Anti-antibodies

Abstract

The present invention relates to anti-serum albumin antibodies and multispecific binding proteins comprising the same. The present invention also relates to pharmaceutical compositions comprising the antibodies or multispecific binding proteins, expression vectors and host cells for making the antibodies or multispecific binding proteins, and methods of using the antibodies or multispecific binding proteins for treating diseases or conditions.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 946,932, filed December 11, 2019, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This invention relates to anti-serum albumin antibodies and multispecific binding proteins comprising such antibodies. The invention also relates to pharmaceutical compositions comprising these antibodies or multispecific binding proteins, expression vectors and host cells for preparing these antibodies or multispecific binding proteins, and methods for treating diseases or conditions using these antibodies or multispecific binding proteins. Background of the Invention

[0005] Serum albumin is the most abundant protein in serum. It exhibits high stability, solubility, and a long circulating half-life. Peptides such as antibodies that bind to serum albumin have been developed to increase the circulating half-life of therapeutic proteins. Despite significant progress, novel and useful anti-serum albumin antibodies and multispecific binding proteins with improved pharmacokinetic properties remain in demand. Invention Overview

[0007] This invention is partly based on the development of novel antibodies that bind to serum albumin. A multispecific binding protein is also provided, comprising a first domain binding to a first target protein expressed on target cells, such as CD19 (e.g., human CD19), and / or a second domain binding to a second target protein expressed on immune effector cells, such as CD3 (e.g., human CD3), and a third domain binding to serum albumin (e.g., human serum albumin), wherein the third domain is derived from these novel antibodies. These domains are linked in a specific manner to achieve favorable therapeutic effects and in vivo half-life. The multispecific binding protein can be used to treat diseases and conditions associated with abnormal cells expressing the first target protein, such as certain B-cell hematologic malignancies.

[0008] Therefore, in one aspect, the present invention provides an antigen-binding site for binding human serum albumin, comprising a VH containing complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 contain amino acid sequences of SEQ ID NO: 184, 409 and 411 respectively, but do not contain amino acid sequences of SEQ ID NO: 129, 133 and 135 respectively.

[0009] In some embodiments, HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of SEQ ID NO: 184, 185, and 187, respectively, but exclude amino acid sequences of SEQ ID NO: 129, 133, and 135, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of SEQ ID NO: 189, 190, and 192, respectively, but exclude amino acid sequences of SEQ ID NO: 129, 133, and 135, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of SEQ ID NO: 189, 193, and 195, respectively, but exclude amino acid sequences of SEQ ID NO: 129, 133, and 135, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of SEQ ID NO: 123, 124, and 126, respectively. In some embodiments, VH comprises at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO:121. In some embodiments, the antigen binding site has a Kx concentration of less than or equal to 10 nM. D Binds to human serum albumin. In some embodiments, the antigen-binding site has a K+ level of less than or equal to 2 nM. D Protein A binding site. The antigen binding site of any one of claims 0-0, wherein the antigen binding site has a melting temperature of 60°C or higher.

[0010] In another aspect, this disclosure provides a multispecific binding protein comprising: (a) a first antigen-binding site that binds to a first target protein (e.g., human CD19) expressed on a target cell; (b) a second antigen-binding site that binds to a second target protein (e.g., human CD3) expressed on an immune effector cell; and (c) a third antigen-binding site that binds to human serum albumin, wherein the third antigen-binding site is the antigen-binding site for human serum albumin disclosed herein.

[0011] In some embodiments, the multispecific binding protein comprises a single polypeptide chain. In some embodiments, the third antigen binding site is not located between the first and second antigen binding sites within the polypeptide chain.

[0012] In some embodiments, the third antigen binding site is located at the N-terminus of the first antigen binding site and the second antigen binding site in the polypeptide chain. In some embodiments, the third antigen binding site is located at the N-terminus of the first antigen binding site in the polypeptide chain, and the first antigen binding site is located at the N-terminus of the second antigen binding site in the polypeptide chain. In some embodiments, the third antigen binding site is located at the N-terminus of the second antigen binding site in the polypeptide chain, and the second antigen binding site is located at the N-terminus of the first antigen binding site in the polypeptide chain.

[0013] In some embodiments, the third antigen binding site is located at the C-terminus of the first and second antigen binding sites in the polypeptide chain. In some embodiments, the first antigen binding site is located at the N-terminus of the second antigen binding site in the polypeptide chain, and the second antigen binding site is located at the N-terminus of the third antigen binding site in the polypeptide chain. In some embodiments, the second antigen binding site is located at the N-terminus of the first antigen binding site in the polypeptide chain, and the first antigen binding site is located at the N-terminus of the third antigen binding site in the polypeptide chain.

[0014] In some embodiments, the first antigen-binding site is located at the N-terminus of the third antigen-binding site in the polypeptide chain, and the third antigen-binding site is located at the N-terminus of the second antigen-binding site in the polypeptide chain. In other embodiments, the second antigen-binding site is located at the N-terminus of the third antigen-binding site in the polypeptide chain, and the third antigen-binding site is located at the N-terminus of the first antigen-binding site in the polypeptide chain.

[0015] In some embodiments, the first antigen-binding site comprises a single-chain variable fragment (scFv). In some embodiments, the third antigen-binding site comprises a single-domain antibody (sdAb). In some embodiments, the second antigen-binding site comprises scFv.

[0016] In some embodiments, the second antigen binding site binds to human CD3ε. In some embodiments, the second antigen binding site has a K+ level in the range of 1-100 nM. D Combined with human CD3ε.

[0017] In some embodiments, the second antigen-binding site comprises a VH containing complementarity-determining regions HCDR1, HCDR2, and HCDR3 and a VL containing complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and LCDR3 comprise amino acid sequences represented by SEQ ID NO:415, 416, 418, 419, 420, and 421, respectively. In some embodiments, VH comprises at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to SEQ ID NO:412, and VL comprises at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to SEQ ID NO:413. In some embodiments, the antigen-binding site comprises the amino acid sequence of SEQ ID NO:422 or 423.

[0018] In some embodiments, the second antigen binding site comprises a VH containing complementarity-determining regions HCDR1, HCDR2, and HCDR3 and a VL containing complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and LCDR3 comprise amino acid sequences represented by SEQ ID NO:415, 416, 426, 419, 420, and 421, respectively. In some embodiments, VH comprises at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:424, and VL comprises at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:413. In some embodiments, the antigen binding site comprises the amino acid sequence of SEQ ID NO:427 or 428.

[0019] In some embodiments, the second antigen binding site comprises a VH containing complementarity-determining regions HCDR1, HCDR2, and HCDR3 and a VL containing complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and LCDR3 comprise amino acid sequences represented by SEQ ID NO:415, 431, 418, 419, 420, and 432, respectively. In some embodiments, VH comprises at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:429, and VL comprises at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:430. In some embodiments, the antigen binding site comprises the amino acid sequence of SEQ ID NO:433 or 434.

[0020] In some embodiments, at least two adjacent antigen-binding sites are connected by a peptide linker. In some embodiments, each adjacent antigen-binding site is connected by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 298, 299, or 302. In some embodiments, the peptide linker consists of the amino acid sequence of SEQ ID NO: 298, 299, or 302.

[0021] In some embodiments, the multispecific binding protein does not contain an antibody Fc region. In some embodiments, the molecular weight of the multispecific binding protein is at least 65 kDa. In some embodiments, the serum half-life of the multispecific binding protein is at least 24, 36, 48, or 60 hours.

[0022] This disclosure also provides antibodies that contain the antigen-binding sites for human serum albumin disclosed herein.

[0023] In another respect, this disclosure provides a pharmaceutical composition comprising: (a) the multispecific binding protein or antibody disclosed herein; and (b) a pharmaceutically acceptable carrier.

[0024] This disclosure also provides isolated polynucleotides encoding the multispecific binding proteins or antibodies disclosed herein. Furthermore, this disclosure provides vectors containing the polynucleotides disclosed herein, and recombinant host cells containing the polynucleotides disclosed herein or the vectors.

[0025] This disclosure also provides a method for generating multispecific binding proteins or antibodies, the method comprising culturing the host cells disclosed herein under suitable conditions that allow for the expression of the multispecific binding proteins or antibodies. In some embodiments, the method further comprises isolating the multispecific binding proteins or antibodies. In some embodiments, the method further comprises co-formulating the isolated multispecific binding proteins or antibodies with a pharmaceutically acceptable carrier.

[0026] In addition, this disclosure provides a method for stimulating an immune response against target cells, the method comprising exposing the cells and T lymphocytes to the multispecific binding protein, antibody or pharmaceutical composition disclosed herein.

[0027] This disclosure also provides a method for treating blood cancers in subjects in need, the method comprising administering to the subject an effective amount of the disclosed multispecific binding protein, antibody, or pharmaceutical composition. In some embodiments, the blood cancer is a B-cell hematologic malignancy.

[0028] Furthermore, this disclosure provides a complex comprising CD3-expressing T cells, CD19-expressing B cells, and a multispecific binding protein disclosed herein, wherein the multispecific binding protein binds to both T cells and B cells. In some embodiments, the complex further comprises serum albumin. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the six domains of a single-chain, multispecific binding protein. The CD19-binding domain (scFv form), the CD3-binding domain (scFv form), and the HSA-binding domain (sdAb form) are connected in different orientations. The top of each construct represents the N-terminus of a given polypeptide chain, and the bottom of each construct represents the C-terminus of a given polypeptide chain. Invention Details

[0031] This invention is partly based on the development of novel antibodies that bind to serum albumin. A multispecific binding protein is also provided, comprising a first domain binding to a first target protein expressed on target cells, such as CD19 (e.g., human CD19), and / or a second domain binding to a second target protein expressed on immune effector cells, such as CD3 (e.g., human CD3), and a third domain binding to serum albumin (e.g., human serum albumin), wherein the third domain is derived from these novel antibodies. These domains are linked in a specific manner to obtain advantageous therapeutic efficacy and in vivo half-life. The multispecific binding protein can be used to treat diseases and conditions associated with abnormal cells expressing the first target protein, such as certain B-cell hematologic malignancies.

[0032] To facilitate understanding of the invention, several terms and phrases are defined below.

[0033] The term "multispecific binding protein" refers to a protein or protein conjugate capable of binding to two or more different targets (e.g., two or more different antigens or two or more different epitopes of the same antigen). For example, a multispecific binding protein can bind to two or more different targets through two or more different binding domains. The structure and / or function of a multispecific binding protein can be based on the structure and / or function of an antibody, such as a full-length or complete immunoglobulin molecule, an antibody heavy chain variable domain (VH) and / or a light chain variable domain (VL), and / or a single-chain antibody. In one example, each binding domain of the multispecific binding protein according to the invention contains a minimum structural requirement for an antibody to allow target binding. This minimum requirement can be defined, for example, by the presence of at least three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH domain) and / or three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL domain). An alternative method for defining the minimum structural requirements of an antibody is to define the epitope of a specific target that the antibody binds to, or by referencing a known antibody that competes with the antibody for binding to the same epitope. The antibodies upon which the constructs according to the invention are based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies.

[0034] Any binding domain of the multispecific binding protein according to the invention may contain the above-described CDR group. Those CDRs may be contained within the framework of VH and / or VL. For example, the Fd fragment has two VH domains and generally retains some antigen-binding function of the complete antigen-binding domain. Other examples of antibody fragments, antibody variants, or binding domain forms include: (1) Fab fragments, which are monovalent fragments having VL, VH, CL, and CH1 domains; (2) F(ab')2 fragments, which are divalent fragments having two Fab fragments linked by disulfide bonds through hinge regions; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains of an antibody single arm; (5) dAb fragments having VH domains (Ward et al., (1989) Nature 341: 544-546); (6) separated complementarity-determining regions (CDRs); (7) single-chain Fv (scFv), which may be derived from, for example, scFv libraries. Exemplary forms of the multispecific binding proteins according to the present invention are described, for example, WO2000006605A2, WO2005040220A1, WO2008119567A2, WO2010037838A2, WO2013026837A1, WO2013026833A1, US20140308285A1, US20140302037A1, WO2014144722A2, WO2014151910A1 and WO2015048272A1.

[0035] The multispecific binding protein according to the present invention may also contain modified fragments of antibodies, also known as antibody variants, such as di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, di-bodies, single-chain di-bodies, tandem di-scFv, tandem tri-scFv, "multivalent antibodies" such as tri-bodies or tetra-bodies, or single-domain antibodies such as nanobodies or single-variable-domain antibodies containing a single variable domain, which may be VH (also known as VHH in the case of sdAb) or VL, which bind antigens or epitopes independently of other V regions or domains.

[0036] As used herein, the terms “single-chain Fv,” “single-chain antibody,” and “scFv” refer to a single-peptide chain antibody fragment containing variable regions from both the heavy and light chains but lacking constant regions. Typically, single-chain antibodies further include peptide linkers connecting the VH and VL domains, enabling them to form the desired structure for binding antigens. Single-chain antibodies are discussed in detail by Pluckthun in *The Pharmacology of Monoclonal Antibodies*, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). Various methods for generating single-chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; and Skerra et al. (1988) Science 242:1038-1041. In specific embodiments, single-chain antibodies may also be bispecific, multispecific, human, humanized, and / or synthetic.

[0037] Furthermore, the “multispecific binding protein” described herein can be a monovalent, divalent, or multivalent construct. Additionally, the “multispecific binding protein” described herein can include molecules consisting of only one polypeptide chain, or molecules consisting of more than one polypeptide chain, wherein these chains can be identical (homodimer, homotrimer, or homooligomer) or different (heterodimer, heterotrimer, or heterooligomer). Examples of the antibodies and their variants or derivatives identified above are described, for example, in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988); Using Antibodies: a laboratory manual, CSHL Press (1999); Kontermann and Dibel, Antibody Engineering, Springer, 2nd ed. 2010; and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0038] The domains of the multispecific binding protein of the present invention can be linked by one or more peptide bonds and / or peptide linkers. According to the present invention, the term "peptide linker" includes an amino acid sequence that connects two domains. Peptide linkers can also be used to fuse a third domain with other domains of the multispecific binding protein of the present invention. A fundamental technical feature of such peptide linkers is that they do not contain any polymerization activity. Suitable peptide linkers include those described in U.S. Patent Nos. 4,751,180 and 4,935,233 or WO198809344A1.

[0039] The multispecific binding protein of the present invention can be an in vitro generated multispecific binding protein. The term "in vitro generated multispecific binding protein" refers to a multispecific binding protein as defined above, wherein all or part of the variable region (e.g., at least one CDR) is generated through non-immune cell selection, such as by in vitro phage display, protein microarray, or any other method that can test the ability of a candidate sequence to bind to an antigen. The multispecific binding protein of the present invention can also be generated through genomic rearrangement in animal immune cells. "Recombinant antibody" is an antibody prepared using recombinant DNA technology or genetic engineering.

