H6) were constructed into scFv-monomer Fc forms and cloned (Dimitrov et al., 2012.) (SEQ ID NO.55, 57, 59, 61, 63, 65). Each of the 6 humanized anti-CD19 scFv monomer Fc fusion proteins was expressed using the ExpiCHO expression system and purified by protein A affinity chromatography. They were named SI-63SF1 (H1), SI-63SF2 (H2), SI-63SF4 (H3), SI-63SF5 (H4), SI-63SF6 (H5) and SI-63SF7 (H6). After the expression and purification process, the physical properties of all 6 proteins were characterized, including yield (titer), purity (%HMW and aSEC), binding affinity to human CD19 (KD, Kon and Kdis) and thermal stability. For the Octet assay, the scFv-monomeric Fc fusion protein was loaded at 10 μg / ml by the AHC sensor and bound to a serial dilution of His-tagged human CD19 (1:2.5 dilution starting from the highest concentration of 200 nM), and the resulting ensemble was fit to a 1:1 binding model. For DLS analysis, the temperature was increased from 25°C to 75°C at 0.5°C / min, while the radius of the scFv-monomeric Fc fusion protein (1 mg / ml) was monitored by a Wyatt DynaPro microplate reader III. The analytical SEC curve is shown in Figure 7 All the measurement results are listed in Table 4.

[0095] The data showed that SI-63SF5 (H4) had the highest DLS melting temperature (Tm) at 51.8°C (Table 4). Due to its higher thermal stability, the humanized anti-CD19 variable region with the H4 peptide was selected for further study in the GNC antibody platform.

[0096] Example 7. Humanized anti-CD19 scFv or Fab domain in GNC antibody

[0097] Guidance and navigation control (GNC) antibodies refer to multispecific antibodies that can bind to an antigen expressed by at least one target cell (including but not limited to tumor cells, immune cells or microbial cells) and an antigen expressed by at least one effector cell (e.g., an immune cell) (see applicant's application WO / 2019 / 005642, which is incorporated herein in its entirety). GNC antibodies contain antibody structures of Fab and Fc regions, which have various additional binding domains, such as one or more single-chain fragment variable domains, also known as scFv, connected to the antibody core. GNC antibodies are able to target tumor antigens, engage immune activation receptors, and guide immune effector cell-mediated tumor killing at a fraction of the cost. For example, tetraspecific GNC (tetra-GNC) antibodies have been shown to exert an ideal multifaceted effect, with structurally and functionally diverse but relatively independent binding domains (see applicant's application WO / 2019 / 191120, which is incorporated herein in its entirety). In this case, the humanized anti-CD19 variable domain can be added to any GNC antibody as a Fab or scFv domain.

[0098] To characterize the humanized CD19 binding domain in the GNC antibody, DNA sequences encoding H4 and H7 were constructed and cloned into the GNC antibody format at one of the five scFv sites and one of the Fab sites, respectively ( Figure 8 The construction scheme is shown in Figure 1). Optionally, the mutation R19S (Kabat numbering) is incorporated into the FR1 region of the humanized (H4) VH domain of the VH3-containing scFv on the GNC light chain, such as SI-55H11. When the VH3-containing scFv is linked to the light chain, V H The domain can bind to the protein A resin during purification, resulting in light chain monomers and dimers contaminating the desired heavy chain-light chain heterotetramer. In order to reasonably destroy the protein A binding of VH3 family members, a structural approach is used to destroy the binding interface. Crystal structure 1DEE (Graille M. et al., Proc. Nat. Acad. Sci. 2000.) shows that the residue R19 (Kabat numbering) in VH3 is in direct contact with the two side chains of the protein A domain D. In particular, the contact with Q32 and D36 can be eliminated to significantly weaken the interaction. Therefore, due to its shorter side chain, it does not form these interactions. In addition, S19 is naturally present in other VH family members, indicating that its immunogenicity may be lower than other substitutions. For hexaspecific GNC antibodies that can contain up to two VVH3 scFvs per light chain, this mutation is particularly important in allowing effective purification of the desired product.

[0099] Table 5 lists six specific GNC antibodies with humanized CD19 binding domain H4 at D1 of SI-77H3 (SEQ ID NO. 67 and 69), D2 (Fab) of SI-77H6 (SEQ ID NO. 71, 73) and D6 of SI-55H11 (SEQ ID NO. 75 and 77); and five specific GNC antibodies with humanized CD19 binding domain H4 at D6 of SI-38P12. The expression vectors encoding these GNC antibodies were transfected and expressed in the ExpiCHO system, and all GNC antibodies were purified by protein A affinity chromatography. The results of yield and purity measured by titer and aSEC showed that GNC antibodies with humanized CD19 binding domains can be expressed and purified as scFv or Fab ( Fig. 9 and Table 6).