[0040] The multispecific binding proteins of this invention can be monoclonal. As used herein, the term "monoclonal" means that the proteins obtained from a population are substantially homogeneous, i.e., individual proteins in the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). In the case of antibodies, monoclonal antibodies are highly specific, targeting a single antigenic side or determinant on the antigen, in contrast to conventional (polyclonal) antibody formulations that typically comprise different antibodies targeting different determinants (or epitopes). The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method.

[0041] The multispecific binding protein or one or more antigen-binding sites of the present invention can be affinity-matured. In immunology, affinity maturation is the process by which B cells produce antibodies with higher affinity for an antigen during an immune response. With repeated exposure to the same antigen, the host produces antibodies with progressively increasing affinity. As with the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Using display methods such as phage display to perform two or three rounds of mutation and selection can produce antibody fragments with affinity in the low nanomolar range.

[0042] Amino acid substitution variations can be introduced into multispecific binding proteins by replacing one or more hypervariable residues of a parent antibody (e.g., humanized or human antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient method for generating such alternative variants involves affinity maturation using phage display. Briefly, several hypervariable sides (e.g., 6-7 sides) are mutated to generate all possible amino acid substitutions on each side. The resulting antibody variants are displayed monovalently from filamentous phage particles as a fusion with the M13 gene III product packaged within each particle. The biological activity (e.g., binding affinity) of the phage-displayed variants is then screened as disclosed herein. To identify candidate hypervariable sides for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Optionally or additionally, analysis of the crystal structure of the antigen-antibody complex to identify contact points between binding domains may be beneficial. Such contact residues and adjacent residues are candidate residues to be replaced according to the techniques described herein. Once such variants are generated, a set of variants are screened as described herein, and antibodies that exhibit superior properties in one or more relevant assays can be selected for further development.

[0043] The multispecific binding proteins of this invention may specifically comprise “chimeric” antibodies (immunoglobulins) or fragments thereof, wherein a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific 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, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-55). Chimeric antibodies of interest herein include “primate-derived” antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) or human constant region sequences. Various methods for preparing chimeric antibodies have been described. See, for example, Morrison et al. (1985) Proc. Natl. Acad. Sci. USA, 81:6851; Takeda et al. (1985) Nature, 314:452; U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397; European Patent No. EP0171496; European Patent Application Publication No. EP0173494; and British Patent No. GB2177096.

[0044] The term "binding domain" or "binding (antigen) domain" used in connection with this invention is characterized by a domain that binds to or interacts with a given target side (specificity) on a given target epitope or target molecule (antigen), such as CD19, serum albumin, and CD3. The structure and function of the first, second, and / or third binding domains can be based on the structure and / or function of the antibody, such as the structure and / or function of a full-length or complete immunoglobulin molecule. The binding domain can be derived from the VH and / or VL or VHH domains of the antibody or fragments thereof. For example, the binding domain may include three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL domain) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH domain). The binding domain may also include VHH CDRs (i.e., CDR1, CDR2, and CDR3 of the VHH region).

[0045] The terms "variable domain" and "variable region" are used interchangeably to refer to the portions of an antibody or immunoglobulin domain that exhibit variability in their sequence and participate in determining the specificity and binding affinity of a particular antibody. This variability is not uniformly distributed throughout the variable domain of an antibody; it is concentrated in subdomains of each heavy and light chain variable region. These subdomains are called "hypervariable regions" or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRMs or FRs) and provide a scaffold for the six CDRs in three-dimensional space to form an antigen-binding surface.

[0046] In this invention, any binding domain of a multispecific binding protein may comprise a single-domain antibody (sdAb). A single-domain antibody comprises a single monomeric antibody variable domain capable of selectively binding to a specific antigen independently of other variable regions or domains. The first single-domain antibody was derived from heavy-chain antibodies discovered in camels; these antibodies were termed VHH fragments. Cartilaginous fishes also possess heavy-chain antibodies (IgNARs), from which VHH fragments can be obtained. NAR Single-domain antibodies are fragments. Another approach is to split the dimeric variable domain from common immunoglobulins (e.g., from humans or rodents) into monomers, thereby obtaining VH or VL as single-domain antibodies. Although most current research on single-domain antibodies is based on heavy chain variable domains, nanobodies derived from light chains have also shown specific binding to target epitopes. Examples of single-domain antibodies include nanobodies and single variable domain antibodies.

[0047] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule or its derivatives or variants that participates in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues in the N-terminal variable region ("V") of the heavy chain ("H") and the light chain ("L"). Three highly divergent segments within the V region of the heavy and light chains are called "hypervariable regions," which are inserted between more conserved flanking segments called "frame regions" or "FRs." Thus, the term "FR" refers to the amino acid sequence naturally present between and near the hypervariable regions of an immunoglobulin. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface that binds the antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity-determining regions" or "CDRs." In some animals (such as camels and cartilaginous fish), the antigen-binding site is formed by a single antibody chain that provides a "single-domain antibody." Antigen binding sites can be present in intact antibodies, in antigen-binding fragments of antibodies that retain the antigen-binding surface, or in recombinant peptides such as scFv that use peptide linkers to connect heavy chain variable domains to light chain variable domains in a single peptide.

[0048] As used herein, the term "antibody" refers to a protein or protein conjugate that contains an antigen-binding site. Antibodies can be monospecific or multispecific (e.g., bispecific).

[0049] As used herein, the terms “a” and “an” mean “one or more” and include plural forms, unless the context is inappropriate.

[0050] As used herein, the terms "subject" and "patient" refer to an organism that will be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., rats, monkeys, equines, bovines, suidae, canines, felines, etc.), and more preferably include humans.

[0051] As used herein, the term "effective amount" means an amount of compound (e.g., the compound of the present invention) sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more applications, doses, or administrations and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treatment" includes any effect that results in improvement of a symptom, disease, disorder, etc., or improvement of its symptoms, such as relief, reduction, regulation, improvement, or elimination.

[0052] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro.

[0053] As used herein, the term "pharmaceuticalally acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. Compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0054] Throughout the specification, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is also contemplated that compositions of the invention consist substantially of or comprise of the listed components, and processes and methods according to the invention consist substantially of or comprise of the listed processing steps.

[0055] Generally, unless otherwise specified, specified percentages of compositions are by weight. Furthermore, if a variable is not specified, its prior specification shall prevail.

[0056] I. Antiserum albumin antibody

[0057] In one aspect, this disclosure provides a serum albumin (e.g., human serum albumin (HSA)) antigen-binding site derived from the single-domain antibodies listed in Table 1. This disclosure also provides antibodies containing this antigen-binding site. Unless indicated by an asterisk (*), CDR sequences are identified according to the Kabat numbering scheme.

[0058] Table 1. Exemplary antibody sequences binding serum albumin

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] In some embodiments, the antigen-binding site of the present invention that binds to serum albumin includes a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as the VH of the antibodies disclosed in Table 1. In some embodiments, the antigen-binding site comprises HCDR1, HCDR2, and HCDR3 of the VH sequence of the antibody disclosed in Table 1, determined according to the following methods: Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262:732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In some embodiments, the antigen-binding site comprises the HCDR1, HCDR2, and HCDR3 sequences of the antibody disclosed in Table 1. In some embodiments, the antigen-binding site comprises the VH sequence of the antibody disclosed in Table 1.

[0066] Series 1 Constructs

[0067] In some embodiments, the antigen-binding site for HSA includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 408, 409, and 410, respectively, wherein the antigen-binding site does not contain the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 128, 133, and 134, respectively. In some embodiments, the HCDR1 sequence is selected from SEQ ID NO: 122, 128, 132, 137, 145, 157, and 169; the HCDR2 sequence is selected from SEQ ID NO: 124, 133, 146, 151, 153, 161, 165, 171, 179, and 181; and / or the HCDR3 sequence is selected from SEQ ID NO: 125, 134, 142, 147, 154, 158, 162, 166, 172, and 176.

[0068] In some embodiments, the antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 184, 409, and 411, respectively, wherein the antigen binding site does not contain the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 129, 133, and 135, respectively. In some embodiments, the HCDR1 sequence is selected from SEQ ID NO: 123, 129, and 170; the HCDR2 sequence is selected from SEQ ID NO: 124, 133, 146, 151, 153, 161, 165, 171, 179, and 181; and / or the HCDR3 sequence is selected from SEQ ID NO: 126, 135, 143, 148, 155, 159, 163, 167, 173, and 177.

[0069] In some embodiments, the antigen binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:121.

[0070] In some embodiments, the antigen-binding site has a higher binding affinity for human serum albumin, cynomolgus monkey serum albumin, mouse serum albumin, and / or protein A compared to an antigen-binding site having the VH sequence shown in SEQ ID NO:196.

[0071] Series 2 Constructs

[0072] In some embodiments, the antigen-binding site for HSA includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 183, 185, and 186, respectively, wherein the antigen-binding site does not contain the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 128, 133, and 134, respectively. In some embodiments, the HCDR1 sequence is selected from SEQ ID NO: 122, 128, 132, 145, 157, and 169; the HCDR2 sequence is selected from SEQ ID NO: 124, 133, 146, 151, 153, 171, 179, and 181; and / or the HCDR3 sequence is selected from SEQ ID NO: 125, 134, 147, 154, 158, 172, and 176.

[0073] In some embodiments, the antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 184, 185, and 187, respectively, wherein the antigen binding site does not contain the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 129, 133, and 135, respectively. In some embodiments, the HCDR1 sequence is selected from SEQ ID NO: 123, 129, and 170; the HCDR2 sequence is selected from SEQ ID NO: 124, 133, 146, 151, 153, 171, 179, and 181; and / or the HCDR3 sequence is selected from SEQ ID NO: 126, 135, 148, 155, 159, 173, and 177.

[0074] In some embodiments, the antigen binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:121.

[0075] In some embodiments, the antigen-binding site has a higher affinity for human serum albumin than an antigen-binding site having the VH sequence shown in SEQ ID NO:196. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site has a Kc of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM. D Binds to human serum albumin C. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site has a K+ level in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM. D It binds to human serum albumin.

[0076] Series 3 Constructs

[0077] In some embodiments, the antigen-binding site for HSA includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 188, 190, and 191, respectively, wherein the antigen-binding site does not contain the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 128, 133, and 134, respectively. In some embodiments, the HCDR1 sequence is selected from SEQ ID NO: 122, 128, 132, and 145; the HCDR2 sequence is selected from SEQ ID NO: 124, 133, and 161; and / or the HCDR3 sequence is selected from SEQ ID NO: 125, 134, 162, 147, and 176.

[0078] In some embodiments, the antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 189, 190, and 192, respectively, wherein the antigen binding site does not contain the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 129, 133, and 135, respectively. In some embodiments, the HCDR1 sequence is selected from SEQ ID NO: 123 and 129; the HCDR2 sequence is selected from SEQ ID NO: 124, 133, and 161; and / or the HCDR3 sequence is selected from SEQ ID NO: 126, 135, 163, 148, and 177.

[0079] In some embodiments, the antigen binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:121.

[0080] In some embodiments, the antigen-binding site has a higher affinity for protein A than an antigen-binding site having the VH sequence shown in SEQ ID NO:196. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site has a Kc of less than or equal to 2.5 nM or 2 nM. D Binding protein A. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site has a K+ level in the range of 1-2.5 nM or 1-2 nM. D Binding protein A.

[0081] Series 4 Constructs

[0082] In some embodiments, the antigen-binding site for HSA includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 188, 193, and 194, respectively, wherein the antigen-binding site does not contain the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 128, 133, and 134, respectively. In some embodiments, the HCDR1 sequence is selected from SEQ ID NO: 122, 128, 132, and 145; the HCDR2 sequence is selected from SEQ ID NO: 124 and 133; and / or the HCDR3 sequence is selected from SEQ ID NO: 125, 134, 147, and 176.

[0083] In some embodiments, the antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 189, 193, and 195, respectively, wherein the antigen binding site does not contain the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 129, 133, and 135, respectively. In some embodiments, the HCDR1 sequence is selected from SEQ ID NO: 123 and 129; the HCDR2 sequence is selected from SEQ ID NO: 124 and 133; and / or the HCDR3 sequence is selected from SEQ ID NO: 126, 135, 148, and 177.

[0084] In some embodiments, the antigen binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:121.

[0085] In some embodiments, the antigen-binding site has a higher affinity for human serum albumin and a higher affinity for protein A compared to an antigen-binding site having the VH sequence shown in SEQ ID NO:196. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site has a Kc of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM. D Combined with human serum albumin C, and at a concentration of K less than or equal to 2.5 nM or 2 nM. DBinding protein A. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site has a K value in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM. D Combined with human serum albumin, and with K in the range of 1-2.5 nM or 1-2 nM. D Binding protein A.

[0086] Individual constructs

[0087] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-101. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:122, 124, and 125, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:123, 124, and 126, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:121. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:121.

[0088] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-102. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 124, and 125, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 124, and 126, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:127. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:127.

[0089] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-103. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:122, 124, and 125, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:123, 124, and 126, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:130. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:130.

[0090] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-104. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:132, 133, and 134, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 133, and 135, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:131. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:131.

[0091] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-105. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:137, 133, and 134, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 133, and 135, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:136. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:136.

[0092] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-106. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 124, and 139, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 124, and 140, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:138. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:138.

[0093] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-107. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 124, and 142, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 124, and 143, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:141. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:141.

[0094] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-108. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:145, 146, and 147, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 146, and 148, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:144. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:144.

[0095] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-109. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:145, 133, and 134, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 133, and 135, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:149. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:149.

[0096] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-110. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 128, 151, and 134, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO: 129, 151, and 135, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO: 150. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO: 150.

[0097] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-111. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 153, and 154, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 153, and 155, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:152. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:152.

[0098] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-112. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:157, 133, and 158, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 133, and 159, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:156. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:156.

[0099] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-113. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 161, and 162, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 161, and 163, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:160. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:160.

[0100] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-114. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 165, and 166, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 165, and 167, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:164. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:164.

[0101] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-115. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:169, 171, and 172, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:170, 171, and 173, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:168. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:168.

[0102] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-116. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 133, and 147, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 133, and 148, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the amino acid sequence of SEQ ID NO:174. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:174.

[0103] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-117. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 133, and 176, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 133, and 177, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:175. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:175.

[0104] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-118. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 179, and 147, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 179, and 148, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:178. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:178.

[0105] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-119. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 181, and 125, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 181, and 126, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:180. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:180.

[0106] In some embodiments, the antigen-binding site for binding serum albumin is derived from CNG-HSA-120. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:128, 133, and 154, respectively. In some embodiments, the antigen-binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:129, 133, and 155, respectively. In some embodiments, the antigen-binding site comprises a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:180. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO:180.

[0107] In some embodiments, antigen binding sites derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 have higher binding affinity for human, cynomolgus monkey, and / or mouse serum albumin than antigen binding sites having the VH sequence shown by SEQ ID NO:196.

[0108] In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K+ of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM. D Binds to human serum albumin. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K+ value in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM. D It binds to human serum albumin.

[0109] In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K+ of less than or equal to 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM. D Combined with cynomolgus monkey serum albumin. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K+ value in the range of 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM. D Combined with cynomolgus monkey serum albumin.

[0110] In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K+ of less than or equal to 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM. DBinds to mouse serum albumin. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, it is derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG- The antigen binding sites of HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 are in the range of 1-100 nM, 1-90 nM, 1-80 nM, 1-70 nM, 1-60 nM, 1-50 nM, 1-40 nM, 1-30 nM, 1-20 nM, or 1-10 nM. D Combined with mouse serum albumin.

[0111] In some embodiments, the antigen binding sites derived from CNG-HSA-101, CNG-HSA-103, CNG-HSA-106, CNG-HSA-107, CNG-HSA-108, CNG-HSA-109, CNG-HSA-111, CNG-HSA-113, CNG-HSA-114, CNG-HSA-115, CNG-HSA-116, CNG-HSA-118, or CNG-HSA-120 are designated with a first K D Combined with human serum albumin and with the second K D Combined with mouse serum albumin, of which the second K D With the first K D The ratio is within the following ranges: 0.5-10, 0.5-9, 0.5-8, 0.5-7, 0.5-6, 0.5-5, 0.5-4, 0.5-3, 0.5-2, 0.9-10, 0.9-9, 0.9-8, 0.9-7, 0.9-6, 0.9-5, 0.9-4, 0.9-3, 0.9-2, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. It should be understood that antigen-binding sites with a ratio closer to 1 have a more similar affinity to mouse serum albumin than human serum albumin, which allows for more precise assessment of the pharmacokinetics of the antigen-binding site or the protein containing that site using mouse models.

[0112] In some embodiments, antigen binding sites derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, or CNG-HSA-117 have a higher affinity for protein A than antigen binding sites having the VH sequence shown in SEQ ID NO:196. It should be understood that the increased affinity for protein A allows for the purification of the antigen binding site or proteins containing the antigen binding site but not the antibody Fc region by protein A chromatography. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, or CNG-HSA-117 has a K+ of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM. D Combined with human serum albumin C, and at a concentration of K less than or equal to 2.5 nM or 2 nM. D Binding protein A. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, K, derived from the CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, or CNG-HSA-117 antigen-binding site, is in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM. D Combined with human serum albumin, and with K in the range of 1-2.5 nM or 1-2 nM. D Binding protein A.

[0113] In some embodiments, antigen binding sites derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, or CNG-HSA-117 exhibit higher binding affinity for human serum albumin and higher affinity for protein A compared to antigen binding sites having the VH sequence shown in SEQ ID NO:196. In some embodiments, when the antigen binding site is present as a monomer, antigen binding sites derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-116, or CNG-HSA-117 exhibit a Kc of less than or equal to 2.5 nM or 2 nM, as measured by SPR.D Binding protein A. In some embodiments, when the antigen-binding site is present as a monomer, as measured by SPR, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-116, or CNG-HSA-117 has a K+ in the range of 1-2.5 nM or 1-2 nM. D Binding protein A.

[0114] The melting temperature represents the thermal stability of the antigen-binding site and can be measured by differential scanning fluorometry, for example, as described in Durowoju et al. (2017) J. Vis. Exp. (121): 55262. The thermal stability of antibodies or fragments thereof can be enhanced by grafting CDRs onto a stable framework, introducing non-standard disulfide bonds, and other mutagenesis, as described in McConnell et al. (2014) MAbs, 6(5): 1274-82; and Goldman et al. (2017) Front. Immunol., 8: 865. In some embodiments, the antigen binding sites derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-105, CNG-HSA-106, CNG-HSA-108, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, CNG-HSA-117, or CNG-HSA-120, as measured by differential scanning fluorometry, have a melting temperature of 60°C or higher. In some embodiments, the antigen binding sites derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-106, or CNG-HSA-120, as measured by differential scanning fluorometry, have a melting temperature of 65°C or higher.

[0115] This disclosure also provides antigen-binding sites that compete with antibody or antigen-binding sites containing the VH sequence provided in Table 1 for binding to serum (e.g., human serum albumin) and / or for binding to protein A.

[0116] II. Multispecific binding protein

[0117] In one aspect, this disclosure provides a multispecific binding protein comprising a domain that binds to a target molecule (e.g., a target protein expressed on a target cell) and an antigen-binding site disclosed in part 0 of the preceding title “Anti-serum albumin antibody”. In some embodiments, the multispecific binding protein comprises a first domain (e.g., a first antigen-binding site) that binds to a first target protein expressed on a target cell; and / or a second domain (e.g., a second antigen-binding site) that binds to a second target protein expressed on an immune effector cell; and a third domain (e.g., a third antigen-binding site) that binds to serum albumin (e.g., HSA), wherein the third domain comprises the antigen-binding site disclosed in part 0 of the preceding title “Anti-serum albumin antibody”. The first target may be a molecule (e.g., a protein) expressed on a target cell (e.g., a cancer cell or a cell in a tumor microenvironment) that is to be cleared, such as CD19, HER2, BCMA, CD33, or EGFR. The second target can be a molecule (e.g., a protein) expressed on immune effector cells (e.g., T cells or NK cells), such as CD3 (e.g., CD3ε (epsilon), CD3δ (delta), and / or CD3γ (gamma)), 4-1BB, NKG2D, or NKp30. It is anticipated that multispecific binding proteins that bind to such first and second targets will promote the clearance of cells expressing the first target.

[0118] In some embodiments, the first, second, and third domains comprise a first antigen-binding site, a second antigen-binding site, and a third antigen-binding site, respectively. Each antigen-binding site of the multispecific binding protein can take various forms, such as a single-chain variable fragment (scFv), a Fab fragment, or a single-domain antibody (sdAb). In some embodiments, the first antigen-binding site comprises an scFv. In some embodiments, the second antigen-binding site comprises an scFv. In some embodiments, the third antigen-binding site comprises an sdAb.

[0119] Alternatively, it has been considered that one or more binding domains may not contain an antigen-binding site. For example, U.S. Patent Application Publication No. US20130316952A1 discloses a polypeptide that binds serum albumin, having the amino acid sequence LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNA LKDEILKA (SEQ ID NO:282). Other exemplary polypeptides binding HSA are described in Dennis et al. (2002) J. Biol. Chem., 277:35035-43; Jacobs et al. (2015) Protein Eng. Des. Sel., 28:385-93; and Zorzi et al. (2017) Nat. Commun., 8:16092.

[0120] In some embodiments, the multispecific binding protein also includes an antibody Fc region. The presence of the Fc region can increase the serum half-life of the multispecific binding protein. Depending on the specific Fc isotype and variant used, the Fc region can also alter the activity (e.g., cytotoxic activity) of the multispecific binding protein.

[0121] In other embodiments, the multispecific binding protein does not contain an antibody Fc region. The absence of the Fc region contributes to a smaller size of the multispecific binding protein, which can exhibit improved tissue penetration and pharmacokinetic properties. In some embodiments, the multispecific binding protein consists of, or is substantially composed of, first, second, and third antigen binding sites and linkers between them.

[0122] In some implementations, the multispecific binding protein binds monovalently to a first target protein, a second target protein, and / or serum albumin. The exclusion of additional binding domains reduces the risk of nonspecific immune cell activation and decreases the size of the multispecific binding protein.

[0123] A. First antigen binding site

[0124] In some embodiments, the first antigen-binding site of the multispecific binding protein binds to CD19 (e.g., human CD19). In some embodiments, the first antigen-binding site of the multispecific binding protein binds to FLT3 (e.g., human FLT3).

[0125] The first antigen-binding site for CD19 can be derived from, for example, MT-103 (a single-chain bispecific CD19 / CD3 antibody; see Hoffman et al. (2005) Int. J. Cancer, 115:98-104; Schlereth et al. (2006) Cancer Immunol. Immunother. 55:503-14), CD19 / CD16 bivalent antibodies (see Schlenzka et al. (2004) Anti-cancer Drugs 15:915-19; Kipriyanov et al. (2002) J. Immunol. 169:137-44), BU12-saponin (see Flavell et al. (1995) Br. J. Cancer 72:1373-79) and anti-CD19 idarubicin (see Rowland et al. (1993) Cancer Immunol. Immunother. 55:503-14). Further exemplary antigen-binding sites for binding CD19 that can derive from the first antigen-binding site of this invention are disclosed in U.S. Patent Application Publications US20170174786A1, US20090042291A1, US20160046730A1, US20070154473A1, US20090142349A1, US20180142018A1, US20090136526A1, US20060257398A1, and US20180230225A1, and PCT Publication WO2019057100A1. For example, in some embodiments, the first antigen-binding site for binding CD19 is derived from the antibodies listed in Table 2.

[0126] Table 2. Exemplary antibody sequences binding to CD19

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] When the VL and LCDR sequences are labeled “N / A”, the antigen binding site is an sdAb that has only VH (e.g., VHH).

[0136] In some embodiments, the first antigen-binding site comprises a VH and a VL, wherein the VH contains at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the amino acid sequence identical to the VH of the same antibody disclosed in Table 2, and the VL contains at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the amino acid sequence identical to the VL of the same antibody disclosed in Table 2. In some embodiments, the antigen binding site comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and / or VL sequences of the antibodies disclosed in Table 2, determined according to the following methods: Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262:732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In some embodiments, the antigen binding site comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of the antibodies disclosed in Table 2. In some embodiments, the antigen binding site comprises the VH and VL sequences of the antibodies disclosed in Table 2.

[0137] Such antigen-binding sites can take the form of scFv. In some embodiments, VH is located at the C-terminus of VL. In some embodiments, VH is located at the N-terminus of VL. In some embodiments, VH and VL are linked by a peptide linker, for example, the linker disclosed in subsection D entitled “Linker” below. To stabilize the scFv, amino acid residues at position 44 of VH and position 100 (according to Kabat numbering) of VL can be substituted with Cys, thereby promoting the formation of a disulfide bond between VH and VL. Thus, in some embodiments, VH and VL contain Cys at positions 100 and 44, respectively.

[0138] In other embodiments, the first antigen-binding site comprises an sdAb containing a VH, which includes complementarity-determining regions HCDR1, HCDR2, and HCDR3. In some embodiments, the VH contains at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the VH of the sdAb antibodies provided in Table 2. In some embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of the antibodies disclosed in Table 2, determined according to the following methods: Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIHP Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262:732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In some embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of the antibodies provided in Table 2. In some implementations, VH comprises the amino acid sequence of VH of sdAb provided in Table 2.

[0139] In some implementations, the first antigen binding site has a K value less than or equal to the following: DBinding to CD19: 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. For example, in some embodiments, the first antigen binding site is at the following K... D Combining with CD19: approximately 10 pM to approximately 1 nM, approximately 10 pM to approximately 0.9 nM, approximately 10 pM to approximately 0.8 nM, approximately 10 pM to approximately 0.7 nM, approximately 10 pM to approximately 0.6 nM, approximately 10 pM to approximately 0.5 nM, approximately 10 pM to approximately 0.4 nM, approximately 10 pM to approximately 0.3 nM, approximately 10 pM to approximately 0.2 nM, approximately 10 pM to approximately 0.1 nM, approximately 10 pM to approximately 50 pM, 0.1 nM to approximately 10 nM, approximately 0.1 nM to approximately 9 nM, approximately 0.1 nM to approximately 8 nM, approximately 0.1 nM to approximately 7 nM, approximately 0.1 nM to approximately 6 nM, approximately 0.1 nM to approximately 5 nM, approximately 0.1 nM to approximately 4 nM, approximately 0 0.1nM - about 3nM, about 0.1nM - about 2nM, about 0.1nM - about 1nM, about 0.1nM - about 0.5nM, about 0.5nM - about 10nM, about 1nM - about 10nM, about 2nM - about 10nM, about 3nM - about 10nM, about 4nM - about 10nM, about 5nM - about 10nM, about 6nM - about 10nM, about 7nM - about 10nM, about 8nM - about 10nM, or about 9nM - about 10nM.

[0140] It should be understood that the binding affinity of a single first antigen-binding site to CD19 may differ from that of the same antigen-binding site in the case of multispecific binding proteins disclosed herein, possibly due to conformational constraints from other domains. Environment-related binding affinities are described in section E below, entitled “Binding Affinities.”

[0141] In some embodiments, when present in Fab form, the first antigen binding site has a melting temperature of at least 60°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. In some embodiments, when present in Fab form, the first antigen binding site has a melting temperature within the following ranges: 60-85°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-85°C, 65-80°C, 65-75°C, 65-70°C, 70-85°C, 70-80°C, 70-75°C, 75-85°C, 75-80°C, or 80-85°C.

[0142] B. Second antigen binding site

[0143] In some embodiments, the second antigen-binding site of the multispecific binding protein binds CD3 (e.g., human CD3 and / or macaque CD3). In some embodiments, the second antigen-binding site binds CD3ε (epsilon). In some embodiments, the second antigen-binding site binds CD3δ (delta). In some embodiments, the second antigen-binding site binds CD3γ (gamma).

[0144] Early The construct binds to a conformational epitope of CD3 and is typically species-specific (see PCT Publication No. WO2008119567A2). Improved Constructs, such as bonatomab (also known as AMG 103; see PCT Publication WO1999054440A1) and soritomab (also known as AMG 110; see PCT Publication WO2005040220A1), bind to an environment-independent epitope at the N-terminus of the CD3ε chain (e.g., amino acid residues 1-27 of the human CD3ε extracellular domain) and exhibit cross-species specificity for the CD3ε chains of humans, common marmosets (Callithrix jacchus), woolly tadpoles (Saguinus Oedipus), and squirrel monkeys (Saimirisciureus) (see ibid.). These constructs do not exhibit cross-species specificity with earlier The same level of nonspecific activation of T cells was observed in the construct, and therefore it is believed to have a lower risk of side effects (see Brischwein et al. (2007) J. Immunother., 30(8): 798-807).

[0145] In some embodiments, the second antigen-binding site of the multispecific binding protein binds to an epitope at the N-terminus of the CD3ε chain. In some embodiments, the second antigen-binding site binds to an epitope located at amino acid residues 1-27 of the extracellular domain of human CD3ε. This epitope or its homologs are also present in certain non-human primates. Therefore, in some embodiments, the second antigen-binding site binds to CD3 in different primates, such as humans, New World primates (e.g., common marmoset (Callithrix jacchus), woolly-crowned tadpole monkey (Saguinus Oedipus), and squirrel monkey (Saimiri sciureus)), Old World primates (e.g., baboons and rhesus macaques), gibbons, and non-human subfamilies. Common marmosets and woolly-crowned tadpole monkeys are New World primates belonging to the family Marmosetidae, while squirrel monkeys are New World primates belonging to the family Sciuridae. In some embodiments, the second antigen-binding site binds to human CD3ε and / or rhesus macaque CD3ε. In some implementations, the second antigen binding site also binds to CD3ε of common marmosets, woolly capuchin monkeys, and / or squirrel monkeys.