[0100] To determine the binding affinity of hexaspecific and pentaspecific GNC antibodies to human CD19, an Octet binding assay was used. The GNC antibodies were loaded at 10 μg / ml by the AHC sensor and bound to His-tagged human CD19 at a serial dilution (1:2.5 dilution starting from the highest concentration of 200 nM) or a single 100 nM concentration. The resulting overall fit to the 1:1 binding model demonstrated that these GNC antibodies bind to CD19 with low nanomolar affinity (Table 6).

[0101] Example 8. Position effect of humanized CD19 binding domain in GNC antibody

[0102] In order to evaluate the antibody-dependent cellular toxicity mediated by humanized CD19 binding domain, peripheral blood mononuclear cells (PBMC) from humans and cynomolgus monkeys were used. T cell engagers were added to humans or cynomolgus monkey PBMCs and cultured for 5 days. After 5 days, cultured cells were collected and survival and non-survival CD20+B cells were counted by FACS. The analysis of surviving single B cells and all B cells (single cells, doublet cells or other cells in the gate) of survival was evaluated independently. Quantitative relative total cell count was performed using the counting beads of spiked-in controls. In this study, the six specific GNC antibodies tested included SI-77H3 (H4 at D1), SI-77H6 (H7 at D2, i.e. Fab), SI-55H11 (H4 at D6), and the control was tetra-specific GNC antibody SI-38E17 (SEQ ID NO.79 and 81), which had human CD19 binding domains (21D4) (Table 5) in Fab region (D2).

[0103] Single cell analysis by FACS tends to lack the influence of T cell engagers on the formation of non-cytolytic complexes, and most of them seem to fall outside the gate of single cells. On the contrary, the analysis including doublet cells covers more events, thereby providing a more complete understanding of cell-cell interactions. Figure 10 shows the results of ADCC analysis using gates on all B cells that survive. Control antibody SI-38E17 shows binding specificity to human CD19 but not to cynomolgus monkey CD19. As a comparison, all three six-specific GNC antibodies show similar responses to human and cynomolgus monkey PBMC. Unexpectedly, SI-77H6 does not seem to mediate ADCC to human PBMC and cynomolgus monkey PBMC, despite the presence of humanized CD19 binding affinity (Table 6). As shown in Table 5, in SI-77H6, the humanized CD19 binding domain is the Fab region of the antibody core structure, while in SI-77H3 and SI-55H11, the humanized CD19 binding domain is an additional scFv domain of the antibody core structure. Hexaspecific GNC antibodies have at least 6 binding specificities and are therefore able to bind to at least two different types of cells simultaneously in vivo, which is different from the case where the affinity of a single binding domain is evaluated. This observed correlation of position and effect suggests that there are biologically different results mediated by the position effect of the humanized anti-CD19 Fab domain in the GNC antibody, and that SI-77H6 may not be able to support the correct formation of cytolytic immune synapses between activated T cells and target B cells. Since CD19 is expressed by both normal B cells and tumor B cells, the positioning effect may be useful when each binding domain is specified for the benefit of treating different types of cancer (i.e., solid tumors versus liquid tumors). For example, SI-77H6-like GNC antibodies can still be used to treat solid tumors that have higher efficacy but lower cytotoxicity against normal B cells. In another embodiment, SI-77H6-like GNC antibodies can be used to help B cells engage T cells in cytolytic interactions against other tumor-associated antigens.

[0104] Example 9. RTCC of Hexaspecific GNC Antibodies with Humanized CD19 Binding Domains

[0105] To demonstrate the cytotoxic effects of the hexaspecific GNC antibodies with humanized CD19 binding domains, redirected T cell cytotoxicity (RTCC) analysis was performed using Raji cells. The Raji line of lymphoblastoid cells is derived from Burkitt's lymphoma. Since each of the three hexaspecific GNC antibodies can bind to multiple tumor antigens other than CD19, such as EGFR, HER3, and PD-L1 (Table 5), Raji cells expressing mKate2 fluorescent protein were stained with labeled monoclonal antibodies against each tumor antigen and analyzed by FACS. The histogram results confirmed that Raji cells expressed CD19, and no expression of EGFR, HER3, or PD-L1 was detected ( Fig.11A ).

[0106] Raji cells expressing mKate2 fluorescent protein were co-cultured with human CD8 T cells at a ratio of 5 T cells / Raji cell in the presence of T cell engagement protein at concentrations ranging from 10 nM to 1 fM in triplicate for 81 hours. Target cell fluorescence signals were evaluated by quantitative microscopy and dose-response curves as a measure of specific cell lysis using Graphpad Prism 8 with a 5-parameter asymmetric sigmoid nonlinear regression and least squares fitting. Fig. 11B As shown, the humanized CD19 binding domains of each of SI-55H11 (EC50, 2pM), SI-77H3 (EC50, 8pM) and SI-77H6 (EC50, 30pM) mediate effective cytolysis of tumor cells, and the potency of SI-55H11 is the same as that of SI-38E17 (EC50, 2pM), a human anti-CD19 antibody (21D4) (Table 6). SI-77H6 exhibits suboptimal cytolysis with reduced potency, a phenomenon parallel to its effect of CD19 binding to normal B cells in the absence of cytolysis induction. SI-77H3 is able to completely kill tumor cells, but with a reduced EC50. Therefore, using optimized configurations and processing conditions (e.g., the ratio of activated T cells to target cells), the disclosed humanized CD19 binding domains can exert the same potency as the human CD19 binding domains in multispecific GNC antibodies, with the additional feature of cross-reactivity to cynomolgus monkey CD19.