[0146] Second antigen-binding sites that bind to cellular ectotopes of human and / or macaque CD3 can be derived from, for example, muromonab-CD3 (OKT3) as described in WO2008101154; otelixizumab (TRX4) as described in WO2007145941; teplizumab (MGA031) as described in WO2013040164; visilizumab (Nuvion) ​​as described in WO2004052397; SP34 as described in WO2015181098; and so on. X35, VIT3, or BMA030 (BW264 / 56) as described in 5006749; CLB-T3 / 3, CRIS7, CLB-T3.4.2, WT32, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, or F101.01 as described in WO2004106383; YTH12.5 or SPv-T3b as described in WO2004106383; and Fl as described in WO2012084895. 11-409, such as TR-66 as described in WO2013158856; UCHT-1 as described in WO2000041474; WT-31 as described in WO2016085889; or antibodies as described in WO2008119567. For example, in some embodiments, the second antigen binding site for binding to CD3 is derived from antibodies listed in Table 3.

[0147] Table 3. Exemplary antibody sequences binding to CD3

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] When the VL and LCDR sequences are labeled “N / A”, the antigen binding site is an sdAb that has only VH (e.g., VHH).

[0157] In some embodiments, the second antigen binding site comprises a VH and a VL, wherein the VH contains at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the amino acid sequence identical to the VH of the same antibody disclosed in Table 3, and the VL contains at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the amino acid sequence identical to the VL of the same antibody disclosed in Table 3. In some embodiments, the antigen binding site comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and / or VL sequences of the antibodies disclosed in Table 3, determined according to the following methods: Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262:732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In some embodiments, the antigen binding site comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of the antibodies disclosed in Table 3. In some embodiments, the antigen binding site comprises the VH and VL sequences of the antibodies disclosed in Table 3.

[0158] In some embodiments, the second antigen binding site for CD3 is derived from CNG-CD3-1. In some embodiments, the second antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:414, 416, and 417, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NO:419, 420, and 421, respectively. In some embodiments, the second antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:415, 416, and 418, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NO:419, 420, and 421, respectively. In some embodiments, the second antigen binding site comprises VH and VL, wherein the VH comprises at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:412, and the VL comprises at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:413. In some embodiments, the VH and VL of the second antigen binding site comprise the amino acid sequences of SEQ ID NO:412 and 413.

[0159] In some embodiments, the second antigen binding site for CD3 is derived from CNG-CD3-2. In some embodiments, the second antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:414, 416, and 425, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NO:419, 420, and 421, respectively. In some embodiments, the second antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:415, 416, and 426, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NO:419, 420, and 421, respectively. In some embodiments, the second antigen binding site comprises VH and VL, wherein the VH comprises at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:424, and the VL comprises at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:413. In some embodiments, the VH and VL of the second antigen binding site comprise the amino acid sequences of SEQ ID NO:424 and 413.

[0160] In some embodiments, the second antigen binding site for CD3 is derived from CNG-CD3-3. In some embodiments, the second antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:414, 431, and 417, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NO:419, 420, and 432, respectively. In some embodiments, the second antigen binding site comprises a VH containing the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NO:415, 431, and 418, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NO:419, 420, and 432, respectively. In some embodiments, the second antigen binding site comprises VH and VL, wherein the VH comprises at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:429, and the VL comprises at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the same amino acid sequence as SEQ ID NO:430. In some embodiments, the VH and VL of the second antigen binding site comprise the amino acid sequences of SEQ ID NO:429 and 430.

[0161] Such antigen-binding sites can take the form of scFv. In some embodiments, VH is located at the C-terminus of VL. In some embodiments, VH is located at the N-terminus of VL. In some embodiments, VH and VL are linked by a peptide linker, for example, the linker disclosed in subsection D entitled “Linker” below. In some embodiments, the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 422, 427, or 433. To stabilize scFv, amino acid residues at position 44 of VH and position 100 of VL (according to Kabat numbering) can be substituted with Cys, thereby promoting the formation of a disulfide bond between VH and VL. Thus, in some embodiments, VH and VL contain Cys at positions 100 and 44, respectively. In some embodiments, the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 423, 428, or 434.

[0162] In other embodiments, the second antigen binding site comprises an sdAb containing a VH, which includes complementarity-determining regions HCDR1, HCDR2, and HCDR3. In some embodiments, the VH contains at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence identical to the VH of the sdAb antibodies provided in Table 3. In some embodiments, the VH contains the HCDR1, HCDR2, and HCDR3 sequences of the antibodies provided in Table 3. In some embodiments, the VH contains the amino acid sequence of the VH of the sdAbs provided in Table 3.

[0163] In some embodiments, the second antigen-binding site competes with antibodies or antigen-binding fragments thereof that include the VH, VL and / or scFv sequences provided in Table 3 for binding to CD3 (e.g., human CD3 and / or macaque CD3).

[0164] In some embodiments, the second antigen-binding site of the multispecific binding protein has a dissociation constant (K0) of about 0.1 nM to about 1 μM. D ) combined with CD3 (e.g., human CD3 and / or macaque CD3). K D It can be measured using methods known in the art. In some implementations, K D SPR is measured by immobilizing CD3 or its extracellular fragments on a chip. In some embodiments, K+ of CD3 expressed on the cell surface is measured by flow cytometry. D For example, as described in Example 6 below.

[0165] In some implementations, the second antigen binding site has a K value less than or equal to the following: D Binding to CD3: 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. For example, in some embodiments, the first antigen binding site is at the following K... DCombining CD3: approximately 10 pM to approximately 1 nM, approximately 10 pM to approximately 0.9 nM, approximately 10 pM to approximately 0.8 nM, approximately 10 pM to approximately 0.7 nM, approximately 10 pM to approximately 0.6 nM, approximately 10 pM to approximately 0.5 nM, approximately 10 pM to approximately 0.4 nM, approximately 10 pM to approximately 0.3 nM, approximately 10 pM to approximately 0.2 nM, approximately 10 pM to approximately 0.1 nM, approximately 10 pM to approximately 50 pM, 0.1 nM to approximately 10 nM, approximately 0.1 nM to approximately 9 nM, approximately 0.1 nM to approximately 8 nM, approximately 0.1 nM to approximately 7 nM, approximately 0.1 nM to approximately 6 nM, approximately 0.1 nM to approximately 5 nM, approximately 0.1 nM to approximately 4 nM, approximately 0 0.1nM - about 3nM, about 0.1nM - about 2nM, about 0.1nM - about 1nM, about 0.1nM - about 0.5nM, about 0.5nM - about 10nM, about 1nM - about 10nM, about 2nM - about 10nM, about 3nM - about 10nM, about 4nM - about 10nM, about 5nM - about 10nM, about 6nM - about 10nM, about 7nM - about 10nM, about 8nM - about 10nM, or about 9nM - about 10nM.

[0166] It should be understood that, in the case of multispecific binding proteins, a larger K... D (i.e., lower affinity for CD3) is likely desirable. It is undesirable to be bound by theory, as the expectation is that multispecific binding proteins with very high affinity for CD3 may lead to excessive cytokine release, thereby narrowing the therapeutic window. Therefore, in some embodiments, the second antigen binding site has a K value higher than or equal to the following... D Binding to CD3 (e.g., human CD3, e.g., human CD3ε): 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. In some embodiments, the second antigen binding site is at the following K... D Combined with CD3: approximately 1 nM to approximately 100 nM, approximately 1 nM to approximately 90 nM, approximately 1 nM to approximately 80 nM, approximately 1 nM to approximately 70 nM, approximately 1 nM to approximately 60 nM, approximately 1 nM to approximately 50 nM, approximately 1 nM to approximately 40 nM, approximately 1 nM to approximately 30 nM, approximately 1 nM to approximately 20 nM, approximately 1 nM to approximately 10 nM, approximately 10 nM to approximately 100 nM, approximately 10 nM to approximately 90 nM, approximately 10 nM to approximately 80 nM, approximately 10 nM to approximately 70 nM, approximately 10 nM to approximately 60 nM, approximately 10 nM to approximately 50 nM, approximately 10 nM to approximately 40 nM, approximately 10 nM to approximately 30 nM, or approximately 10 nM to approximately 20 nM.

[0167] It should be understood that the binding affinity of a single second antigen binding site to CD3 may differ from that of the same antigen binding site in the case of multispecific binding proteins disclosed herein, possibly due to conformational constraints from other domains. Environment-related binding affinities are described in subsection E, entitled “Binding Affinities” below.

[0168] In some embodiments, when present in Fab form, the second antigen binding site has a melting temperature of at least 60°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. In some embodiments, when present in Fab form, the second antigen binding site has a melting temperature within the following ranges: 60-85°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-85°C, 65-80°C, 65-75°C, 65-70°C, 70-85°C, 70-80°C, 70-75°C, 75-85°C, 75-80°C, or 80-85°C.

[0169] C. Construct Form

[0170] The first, second, and third antigen binding sites can take various forms. In some embodiments, the first, second, and / or third antigen binding sites comprise two antibody variable domains (e.g., VH and VL). VH and VL can be mutated to introduce disulfide bonds at the stable antigen binding site (e.g., between H44 and L100) (see Zhao et al. (2010) Int. J. Mol. Sci., 12(1): 1-11). In some embodiments, the first, second, and / or third antigen binding sites comprise a single antibody variable domain (e.g., sdAb).

[0171] In an antigen-binding site containing VH and VL, VH and VL can be linked to form an scFv. VH can be located at the N-terminus or C-terminus of VL. VH and VL are typically linked via a linker such as a peptide linker. Exemplary sequences of peptide linkers are provided in subsection D, hereinafter titled "Linker". In some embodiments, the VH of the antigen-binding domain is linked to the VL of the antigen-binding domain via a peptide linker having the amino acid sequences listed in Table 4. In a particular embodiment, the VH of the antigen-binding domain is linked to the VL of the antigen-binding domain via a peptide linker having the amino acid sequence of SEQ ID NO: 298, 299, or 302, wherein VH is located at the N-terminus of VL. In other particular embodiments, the VH of the antigen-binding domain is linked to the VL of the antigen-binding domain via a peptide linker having the amino acid sequence of SEQ ID NO: 298, 299, or 302, wherein VH is located at the C-terminus of VL.

[0172] Alternatively, VH and VL can be located on different polypeptide chains, and the formation of the VH-VL complex can be facilitated by additional domains such as the antibody constant region CH1 and CL. Therefore, in some embodiments, the multispecific binding protein comprises a Fab containing the VH and VL disclosed herein.

[0173] In some embodiments, the multispecific binding protein of the present invention comprises a first antigen-binding site containing a single antibody variable domain, a second antigen-binding site containing a single antibody variable domain, and a third antigen-binding site containing a single antibody variable domain. In some embodiments, the multispecific binding protein comprises a first antigen-binding site in the form of an sdAb, a second antigen-binding site in the form of an sdAb, and a third antigen-binding site in the form of an sdAb.

[0174] In some embodiments, the multispecific binding protein of the present invention comprises a first antigen-binding site containing a single antibody variable domain, a second antigen-binding site containing a single antibody variable domain, and a third antigen-binding site containing two antibody variable domains. In some embodiments, the multispecific binding protein comprises a first antigen-binding site in the form of an sdAb, a second antigen-binding site in the form of an sdAb, and a third antigen-binding site in the form of an scFv.

[0175] In some embodiments, the multispecific binding protein of the present invention comprises a first antigen-binding site containing a single antibody variable domain, a second antigen-binding site containing two antibody variable domains, and a third antigen-binding site containing a single antibody variable domain. In some embodiments, the multispecific binding protein comprises a first antigen-binding site in the form of an sdAb, a second antigen-binding site in the form of an scFv, and a third antigen-binding site in the form of an sdAb.

[0176] In some embodiments, the multispecific binding protein of the present invention comprises a first antigen-binding site containing a single antibody variable domain, a second antigen-binding site containing two antibody variable domains, and a third antigen-binding site containing two antibody variable domains. In some embodiments, the multispecific binding protein comprises a first antigen-binding site in the form of sdAb, a second antigen-binding site in the form of scFv, and a third antigen-binding site in the form of scFv.

[0177] In some embodiments, the multispecific binding protein of the present invention comprises a first antigen-binding site containing two antibody variable domains, a second antigen-binding site containing a single antibody variable domain, and a third antigen-binding site containing a single antibody variable domain. In some embodiments, the multispecific binding protein comprises a first antigen-binding site in the form of scFv, a second antigen-binding site in the form of sdAb, and a third antigen-binding site in the form of sdAb.

[0178] In some embodiments, the multispecific binding protein of the present invention comprises a first antigen-binding site containing two antibody variable domains, a second antigen-binding site containing a single antibody variable domain, and a third antigen-binding site containing two antibody variable domains. In some embodiments, the multispecific binding protein comprises a first antigen-binding site in the form of scFv, a second antigen-binding site in the form of sdAb, and a third antigen-binding site in the form of scFv.

[0179] In some embodiments, the multispecific binding protein of the present invention comprises a first antigen-binding site containing two antibody variable domains, a second antigen-binding site containing two antibody variable domains, and a third antigen-binding site containing a single antibody variable domain. In some embodiments, the multispecific binding protein comprises a first antigen-binding site in the form of scFv, a second antigen-binding site in the form of scFv, and a third antigen-binding site in the form of sdAb.

[0180] In some embodiments, the multispecific binding protein of the present invention comprises a first antigen-binding site containing two antibody variable domains, a second antigen-binding site containing two antibody variable domains, and a third antigen-binding site containing two antibody variable domains. In some embodiments, the multispecific binding protein comprises a first antigen-binding site in the form of an scFv, a second antigen-binding site in the form of an scFv, and a third antigen-binding site in the form of an scFv.

[0181] The three antigen-binding sites of a multispecific binding protein can be linked in any of the following directions along the amino-carboxyl group:

[0182] (i) First antigen binding site (e.g., CD19 binding domain) - second antigen binding site (e.g., CD3 binding domain) - third antigen binding site (serum albumin binding domain);

[0183] (ii) First antigen binding site (e.g., CD19 binding domain) - third antigen binding site (serum albumin binding domain) - second antigen binding site (e.g., CD3 binding domain);

[0184] (iii) Second antigen binding site (e.g., CD3 binding domain) - first antigen binding site (e.g., CD19 binding domain) - third antigen binding site (serum albumin binding domain);

[0185] (iv) Second antigen binding site (e.g., CD3 binding domain) - third antigen binding site (serum albumin binding domain) - first antigen binding site (e.g., CD19 binding domain);

[0186] (v) Third antigen binding site (serum albumin-binding domain) - first antigen binding site (e.g., CD19 binding domain) - second antigen binding site (e.g., CD3 binding domain); and

[0187] (vi) Third antigen binding site (serum albumin binding domain) - second antigen binding site (e.g., CD3 binding domain) - first antigen binding site (e.g., CD19 binding domain), wherein the dash above represents a peptide bond and / or linker (e.g., peptide linker).