[0107] Although the present disclosure has been described with reference to specific embodiments or examples, it is understood that the embodiments are illustrative and the scope of the present disclosure is not limited thereto. Alternative embodiments of the present disclosure may become apparent to those of ordinary skill in the art to which the present disclosure belongs. Such alternative embodiments are considered to be included within the scope of the present disclosure. Therefore, the scope of the present disclosure is limited by the appended claims and supported by the foregoing description. All references cited or mentioned in the present disclosure are incorporated herein by reference in their entirety.

[0108] References:

[0109] 1. Watkins MP, Bartlett NL. CD19-targeted immunotherapies for treatment of patients with non-Hodgkin B-cell lymphomas. Expert Opin InvestigDrugs. 2018; 27(7):601–611. doi:10.1080 / 13543784.2018.1492549.

[0110] 2.( https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5058631 / .)

[0111] 3.McDonagh,Charlotte et.Al.CD19 Binding agents and UsesThereof.US20090136526A1.

[0112] 4.Rao-Naik et.al.CD19 Antibodies and their uses.US 20090142349 A1.

[0113] 5. Dimitrov et al. 2012: https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2017.01545 / full .

[0114] 6. (Graille M. et al. Proc. Nat. Acad. Sci. 2000.) 2000.)

[0115] sheet

[0116] Table 1. Total MHCII binding scores and humanization scores of humanized variable domains calculated using MixMHC2pred and Z-score analysis algorithms, respectively.

[0117]

[0118] Table 2. Purity, binding affinity and thermal stability of SI-63C1 (with mouse parental BU12 variable sequence), SI-63C2 (with H1 humanized anti-CD19 variable sequence), SI-34C1 (with 21D4 human anti-CD19 variable sequence) and SI-63R1 (humanized anti-CD19 scFv-His tag protein).

[0119]

[0120] Table 3. Cross-reactivity of recombinant antibodies (SI-63C1 and SI-63C2) and mouse anti-human CD19 antibodies (SJ25C, LT19, HIB19 and 4G7) on CD20+ lymphocytes of human, cynomolgus and rhesus origin as measured by EC50.

[0121]

[0122] Table 4 Purity, binding affinity and thermal stability of humanized anti-CD19 scFv monomer Fc fusion proteins SI-63SF1 (H1), SI-63SF2 (H2), SI-63SF4 (H3), SI-63SF5 (H4), SI-63SF6 (H5) and SI-63SF7 (H6).

[0123]

[0124]

[0125] Table 5. Location of humanized CD19 binding domain and other antigen binding domains in GNC antibodies.

[0126]

[0127] Table 6. Physical and functional properties of GNC antibodies with humanized anti-CD19 scFv domain or Fab region.

[0128]

[0129] Sequence Listing

[0130]

[0131]

Claims

1. A peptide with binding specificity to human CD19, comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 91 or 93.

2. A scFv peptide comprising the peptide according to claim 1.

3. The scFv peptide according to claim 2, which has a KD of no greater than 10 nM for binding affinity to human CD19.

4. An antibody having binding specificity to human CD19, wherein the antibody comprises the peptide of claim 1. The antibody according to claim 4 , wherein the antibody is a multispecific antibody.

6. The antibody of claim 4, comprising a scFv, wherein the scFv comprises the peptide of claim 1.

7. The antibody according to claim 4, comprising Fab, wherein the Fab comprises the peptide according to claim 1.

8. A multispecific antibody-like protein comprising the peptide of claim 1, wherein the multispecific antibody-like protein has an N-terminus and a C-terminus, and the multispecific antibody-like protein comprises, from the N-terminus to the C-terminus, a first binding domain (D1) at the N-terminus, a second binding domain (D2) comprising a light chain portion, an Fc region, a third binding domain (D3) and a fourth binding domain (D4) at the C-terminus, wherein the light chain portion comprises a fifth binding domain (D5) covalently linked to the C-terminus, a sixth binding domain (D6) covalently linked to the N-terminus, or both, and Wherein D1, D2, D3, D4, D5 and D6 each have binding specificity to a tumor antigen, an immune signaling antigen or a combination thereof. 9 . The multispecific antibody-like protein according to claim 8 , wherein D1 comprises the peptide according to claim 1 . 10 . The multispecific antibody-like protein according to claim 8 , wherein D1 comprises a peptide having an amino acid sequence having 95% sequence identity with SEQ ID NO. 7 or 19.

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