[0188] In some embodiments, the third antigen-binding site is not located between the first and second antigen-binding sites. Constructs having this form are expected to have good therapeutic efficacy and in vivo half-life. In some embodiments, the third antigen-binding site is located at the N-terminus of both the first and second antigen-binding sites or at the C-terminus of both the first and second antigen-binding sites. In some embodiments, the third antigen-binding site is located at the N-terminus of both the first and second antigen-binding sites. In some embodiments, the third antigen-binding site is located at the C-terminus of both the first and second antigen-binding sites.

[0189] If a single polypeptide chain contains two antigen-binding sites, the position (N-terminus or C-terminus) of one antigen-binding site relative to the other is determined according to the known definitions of "N-terminus" and "C-terminus" in the art. It should be understood that if an antigen-binding site contains two separate polypeptide chains, its position (N-terminus or C-terminus) relative to another antigen-binding site (having one or two polypeptide chains) can be similarly determined (if the separate polypeptide chains contain at least one of the former and at least one of the latter). Further, if antigen-binding site A is at the N-terminus of antigen-binding site B, and antigen-binding site B is at the N-terminus of antigen-binding site C, then antigen-binding site A is considered to be located at the N-terminus of antigen-binding site C, even if antigen-binding sites A and C are not present in any single common polypeptide chain. More complex structures of multispecific binding proteins are also considered, some of which may have orientations that are difficult to characterize using the terms "N-terminus" and "C-terminus" as described above, for example, due to the different relative positions of two antigen-binding sites on one polypeptide chain relative to another polypeptide chain, or the presence of loop structures.

[0190] According to the present invention, the multispecific binding protein and its constituent binding domains are in the form of one or more polypeptides. Such polypeptides may include protein and non-protein portions (e.g., chemical linkers or chemical cross-linking agents such as glutaraldehyde). In some embodiments, the multispecific binding protein of the present invention includes a first antigen binding site, a second antigen binding site, and a third antigen binding site, all linked together to form a single polypeptide chain. In some embodiments, the first, second, and third antigen binding sites are in the form of scFv and / or sdAb, for example, in a combination as described above, to form a single polypeptide chain.

[0191] D. Connector

[0192] As described above, the antigen-binding sites of the multispecific binding proteins of the present invention can be linked by peptide bonds or linkers (e.g., peptide linkers). In some embodiments, at least two adjacent antigen-binding sites are linked by linkers (e.g., peptide linkers). In some embodiments, every two adjacent antigen-binding sites are linked by linkers (e.g., peptide linkers).

[0193] In some embodiments, the three antigen-binding sites of a multispecific binding protein can be linked in either of the following directions in the amino-to-carboxyl direction via linkers (e.g., peptide linkers) denoted as L1 and L2:

[0194] (i) First antigen binding site (e.g., CD19 binding domain) - L1 - Second antigen binding site (e.g., CD3 binding domain) - L2 - Third antigen binding site (serum albumin binding domain);

[0195] (ii) First antigen binding site (e.g., CD19 binding domain) - L1 - Third antigen binding site (serum albumin binding domain) - L2 - Second antigen binding site (e.g., CD3 binding domain);

[0196] (iii) Second antigen binding site (e.g., CD3 binding domain) - L1 - First antigen binding site (e.g., CD19 binding domain) - L2 - Third antigen binding site (serum albumin binding domain);

[0197] (iv) Second antigen binding site (e.g., CD3 binding domain) - L1 - Third antigen binding site (serum albumin binding domain) - L2 - First antigen binding site (e.g., CD19 binding domain);

[0198] (v) Third antigen binding site (serum albumin-binding domain) - L1 - First antigen binding site (e.g., CD19 binding domain) - L2 - Second antigen binding site (e.g., CD3 binding domain); and

[0199] (vi) Third antigen binding site (serum albumin binding domain) - L1 - Second antigen binding site (e.g., CD3 binding domain) - L2 - First antigen binding site (e.g., CD19 binding domain).

[0200] It is understandable that in a given construct, L1, L2, or both L1 and L2 can be replaced by peptide bonds.

[0201] It can be understood that if a single polypeptide chain contains two adjacent antigen-binding sites, the peptide linker connecting these two antigen-binding sites represents the amino acid sequence between them. If an antigen-binding site contains two separate polypeptide chains, one of which exists in a single common polypeptide that serves as an adjacent antigen-binding site or its polypeptide chain, the peptide linker connecting these two antigen-binding sites represents the amino acid sequence between them in the single common polypeptide.

[0202] In some embodiments, linkers L1 and L2 are peptide linkers. The appropriate lengths of L1 and L2 can be chosen independently. For example, in some embodiments, L1 and / or L2 are about 50 or fewer amino acid residues in length. In some embodiments, L1 consists of about 50 or fewer amino acid residues. In some embodiments, L1 consists of about 20 or fewer amino acid residues. In some embodiments, L2 consists of about 50 or fewer amino acid residues. In some embodiments, L2 consists of about 20 or fewer amino acid residues. In some embodiments, L1 and L2 independently consist of about 50 or fewer amino acid residues. In some embodiments, L1 and L2 independently consist of about 20 or fewer amino acid residues.

[0203] In some embodiments, peptide linkers L1 and L2 have optimized length and / or amino acid composition. In some embodiments, L1 and L2 have the same length and the same amino acid composition. In other embodiments, L1 and L2 are different. In some embodiments, L1 and / or L2 are "short," i.e., composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Therefore, in some cases, the linker consists of about 12 or fewer amino acid residues. In some embodiments, L1 and / or L2 are "long," for example, composed of 15, 20, or 25 amino acid residues. In some embodiments, L1 and / or L2 consist of about 3 to about 15, for example, 8, 9, or 10 consecutive amino acid residues.

[0204] Regarding the amino acid composition of L1 and L2, peptides were selected that possess properties that confer flexibility to the multispecific binding proteins of this invention, do not interfere with the binding domains, and resist protease cleavage. For example, glycine and serine residues typically provide protease resistance. Examples of linkers suitable for connecting domains in multispecific binding proteins include, but are not limited to, (GS). n The sequences are (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, and (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, L1 and / or L2 are independently selected from the peptide sequences listed in Table 4. In some embodiments, L1 and / or L2 are independently selected from SEQ ID NO: 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, or 302. In some embodiments, L1 and / or L2 are independently selected from SEQ ID NO: 298, 299, and 302. In some embodiments, L1 and / or L2 comprise the amino acid sequences of SEQ ID NO: 298, 299, and 302. In some embodiments, L1 and / or L2 consist of the amino acid sequences of SEQ ID NO:298, 299, and 302. In some embodiments, L1 and / or L2 each comprise the amino acid sequences of SEQ ID NO:298, 299, and 302. In some embodiments, L1 and / or L2 each consist of the amino acid sequences of SEQ ID NO:298, 299, and 302.

[0205] Table 4. Exemplary peptide linker sequences

[0206]

[0207] Linkers, such as the peptide linkers disclosed herein, can also be used to connect the VH and VL of scFv, as described in section C above, which is titled “Construct Forms”.

[0208] In some embodiments, the multispecific binding protein further comprises a tag peptide, such as a Flag tag, a 6×His tag, or a 10×His tag (HHHHHHHHHH, SEQ ID NO: 711). Such tag peptides can be used to purify the multispecific binding protein. In some embodiments, the tag peptide (e.g., a 10×His tag) is located at the C-terminus of the multispecific binding protein. In some embodiments, the tag peptide (e.g., a 10×His tag) is located at the N-terminus of the multispecific binding protein.

[0209] E. Combining affinity

[0210] In some embodiments, the multispecific binding protein uses K+ in the range of about 0.1 nM to about 100 μM. D Combined with CD19 (e.g., human CD19), CD3 (e.g., human CD3 and / or macaque CD3) and / or serum albumin (e.g., HSA). K D It can be measured by methods known in the art, such as by SPR or by flow cytometry as described in Examples 1 or 6 below.

[0211] In some embodiments, multispecific binding proteins bind at a K value less than or equal to (i.e., binding stronger than or equal to) below. D Binding to CD19, CD3, and / or serum albumin: 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. For example, in some embodiments, the multispecific binding protein is in the following range of K D Combined with CD19, CD3, and / or serum albumin: approximately 10 pM to approximately 1 nM, approximately 10 pM to approximately 0.9 nM, approximately 10 pM to approximately 0.8 nM, approximately 10 pM to approximately 0.7 nM, approximately 10 pM to approximately 0.6 nM, approximately 10 pM to approximately 0.5 nM, approximately 10 pM to approximately 0.4 nM, approximately 10 pM to approximately 0.3 nM, approximately 10 pM to approximately 0.2 nM, approximately 10 pM to approximately 0.1 nM, approximately 10 pM to approximately 50 pM, 0.1 nM to approximately 10 nM, approximately 0.1 nM to approximately 9 nM, approximately 0.1 nM to approximately 8 nM, approximately 0.1 nM to approximately 8 nM, approximately 0.1 nM to approximately 10 nM, approximately 0.1 nM to approximately 9 nM, approximately 0.1 nM to approximately 8 nM, approximately 0.1 nM to approximately 10 nM, approximately 0.1 nM to approximately 0.2 nM, approximately 10 pM to approximately 0.1 nM, approximately 0.1 nM to approximately 0.2 ... Approximately 7 nM, approximately 0.1 nM to approximately 6 nM, approximately 0.1 nM to approximately 5 nM, approximately 0.1 nM to approximately 4 nM, approximately 0.1 nM to approximately 3 nM, approximately 0.1 nM to approximately 2 nM, approximately 0.1 nM to approximately 1 nM, approximately 0.1 nM to approximately 0.5 nM, approximately 0.5 nM to approximately 10 nM, approximately 1 nM to approximately 10 nM, approximately 2 nM to approximately 10 nM, approximately 3 nM to approximately 10 nM, approximately 4 nM to approximately 10 nM, approximately 5 nM to approximately 10 nM, approximately 6 nM to approximately 10 nM, approximately 7 nM to approximately 10 nM, approximately 8 nM to approximately 10 nM, or approximately 9 nM to approximately 10 nM.

[0212] In some implementations, multispecific binding proteins bind at a K value higher than or equal to (i.e., binding weaker than or equal to) below. DBinding to CD19, CD3, and / or serum albumin: 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. For example, in some embodiments, the multispecific binding protein is in the following range of K... D Combined with CD19, CD3, and / or serum albumin: approximately 10 nM to approximately 1000 nM, approximately 10 nM to approximately 900 nM, approximately 10 nM to approximately 800 nM, approximately 10 nM to approximately 700 nM, approximately 10 nM to approximately 600 nM, approximately 10 nM to approximately 500 nM, approximately 10 nM to approximately 400 nM, approximately 10 nM to approximately 300 nM, approximately 10 nM to approximately 200 nM, approximately 10 nM to approximately 100 nM, approximately 10 nM - Approximately 50 nM, approximately 50 nM to approximately 1000 nM, approximately 100 nM to approximately 1000 nM, approximately 200 nM to approximately 1000 nM, approximately 300 nM to approximately 1000 nM, approximately 400 nM to approximately 1000 nM, approximately 500 nM to approximately 1000 nM, approximately 600 nM to approximately 1000 nM, approximately 700 nM to approximately 1000 nM, approximately 800 nM to approximately 1000 nM, or approximately 900 nM to approximately 1000 nM.

[0213] In some implementations, K combined with CD19 or CD3 D Measured in the absence of serum albumin (e.g., HSA). In some embodiments, K, which binds to CD19 or CD3, is used. D Measured in the absence of serum albumin (e.g., HSA). In some embodiments, K, which binds to CD19 or CD3, is used. D Measured in the presence of serum albumin (e.g., HSA), for example, in the presence of serum albumin (e.g., HSA) at concentrations of about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL.

[0214] In some embodiments, the multispecific binding protein of this disclosure is similar to K-type antigens that bind to individual antigen binding sites or monoclonal antibodies having the same antigen binding site. D The value binds to CD19, CD3, and / or serum albumin. In some embodiments, the multispecific binding protein binds to the K+ of CD19, CD3, and / or serum albumin. DThe value is no more than 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times higher than the KD value of individual antigen-binding sites or monoclonal antibodies binding to CD19, CD3, and / or serum albumin.

[0215] In some embodiments, the multispecific binding protein of this disclosure binds to the K+ of CD19 and / or CD3 in the presence of serum albumin. D Values ​​of K binding to CD19 and / or CD3 in the absence or substantial absence of serum albumin D Similar values. In some embodiments, the multispecific binding protein binds to the K+ of CD19 and / or CD3 in the presence of serum albumin. D The value compared to the K binding CD19 and / or CD3 in the absence or near absence of serum albumin. D The value increases by no more than 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or 50 times.

[0216] F. Therapeutic activity

[0217] The multispecific binding protein disclosed herein is designed to bind to both B cells and T cells simultaneously. T cell recruitment promotes the formation of cytolytic synapses and the lysis of B cells involved in perforin and granzyme delivery. The bound T cells are capable of continuous target cell lysis and are unaffected by immune escape mechanisms that interfere with peptide antigen processing and presentation or clonal T cell differentiation; see, for example, WO2007042261A2. Therefore, the binding of the multispecific binding protein to target B cells disrupts target cell activity and / or impairs the progression of B cell-related diseases.

[0218] The cytotoxicity mediated by the multispecific binding protein of this invention can be measured in vitro in various ways. Effector cells can be, for example, stimulated enriched (human) CD8-positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). If the target cells are of macaque origin or expressed or transfected with a macaque target cell surface antigen bound to the first domain, the effector cells should also be of macaque origin, such as a macaque T cell line like 4119LnPx. Target cells should express a protein targeted by the first antigen-binding site, such as CD19, HER2, BCMA, CD33, or EGFR. In some embodiments, the target cells should express CD19, such as human or macaque CD19.

[0219] In some embodiments, the target cells can be cell lines stably or transiently transfected with CD19 (such as CHO). Alternatively, in some embodiments, the target cells can be cell lines naturally expressing CD19, such as B lymphocytes. The effector cell to target cell (E:T) ratio is typically about 10:1, but can vary. Killing of target cells can... 51 Cr release assay (incubation time approximately 18 hours) or FACS-based cytotoxicity assay (incubation time approximately 48 hours) can be performed. Other methods for measuring cell death are well known to those skilled in the art, such as MTT or MTS assays, ATP-based assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, colony formation assays, and ECIS techniques.

[0220] In some embodiments, the cytotoxic activity mediated by the multispecific binding proteins disclosed herein is measured in the cell-based cytotoxicity assay described above. This is achieved by EC... 50 The value indicates that it corresponds to half of the maximum effective concentration (the concentration of the multispecific binding protein that induces a cytotoxic response between the baseline and the maximum value). In some embodiments, the EC50 of the multispecific binding protein... 50 Values ​​are ≤5000pM, for example, ≤4000pM, ≤3000pM, ≤2000pM, ≤1000pM, ≤500pM, ≤400pM, ≤300pM, ≤200pM, ≤100pM, ≤50pM, ≤20pM, ≤10pM, ≤5pM, ≤4pM, ≤3pM, ≤2pM, or ≤1pM.

[0221] It should be understood that, compared to unstimulated PBMCs, when using stimulated / enriched CD8+... + When T cells act as effector cells, EC 50 Values ​​are typically low. To further understand, compared to low levels of target antigens, when target cells express high levels of target cell surface antigens, EC values ​​are lower. 50 Values ​​are typically low. For example, when a person's CD8 is stimulated / enriched... + When T cells are used as effector cells (and when cells transfected with target cell surface antigens, such as CHO cells or target cell surface antigen-positive human cell lines, are used as target cells), the EC of multispecific binding proteins... 50 Values ​​≤1000 pM, such as ≤500 pM, ≤250 pM, ≤100 pM, 50 pM, ≤10 pM, or ≤5 pM. When human PBMCs are used as effector cells, the EC of multispecifically binding proteins... 50Values ​​≤5000 pM, such as ≤4000 pM, ≤2000 pM, ≤1000 pM, ≤500 pM, ≤200 pM, ≤150 pM, ≤100 pM, ≤50 pM, ≤10 pM, or ≤5 pM. When macaque T cell lines such as LnPx4119 are used as effector cells, and when macaque target cell surface antigen transfected cell lines such as CHO cells are used as target cell lines, the EC of multispecific binding proteins... 50 Values ​​≤2000pM, for example, ≤1500pM, ≤1000pM, ≤500pM, ≤300pM, ≤250pM, ≤100pM, ≤50pM, ≤10pM or ≤5pM.

[0222] Therefore, in some implementations, human CD8 stimulation / enrichment is used. + T cells were used as effector cells to measure ECG. 50 Value. In some implementations ， Using human PBMCs as effector cells to measure EC 50 Value. In some implementations, rhesus monkey T cell lines such as LnPx4119 are used as effector cells and cells engineered to express rhesus monkey CD19 (e.g., CHO cells) are used as target cells to measure EC. 50 value.

[0223] In some embodiments, the multispecific binding protein of the present invention does not induce or mediate the lysis of cells that do not express CD19. The terms "does not induce lysis" or "does not mediate lysis," or their grammatical equivalents, mean that the multispecific binding protein, at concentrations up to 500 nM, does not induce or mediate more than 30% lysis of cells that do not express CD19, for example, no more than 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5%, wherein the lysis of CD19-expressing cell lines is set to 100%.

[0224] In some embodiments, the multispecific binding protein disclosed herein is more effective in killing CD19-expressing cells (e.g., cancer cells) than the respective anti-CD19 or anti-CD3 monoclonal antibodies at the same molar concentration. In some embodiments, the multispecific binding protein is more effective in killing CD19-expressing cells (e.g., cancer cells) than combinations of the respective anti-CD19 and anti-CD3 monoclonal antibodies at the same molar concentration.

[0225] The cytotoxic activity of multispecific binding proteins can be measured in the presence or absence of serum albumin (e.g., HSA). In some embodiments, the cytotoxic activity disclosed above is measured in the absence of serum albumin (e.g., HSA). In some embodiments, the cytotoxic activity disclosed above is measured in the substantially absence of serum albumin (e.g., HSA). In some embodiments, the cytotoxic activity disclosed above is measured in the presence of serum albumin (e.g., HSA), for example, in the presence of about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL of serum albumin (e.g., HSA).

[0226] In some embodiments, the multispecific binding protein of this disclosure exhibits an EC50 similar to that in the absence or substantial absence of serum albumin in the presence of serum albumin. 50 The multispecific binding protein kills CD19-expressing cells in the presence of serum albumin. In some implementations, the multispecific binding protein kills CD19-expressing ECs in the presence of serum albumin. 50 Compared to EC50 cells that kill CD19-expressing cells in the absence or near absence of serum albumin, the value is significantly lower. 50 The increase in values ​​should not exceed 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times. It should be understood that the presence of serum albumin (e.g., approximately 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL serum albumin) can also nonspecifically alter the EC50 of multispecific binding proteins. 50 Value. EC50 can be compared between control proteins lacking serum albumin-binding domains in the presence and absence of serum albumin. 50 The value is used to assess nonspecific effects. In some embodiments, the fold change is offset by the nonspecific effect of serum albumin on control proteins such as bispecific proteins that bind CD19 and CD3.

[0227] G. Component Size

[0228] In some embodiments, the molecular weight of the multispecific binding protein is from about 40 kD to about 100 kD. In some embodiments, the molecular weight of the multispecific binding protein is at least 60 kD, at least 65 kD, at least 70 kD, at least 75 kD, at least 80 kD, at least 85 kD, at least 90 kD, or at least 95 kD. It is understood that smaller size generally facilitates faster diffusion and tissue penetration, but for indications where a large number of target cells (e.g., cancer cells) are present in the bloodstream, size reduction may not be as important.

[0229] In some embodiments, the molecular weight of the multispecific binding protein is from about 40 kD to about 90 kD, from about 40 kD to about 80 kD, from about 40 kD to about 70 kD, from about 40 kD to about 60 kD, from about 40 kD to about 50 kD, from about 50 kD to about 100 kD, from about 50 kD to about 90 kD, from about 50 kD to about 80 kD, from about 50 kD to about 70 kD, from about 50 kD to about 60 kD, from about 60 kD to about 100 kD, from about 6 The molecular weight ranges from about 0 kD to about 90 kD, from about 60 kD to about 80 kD, from about 60 kD to about 70 kD, from about 65 kD to about 100 kD, from about 65 kD to about 90 kD, from about 65 kD to about 80 kD, from about 65 kD to about 70 kD, from about 70 kD to about 100 kD, from about 70 kD to about 90 kD, from about 70 kD to about 80 kD, from about 80 kD to about 100 kD, from about 80 kD to about 90 kD, or from about 90 kD to about 100 kD. In some embodiments, the molecular weight of the multispecific binding protein is less than 40 kD. In some embodiments, the molecular weight of the multispecific binding protein is about 50 kD to about 90 kD, about 50 kD to about 80 kD, about 50 kD to about 70 kD, about 50 kD to about 60 kD, about 60 kD to about 90 kD, about 60 kD to about 80 kD, about 60 kD to about 70 kD, about 65 kD to about 90 kD, about 65 kD to about 80 kD, about 65 kD to about 70 kD, about 70 kD to about 90 kD, or about 70 kD to about 80 kD.

[0230] H. Serum half-life

[0231] Fusion proteins have been developed to increase the in vivo half-life of small proteins, particularly antibody fragments. For example, fusions with the Fc region of heterodimerized antibodies (such as Fc with one or more mutations that extend the in vivo half-life) are described in U.S. Patent Application Publications US20140302037A1, US20140308285A1, and PCT Publications WO2014144722A2, WO2014151910A1, and WO2015048272A1. Another strategy is fusion with human serum albumin (HSA) or an HSA-binding peptide (see, for example, PCT Publications WO2013128027A1 and WO2014140358A1). Neonatal Fc receptors (FcRn) appear to be associated with extending the lifetime of circulating albumin (see Chaudhury et al. (2003) J. Exp. Med., 3:315-22). Albumin and IgG bind non-cooperatively to different sites of FcRn to form a trimolecule (see ibid.). The binding of human FcRn to HSA and human IgG is pH-dependent, stronger at acidic pH and weaker at neutral or physiological pH (see ibid.). This observation suggests that, similar to proteins and protein complexes containing IgG (especially Fc), proteins and protein complexes containing albumin are protected from degradation through pH-sensitive interactions with FcRn (see ibid.). The ability of individual HSA domains to bind immobilized soluble human FcRn was measured using surface plasmon resonance (SPR), and the results showed that FcRn and albumin interact in a pH-dependent manner at sites different from the IgG binding site via albumin's D-III domain (see Chaudhury et al. (2006) Biochemistry 45:4983-90 and PCT Publication No. WO2008068280A1).

[0232] This disclosure provides multispecific binding proteins with extended half-lives. In some embodiments, the multispecific binding protein has a serum half-life of at least 24, 36, 48, 60, 72, 84, or 96 hours. In some embodiments, the multispecific binding protein has a serum half-life of at least about 50 hours. In some embodiments, the multispecific binding protein has a serum half-life of at least about 100 hours. Methods for measuring serum half-life are known in the art, and exemplary methods are described in Example 5. In some embodiments, the serum half-life is measured in non-human primates. In some embodiments, the serum half-life is measured in humans.

[0233] In some embodiments, 50 hours after intravenous administration to the subject, the serum concentration of the multispecific binding protein is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the serum concentration of the multispecific binding protein 1 hour after administration to the subject.

[0234] In some embodiments, the multispecific binding protein has a serum half-life at least 20% longer than that of the control multispecific binding protein, wherein the control multispecific binding protein includes a first domain identical to the first antigen-binding site of the multispecific binding protein, a second domain identical to the second antigen-binding site of the multispecific binding protein, but excludes a third domain identical or substantially identical to the third antigen-binding site of the multispecific binding protein. In some embodiments, the control multispecific binding protein is identical to the multispecific binding protein except for the absence of a third antigen-binding site. In some embodiments, the serum half-life of the multispecific binding protein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% longer than that of the control multispecific binding protein. In some embodiments, the serum half-life of the multispecific binding protein is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times longer than that of the control multispecific binding protein.

[0235] III. Preparation Method

[0236] The aforementioned antibodies and multispecific binding proteins can be prepared using recombinant DNA techniques well known to those skilled in the art. For example, one or more isolated polynucleotides encoding the antibody or multispecific binding protein can be linked to other suitable nucleotide sequences, including, for example, constant region coding sequences and expression control sequences, to produce conventional gene expression constructs (i.e., expression vectors) encoding the desired antibody or multispecific binding protein. The generation of the defined gene constructs is within the scope of conventional techniques in this art.

[0237] Nucleic acids encoding desired antibodies or multispecific binding proteins can be introduced (ligated) into expression vectors, which can then be introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells include *E. coli* cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not produce IgG proteins. Transformed host cells can grow under conditions that allow the host cells to express genes encoding antibodies or multispecific binding proteins.

[0238] Specific expression and purification conditions will vary depending on the expression system used. For example, if a gene is to be expressed in *E. coli*, it is first cloned into an expression vector by positioning the engineered gene downstream of a suitable bacterial promoter (e.g., Trp or Tac) and a prokaryotic signaling sequence. The expressed protein can be secreted. The expressed protein can accumulate in refractive bodies or inclusion bodies, which can be harvested after disrupting the cells by French pressing or sonication. The refractive bodies are then dissolved, and the protein can be refolded and / or cleaved using methods known in the art.

[0239] If the engineered gene is to be expressed in a eukaryotic host cell, such as a CHO cell, it is first inserted into an expression vector containing a suitable eukaryotic promoter, secretion signal, poly A sequence, and stop codon. Optionally, the vector or gene construct may contain enhancers and introns. In embodiments involving fusion proteins containing antibodies or portions thereof, the expression vector optionally contains a sequence encoding all or part of a constant region, enabling the expression of the entire or part of the heavy or light chain. The gene construct can be introduced into eukaryotic host cells using conventional techniques.

[0240] The antibodies or multispecific binding proteins disclosed herein may comprise a single polypeptide chain. In this case, host cells can be transfected with a single vector expressing the polypeptide (e.g., containing an expression control sequence operatively linked to the nucleotide sequence encoding the polypeptide). Alternatively, the antibodies or multispecific binding proteins disclosed herein may comprise two or more polypeptides. In this case, host cells can be co-transfected with more than one expression vector, such as one expression vector expressing each polypeptide. Host cells may also be transfected with a single expression vector expressing two or more polypeptides. For example, the coding sequences of two or more polypeptides may be operatively linked to different expression control sequences (e.g., promoters, enhancers, and / or internal ribosome entry sites (IRES)). The coding sequences of two or more polypeptides may also be separated by ribosome skipping sequences or self-cleaving sequences (such as peptide 2A).

[0241] In some embodiments, the protein construct includes an N-terminal signal sequence in order to express an antibody or multispecific binding protein. Exemplary N-terminal signal sequences include signal sequences from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin.

[0242] Following transfection, individual clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix. Clones can be cultured under conditions suitable for bioreactor scale-up and maintenance of antibody or multispecific binding protein expression.

[0243] Antibodies or multispecific binding proteins can be isolated and purified using methods known in the art, including centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw cycles, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0244] IV. Pharmaceutical Compositions

[0245] This disclosure is further characterized by a pharmaceutical composition comprising a therapeutically effective amount of the antibody or multispecific binding protein described herein. This composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for use in appropriate formulations. Suitable formulations for use in this disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of drug delivery methods, see, for example, Langer (Science 249:1527-1533, 1990).

[0246] In some embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); swelling agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); and low molecular weight polymers. Substances include: polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspensions; surfactants or wetting agents (e.g., pluronics, PEG, dehydrated sorbitol esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tetrabutylphenol); stability enhancers (e.g., sucrose or sorbitol); tension enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol or sorbitol); delivery carriers; diluents; excipients and / or adjuvants (see Remington's...). Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0247] In some embodiments, the pharmaceutical composition may contain nanoparticles, such as polymer nanoparticles, liposomes or microparticles (see Anselmo et al. (2016) Bioeng. Transl. Med. 1: 10-29).

[0248] In some embodiments, the pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery methods, such as liposome carriers, bio-erosive microparticles or porous beads, and long-acting injectables, are also known to those skilled in the art. Sustained-release formulations may include a semi-permeable polymer matrix, such as a film or microcapsule, in the form of porous polymer microparticles or molded articles. Sustained-release matrices may include polyesters, hydrogels, polylactide, copolymers of L-glutamic acid and γ-L-glutamic acid ethyl ester, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0249] Pharmaceutical compositions containing the antibodies or multispecific binding proteins disclosed herein may be present in dose units and may be prepared by any suitable method. The pharmaceutical composition shall be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. In some embodiments, the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered via IV infusion. In some embodiments, the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered via intratumoral injection. Useful formulations may be prepared by methods known in the pharmaceutical industry. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Components suitable for parenteral administration include sterile diluents such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetate, citrate or phosphate; and agents for adjusting tension such as sodium chloride or dextrose.

[0250] For intravenous administration, suitable carriers include normal saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier should be stable under manufacturing and storage conditions and should be protected against microbial contamination. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0251] Intravenous formulations may be contained in syringes, pens, or bags. In some embodiments, the bag may be connected to a channel containing tubing and / or a needle. In some embodiments, the formulation may be a lyophilized or liquid formulation. In some embodiments, the formulation may be lyophilized (freeze-dried) and contained in about 12-60 vials. In some embodiments, the formulation may be lyophilized and 45 mg of the lyophilized formulation may be contained in one vial. In some embodiments, about 40 mg to about 100 mg of the lyophilized formulation may be contained in one vial. In some embodiments, lyophilized formulations from 12, 27, or 45 vials are combined to obtain a therapeutic dose of the protein in the intravenous drug formulation. In some embodiments, the formulation may be a liquid formulation and stored in the form of about 250 mg / vial to about 1,000 mg / vial. In some embodiments, the formulation may be a liquid formulation and stored in the form of about 600 mg / vial. In some embodiments, the formulation may be a liquid formulation and stored in the form of about 250 mg / vial.

[0252] These compositions can be sterilized using conventional sterilization techniques or by aseptic filtration. The resulting aqueous solutions can be used as is or lyophilized, with the lyophilized formulation combined with a sterile aqueous carrier prior to administration. The pH of the formulation is typically between 3 and 11, more preferably between 5 and 9 or 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting solid-form compositions can be packaged in multiple single-dose units, each unit containing a fixed amount of one or more of the aforementioned pharmaceutical agents. The solid-form compositions can also be packaged in containers to provide flexible dosage.

[0253] In some embodiments, this disclosure provides formulations having an extended shelf life comprising the protein of this disclosure in combination with: mannitol, citrate monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0254] In some embodiments, an aqueous formulation comprising the protein of this disclosure is prepared in a pH buffer solution. The buffers of this invention may have a pH in the range of about 4 to about 8, for example, about 4.5 to about 6.0, or about 4.8 to about 5.5, or may have a pH in the range of about 5.0 to about 5.2. The intermediate pH ranges described above are also intended to be part of this disclosure. For example, it is intended to include value ranges using any combination of the above values ​​as upper and / or lower limits. Examples of buffers that control the pH within this range include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconates, histidines, citrates, and other organic acid buffers.

[0255] In some embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain a pH in the range of about 4 to about 8. In some embodiments, the pH range may be about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or about 5.0 to about 5.2. In some embodiments, the buffer system includes citrate monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In some embodiments, the buffer system includes about 1.3 mg / mL of citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL of sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL of disodium phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL of sodium dihydrogen phosphate dihydrate (e.g., 0.86 mg / mL), and about 6.2 mg / mL of sodium chloride (e.g., 6.165 mg / mL). In some embodiments, the buffer system comprises 1-1.5 mg / mL citric acid, 0.25-0.5 mg / mL sodium citrate, 1.25-1.75 mg / mL disodium phosphate dihydrate, 0.7-1.1 mg / mL sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg / mL sodium chloride. In some embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0256] Polyols, acting as tonicants, can stabilize antibodies or multispecific binding proteins and may be included in the formulation. Polyols can be added to the formulation in amounts that vary depending on the desired isotonicity. In some embodiments, aqueous formulations may be isotonic. The amount of polyol added may also vary depending on the molecular weight of the polyol. For example, a lower amount of monosaccharide (e.g., mannitol) may be added compared to a disaccharide (e.g., trehalose). In some embodiments, the polyol that can be used as a tonicant in the formulation is mannitol. In some embodiments, the concentration of mannitol may be from about 5 to about 20 mg / mL. In some embodiments, the concentration of mannitol may be from about 7.5 to 15 mg / mL. In some embodiments, the concentration of mannitol may be from about 10-14 mg / mL. In some embodiments, the concentration of mannitol may be from about 12 mg / mL. In some embodiments, the polyol sorbitol may be included in the formulation.

[0257] Detergents or surfactants may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbates 20, 80, etc.) or poloxamer (e.g., poloxamer 188). The amount of detergent added reduces the aggregation of the formulated antibody and / or minimizes particle formation and / or reduces adsorption in the formulation. In some embodiments, the formulation may include a surfactant, which is a polysorbate. In some embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitol monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th thedi., 1996). In some embodiments, the formulation may contain about 0.1 mg / mL to about 10 mg / mL of polysorbate 80, or about 0.5 mg / mL to about 5 mg / mL. In some embodiments, about 0.1% of polysorbate 80 may be added to the formulation.

[0258] In some embodiments, the protein product of this disclosure is formulated as a liquid preparation. The liquid preparation may be present at a concentration of 10 mg / mL in a USP / Ph Eur Type I 50R vial, which is sealed with a rubber stopper and an aluminum crimp seal. The stopper may be made of an elastomer conforming to USP and Ph Eur standards. In some embodiments, the liquid preparation may be diluted with a 0.9% saline solution.

[0259] In some embodiments, the liquid formulation of this disclosure can be prepared as a solution with a concentration of 10 mg / mL, combined with a stable level of sugar. In some embodiments, the liquid formulation can be prepared in an aqueous carrier. In some embodiments, a stabilizer can be added in an amount not exceeding the viscosity that would result in undesirable or unsuitable intravenous administration. In some embodiments, the sugar can be a disaccharide, such as sucrose. In some embodiments, the liquid formulation may also include one or more of a buffer, a surfactant, and a preservative.

[0260] In some embodiments, the pH of the liquid formulation can be set by adding a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the base may be sodium hydroxide.

[0261] The aqueous carriers of interest in this article are pharmaceutically acceptable (safe and non-toxic for human administration) and can be used to prepare liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextran solution.

[0262] Preservatives may optionally be added to the formulations described herein to reduce bacterial activity. For example, the addition of preservatives may facilitate the production of multipurpose (multi-dose) formulations.

[0263] Antibodies or multispecific binding proteins can be lyophilized to produce a lyophilized formulation comprising the protein and a lyophilization protectant. The lyophilization protectant can be a sugar, such as a disaccharide. In some embodiments, the lyophilization protectant can be sucrose or maltose. The lyophilized formulation may also include one or more of buffers, surfactants, fillers, and / or preservatives.

[0264] The amount of sucrose or maltose used to stabilize the lyophilized pharmaceutical product may be at least a protein-to-sucrose or maltose weight ratio of 1:2. In some embodiments, the protein-to-sucrose or maltose weight ratio may be 1:2 to 1:5. In some embodiments, the pH of the formulation prior to lyophilization may be set by adding a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the pharmaceutically acceptable base may be sodium hydroxide. Prior to lyophilization, the pH of the solution containing the protein of the present invention may be adjusted to between 6 and 8. In some embodiments, the pH range of the lyophilized pharmaceutical product may be 7 to 8.

[0265] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition and administration method, without being toxic to the patient.

[0266] The specific dosage can be a uniform dose for each patient, such as 50-5,000 mg of protein. Optionally, the dosage can be adjusted based on the patient's approximate weight or body surface area. Other factors for determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Those skilled in the art will typically refine the calculations required to determine the appropriate dosage for treatment, particularly based on the dosage information and assays disclosed herein. The dosage can also be determined using known assays that determine the dosage for incorporating appropriate dose-response data. The dosage for an individual patient can be adjusted as the disease progresses. Blood levels of the targetable construct or complex can be measured in the patient to see if dosage adjustments are needed to achieve or maintain effective concentrations. Pharmacogenomics can be used to determine which targetable constructs and / or complexes and their dosages are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

[0267] Typically, body weight-based dosages range from about 0.01 μg to about 100 mg / kg body weight, such as about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight, and about 0.01 μg to about 0.1 μg / kg. Body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0.1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg to about 100 mg / kg body weight. μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to About 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight.

[0268] Dosage can be administered daily, weekly, monthly, or annually, or even once every 2 to 20 years. Those skilled in the art can readily estimate the repetition rate of administration based on measured residence time and the concentration of the targetable construct or complex in body fluids or tissues. Administration of this invention can be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, via catheter infusion, or via direct intralesional injection. It can be administered once or more daily, once or more weekly, once or more monthly, or once or more annually.

[0269] V. Therapeutic Applications

[0270] Antibodies or multispecific binding proteins are expected to be used alone or in combination with other therapeutic agents.

[0271] A. Indications

[0272] This disclosure provides methods for treating or improving proliferative diseases, neoplastic diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host disease, or host-versus-graft disease in subjects in need, the methods comprising administering the multispecific binding protein or antibody disclosed herein. In some embodiments, the disease is associated with the expression or overexpression of a target protein expressed on target cells.

[0273] In some implementations, the cancer to be treated is non-Hodgkin lymphoma, such as B-cell lymphoma. In some implementations, the non-Hodgkin lymphoma is B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, lymph node marginal zone B-cell lymphoma, spleen marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, or primary central nervous system lymphoma. In some other implementations, the cancer to be treated is multiple myeloma. In some other implementations, the cancer to be treated is acute lymphoblastic leukemia (ALL). In some implementations, ALL is relapsed / refractory adult and pediatric ALL.

[0274] B. Combination Therapy

[0275] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or forms. As used herein, the term “combined” administration should be understood as the delivery of two (or more) different treatments to a subject during the course of the subject’s illness, such that the effects of the treatments on the patient overlap at some point in time. In some embodiments, the delivery of one treatment is still in progress when the second treatment begins, thus there is an overlap in administration. This is sometimes referred to herein as “simultaneous” or “simultaneous delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective due to combined administration. For example, the second treatment is more effective, for instance, less of the second treatment can be observed to have the same effect compared to the second treatment without the first treatment, or the second treatment alleviates symptoms to a greater extent; or a similar situation exists with the first treatment. In some embodiments, the delivery results in a reduction in symptoms or other parameters related to the illness greater than that observed with the first treatment in the absence of the other treatment. The effects of the two treatments can be partially additive, completely additive, or greater than additive. Delivery allows the effects of the first treatment to remain detectable when the second treatment is delivered.

[0276] In one aspect, this disclosure provides a method for treating a subject by co-administering a second therapeutic agent with one or more of the multispecific binding proteins and / or antibodies that bind CD19 disclosed herein.

[0277] Exemplary therapeutic agents that can be used as part of a combination therapy for cancer treatment include, for example, radiation, mitomycin, retinoic acid, bendamustine, gemcitabine, vincristine, etoposide, cladribine, dibromomannitol, methotrexate, doxorubicin, carboquinone, pentostatin, diammoniazolinone, fentostatin, cetirizine, letrozole, raltitrexed, daunorubicin, faldrozole, formusine, thymosin, sobuzosen, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesirimelone, aminoglutethimide, acridine, proglutamine, eletine, ketoserin, deoxyfluorouracil, etretinate, isotretinoin, streptozotocin, nimustine, vindesine. Flutamicil, flutamide, glycinol, carmoflurane, razorizine, sizofilan, carboplatin, dibromo-eugenol, fenfluridine, ifosfamide, prednimustine, streptomycin preparations, levamisole, teniposide, inprofen, enoxabin, ergot urea, oxymetholone, tamoxifen, progesterone, methanogen, cyclothioranolol, formestan, interferon-α, interferon-2α, interferon-β, interferon-γ, colony-stimulating factor-1, colony-stimulating factor-2, desniform, interleukin-2, luteinizing hormone-releasing factor, and the above drugs can exhibit variants that bind differentially to their homologous receptors and increase or decrease serum half-life.

[0278] Another class of drugs that can be used as part of combination therapy for cancer treatment are immune checkpoint inhibitors. Checkpoint inhibitors can be selected, for example, from PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, adenosine A2A receptor antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, KIR antagonists, LAG3 antagonists, TIM-3 antagonists, VISTA antagonists, or TIGIT antagonists.

[0279] In some embodiments, the checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. PD-1 is a receptor present on the surface of T cells that acts as an immune system checkpoint, inhibiting or otherwise modulating T cell activity at appropriate times to prevent an overactive immune response. However, cancer cells can exploit this checkpoint by expressing a ligand (e.g., PD-L1) that interacts with PD-1 on the surface of T cells to shut down or modulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapeutic agents. Exemplary treatment methods employing PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994 and European Patent No. 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described in, for example, U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (… Bristol-Myers Squibb Co., pembrolizumab ( Merck Sharp & Dohme Corp., PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (… Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0280] In some embodiments, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of CTLA-4 on T cells with ligands (e.g., CD80 (also known as B7-1) and CD86) on the surface of antigen-presenting cells (rather than cancer cells) leads to T cell suppression. Exemplary CTLA-4-based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815 and 8,883,984, International (PCT) Publications Nos. WO98 / 42752, WO00 / 37504 and WO01 / 14424, and European Patent No. EP 1212422B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0281] In some embodiments, the methods or compositions described herein are administered in combination with (i) a PD-1 or PD-L1 inhibitor, such as the PD-1 or PD-L1 inhibitors disclosed herein, and (ii) a CTLA-4 inhibitor, such as the CTLA-4 inhibitors disclosed herein.

[0282] In some embodiments, the methods or compositions described herein are administered in combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-tryptophan (called indoximod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205.

[0283] Other agents that can be used as part of combination therapy for cancer treatment are monoclonal antibody agents that target non-checkpoint targets (such as Herceptin) and non-cytotoxic agents (such as tyrosine kinase inhibitors).

[0284] Other classes of anticancer agents include, for example: (i) inhibitors selected from the following: ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, DNA-PK and mTOR inhibitors, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chlorodeoxyadenosine, HDAC inhibitors, Hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors. (i) mTOR inhibitors, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositol 3-kinase inhibitors, PARP1 and DHODH inhibitors, proteasome inhibitors, topoisomerase-II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors and WEE1 inhibitors; (ii) agonists of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25 or ICOS; (iii) cytokines selected from the following: IL-12, IL-15, GM-CSF and G-CSF.

[0285] It should be understood that the antibodies or multispecific binding proteins disclosed herein designed to activate T lymphocytes may cause side effects such as neurotoxicity. Therefore, in some embodiments, a second therapeutic agent that can be used in combination with an antibody or multispecific binding protein contains an agent that mitigates the side effects of the antibody or multispecific binding protein (e.g., reduces neurotoxicity). In some embodiments, the second therapeutic agent inhibits T cell transport, for example, by reducing or inhibiting immune cells from crossing the blood-brain barrier. Non-limiting examples of such therapeutic agents include antagonists of adhesion molecules on immune cells (e.g., α4 integrin) (e.g., antagonistic antibodies), such as nastatinumab. In some embodiments, the second therapeutic agent increases the internalization of sphingosine monophosphate (SIP) receptors (e.g., S1PR1 or S1PR5), such as fingolimod or ozamod. In some embodiments, the second therapeutic agent is a nitric oxide synthase (NOS) inhibitor, such as ronopterin, cindunistat, A-84643, ONO-1714, L-NOARG, NCX-456, VAS-2381, GW-273629, NXN-462, CKD-712, KD-7040, or guanidinoethyl disulfide. In some embodiments, the second therapeutic agent is an antagonist of CSF1 or CSF1R, such as pexidartinib, emactuzumab, cabilalizumab, LY-3022855, JNJ-40346527, or MCS110. Other non-limiting examples of second therapeutic agents include pentosan polysulfate, minocycline, anti-ICAM-1 antibody, anti-P-selectin antibody, anti-CD11a antibody, anti-CD162 antibody, and anti-IL-6R antibody (e.g., tocilizumab).

[0286] The amount of antibody or multispecific binding protein and the relative timing of administration can be selected to achieve the desired combined therapeutic effect. For example, when administering combination therapy to a patient requiring such administration, the therapeutic agents in the combination, or one or more pharmaceutical compositions containing therapeutic agents, can be administered in any order, such as sequentially, together, simultaneously, etc. Furthermore, for example, antibodies or multispecific binding proteins can be administered while the other therapeutic agent exerts its preventive or therapeutic effect, or vice versa.

[0287] Throughout the specification, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is also contemplated that compositions of the invention consist substantially of or comprise of the listed components, and processes and methods according to the invention consist substantially of or comprise of the listed processing steps.

[0288] In this application, when an element or component is referred to as being included in and / or selected from the list of listed elements or components, it should be understood that the element or component can be any one of the listed elements or components, or the element or component can be selected from a group consisting of two or more listed elements or components.

[0289] Furthermore, it should be understood that, without departing from the spirit and scope of the invention, elements and / or features of the compositions or methods described herein, whether express or implied, can be combined in various ways. For example, when a particular compound is referred to, that compound can be used in various embodiments of the compositions of the invention and / or the methods of the invention, unless otherwise understood from the context. In other words, in this application, embodiments have been described and depicted in a manner that enables the application to be written and drawn clearly and concisely; however, it is intended and will be understood that embodiments can be combined or separated in various ways without departing from the teachings and invention. For example, it should be understood that all features described and depicted herein are applicable to all aspects of the invention described and depicted herein.

[0290] It should be understood that, unless otherwise understood from the context and usage, the expression "at least one" includes each of the enumerated objects following the expression, as well as various combinations of two or more enumerated objects. Unless otherwise understood from the context, the expression "and / or" relating to three or more enumerated objects should be understood to have the same meaning.

[0291] The use of the terms “including,” “contains,” “has,” “exists,” “includes,” “includes,” or “contains,” including their grammatical equivalents, should be understood as generally open-ended and non-restrictive, such as not excluding additional unlisted elements or steps, unless otherwise specifically stated or understood from the context.

[0292] Where the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless otherwise explicitly stated. As used herein, unless otherwise stated or inferred, the term "about" refers to a variation of ±10% from the nominal value.

[0293] It should be understood that the order of steps or the sequence of certain actions is irrelevant, as long as the invention remains operable. Furthermore, two or more steps or operations can be performed simultaneously.

[0294] Any and all examples or exemplary language used herein, such as “e.g.” or “including,” are intended only to better illustrate the invention and do not constitute a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0295] The foregoing description illustrates various aspects and embodiments of the invention. This patent application particularly considers all combinations and arrangements of these aspects and embodiments. Example

[0296] The invention will now be described in general terms, and will be more readily understood by referring to the following embodiments. These embodiments are merely illustrative of certain aspects and implementations of the invention and are not intended to limit the invention.

[0297] Example 1. Characterization of novel anti-serum albumin antibody

[0298] This embodiment describes novel anti-serum albumin antibodies CNG-HSA-101 to CNG-HSA-120. The amino acid sequences of these antibodies are provided in Table 1 above.

[0299] CNG-HSA-101 to CNG-HSA-120 were optimized from the parental antibody CNG-HSA-1 (a single-domain antibody) by introducing diversity into the heavy chain variable region, generating random mutations via error-prone PCR, and shuffling the VH fragment. Antibody clones with increased binding affinity to biotinylated human serum albumin relative to the parent antibody were selected. Furthermore, thermal selection pressure was employed in the VH shuffling optimization cycle. Thermal selection pressure was applied by incubating the library at different temperatures and then selecting antibodies that retained antigen binding after heat incubation. The selected antibodies were then produced from yeast cells and purified using a Protein A column.

[0300] As previously described, the binding affinity of antibodies to isolated serum albumin was measured using the ForteBio Octet HTX system via surface plasmon resonance (see, for example, Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278 (2013)). In short, ForteBio affinity measurements were performed by loading heavy chain antibodies (HCAbs) online onto the AHC sensor. The sensor was equilibrated offline in assay buffer for 30 minutes, then monitored online for 60 seconds to establish a baseline. The HCAb-loaded sensor was exposed to 100 nM human serum albumin for 3 minutes, then transferred to assay buffer for 3 minutes for off-rate measurement. All kinetics were analyzed using a 1:1 binding model.

[0301] The melting temperature (Tm) of the VHH fragment was measured using dynamic scanning fluorescence (DSF). Briefly, 10 μL of 20X Sypro Orange dye was added to 20 μL of 0.2–1 mg / mL HCAb. A BioRad CFX96 RT PCR machine was used to increase the sample plate temperature from 40°C to 95°C in 0.5°C increments, with each temperature equilibrated for 2 minutes. The negative value of the first derivative of the raw data was used to extract Tm.

[0302] Table 5. Binding of anti-serum albumin antibodies to serum albumin and protein A

[0303]

[0304]

[0305] 1 NB indicates that no binding was detected under the measurement conditions.

[0306] 2 ND indicates not measured.

[0307] As shown in Table 5, CNG-HSA-101 to CNG-HSA-120 exhibited higher binding affinity to human serum albumin, cynomolgus monkey serum albumin, mouse serum albumin, and / or protein A compared to CNG-HSA-1. In particular, all these antibodies showed lower Kb than CNG-HSA-1. D Values ​​were correlated with mouse serum albumin. CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, and CNG-HSA-120 had lower K values ​​than CNG-HSA-1. D Values ​​bound to human serum albumin and cynomolgus monkey serum albumin. CNG-HSA-101, CNG-HSA-103, CNG-HSA-106, CNG-HSA-107, CNG-HSA-108, CNG-HSA-109, CNG-HSA-111, CNG-HSA-113, CNG-HSA-114, CNG-HSA-115, CNG-HSA-116, CNG-HSA-118, and CNG-HSA-120 were compared with the K of the same antibody bound to human serum albumin. D K is less than 4 times higher DValues ​​were correlated with mouse serum albumin. CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, and CNG-HSA-117 had lower K values ​​than CNG-HSA-1. D Value-binding protein A. CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-105, CNG-HSA-106, CNG-HSA-108, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, CNG-HSA-117, and CNG-HSA-120 exhibit melting temperatures above or equal to 60 °C, of ​​which CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-106, and CNG-HSA-120 exhibit melting temperatures above or equal to 65 °C.

[0308] Example 2. Generation of multispecific binding proteins

[0309] This example describes the generation and purification of multispecific binding proteins.

[0310] Nucleic acid encoding a single-stranded, multispecific binding protein (see Table 6) was constructed and codon-optimized for expression in human cells, and cloned into a mammalian expression vector according to standard procedures. After sequence validation, sufficient quantities of the expression vector in plasmid form were prepared for transfection using the Plasmid Plus purification kit (Qiagen). Human embryonic kidney 293 (HEK 293) cells were passaged to appropriate densities for transient transfection. Cells were transiently transfected with the expression vector and cultured for six days.

[0311] The amino acid sequences of various multispecific binding proteins are summarized in Table 6. Constructs tAb0027 to tAb0032 each contain an anti-CD19 scFv with the amino acid sequence shown in SEQ ID NO:9, an anti-CD3 scFv with the amino acid sequence shown in SEQ ID NO:105, and an anti-HSA sdAb with the amino acid sequence shown in SEQ ID NO:121. Constructs tAb0033 to tAb0038 each contain an anti-CD19 scFv with the amino acid sequence shown in SEQ ID NO:18, an anti-CD3 scFv with the amino acid sequence shown in SEQ ID NO:105, and an anti-HSA sdAb with the amino acid sequence shown in SEQ ID NO:121.

[0312] Table 6. Exemplary Multispecific Binding Proteins

[0313]

[0314]

[0315]

[0316]

[0317]

[0318] Cultures were collected by centrifugation at 4000 rpm, and the supernatant was filtered through a 0.22 mm filter. The multispecific binding protein with a 10×His tag at the C-terminus was purified in two steps. The first step was nickel affinity chromatography, eluting with PBS containing 400 mM imidazole. The second step was size exclusion chromatography, eluting in PBS (phosphate-buffered saline) at pH 7.2. The concentration of the multispecific binding protein was determined by UV spectroscopy, and the protein sample was concentrated if necessary. Protein purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Specifically, HPLC was performed on an Agilent 1100 series instrument using a MabPac size exclusion column running at 0.2 mL / min in PBS. Fractions with elution times of approximately 225–240 min were collected for further characterization.

[0319] As described above, the resulting construct contains anti-CD19scFv having the amino acid sequence shown in SEQ ID NO:9 or 18. The binding affinity of the two CD19 binding domains to CD19 was measured by SPR using the monomeric CD19 extracellular domain fused to human IgG1 Fc and the dimer CD19 extracellular domain. Binding kinetic parameters were typically measured using a ForteBio instrument as previously described (see Estep et al. (2013) MAbs, 5(2):270-78). When measured with the monomeric CD19 protein, the K of the CD19 binding domain having the sequence shown in SEQ ID NO:9... D The KD value of the CD19 binding domain of the sequence with SEQ ID NO:18 is 7 nM, and the KD value is 11 nM. When measured with the dimer CD19 protein, the KD value of the CD19 binding domain of the sequence with SEQ ID NO:9 is 5 nM, and the KD value of the CD19 binding domain of the sequence with SEQ ID NO:18 is 7 nM. D The value is 15nM.

[0320] Example 3. Multispecific binding protein-induced targeting of CD19 + T cell cytotoxicity of target cells

[0321] This example describes the cytotoxic activity of a multispecific binding protein.

[0322] The T cell redirection activity of multispecific binding proteins and BiTE proteins was assessed using the KILR Raji cell model. Briefly, pan-T cells were isolated from primary human PBMCs from a single healthy donor using a commercial kit (e.g., the Easy Sep Human T Cell Enrichment Kit, StemCell Technologies) via negative selection. T cells were maintained in RPMI 1640 medium supplemented with 10% serum and 300 IU / mL IL-2 to expand the T cells. The harvested T cells were washed twice to remove any serum.

[0323] KILR Raji cells expressing CD19 were used as target cells. To condition the target cells, each multispecific binding protein or BiTE (see Table 6) was incubated with the target cells for 30 minutes at 37°C in RPMI 1640 medium supplemented with 5% heat-inactivated low-IgG fetal bovine serum and penicillin-streptomycin-glutamine. Proteins were added in 10 different serial dilutions, with each dose being repeated. Human serum albumin was added to the medium for specific samples at a final concentration of 15 mg / mL. CD19-negative KILR SKOV3 cells were also used as a negative control to evaluate the selectivity of the proteins.

[0324] After conditioning, target cells and pan-T cells were incubated together at 37°C for 6 hours at an effector cell to target cell ratio of 10:1 (E:T). Killing of KILR Raji cells resulted in the release of tagged housekeeping proteins from these cells into the culture medium, which was quantified by adding KILR assay reagent (DiscoverX). The luminescence signals from all wells were read on an Envision plate reader. Spontaneous release and total lysis controls were included on each plate to calculate the percentage of cell kill.

[0325] The percentage of lethality is calculated from the luminous signal value using the following formula:

[0326] Kill % = (Value from test protein sample - Average value from spontaneous release control) / (Average value from total cleavage control - Average value from spontaneous release control) × 100.

[0327] EC50 values ​​were calculated from the percentage of lethality by fitting dose-response curves using GraphPad Prism software.

[0328] Table 7 lists the EC50 values ​​of exemplary multispecific binding proteins and control anti-CD19 BiTE protein for T cell redirection killing in the absence and presence of human serum albumin. No significant killing effect was observed against CD19-negative KILR SKOV3 cells.

[0329] Table 7. Cytotoxic activity of multispecific binding proteins

[0330]

[0331] As shown in Table 7, regardless of the construct form, CD3-binding domain, HSA-binding domain, or the presence of HSA in the assay medium, the multispecific binding protein against CD19 scFv containing the amino acid sequence SEQ ID NO: 9 exhibited stronger cytotoxic activity than the multispecific binding protein against CD19 scFv containing the amino acid sequence SEQ ID NO: 18. Based on this data, it is expected that this construct containing anti-CD19 scFv with a higher binding affinity for CD19 will exhibit stronger therapeutic activity compared to other constructs containing lower binding affinity for anti-CD19 scFv.

[0332] Furthermore, all tested multispecific binding proteins showed lower EC in the absence of HSA compared to the presence of HSA. 50 Value (i.e., stronger ability to induce cytotoxicity). Not wanting to be limited by theory, it appears that the presence of HSA causes changes in the protein complex, changes that are specific to multispecific binding proteins containing the HSA-binding domain, rather than the nonspecific effects observed with bonnetumab. EC in the presence of HSA 50 Value and EC when HSA is not present 50 The ratio of values, also referred to as the “fold change” in this paper, was used to assess the impact of HSA on the potential therapeutic activity of multispecific binding proteins. As shown in Table 7, constructs with HSA-binding domains located at the N-terminus of both the CD19-binding domain and the CD3-binding domain (i.e., tAb0031, tAb0032, tAb0037, and tAb0038) showed lower fold changes than other constructs, regardless of which CD19-binding domain was used in the construct.

[0333] Furthermore, among constructs with the same CD19 binding domain, CD3 binding domain, and HSA binding domain, constructs of the form CD19:CD3:HSA (i.e., the CD19 binding domain is located at the N-terminus of the CD3 binding domain, and the CD3 binding domain is located at the N-terminus of the HSA binding domain), namely tAb0027 and tAb0033, showed the lowest or second-lowest EC in both the absence and presence of HSA. 50 value.

[0334] Example 4. Multispecific binding protein on CD19 + cytotoxicity of target cells

[0335] This embodiment provides an alternative method for determining the cytotoxic activity of multispecific binding proteins.

[0336] The multispecific binding proteins disclosed herein can be evaluated in vitro in their mediating T cell-dependent cytotoxicity against B cell antigen-positive target cells. For example, the multispecific binding protein for CD19 disclosed herein can be evaluated in vitro in its effect on CD19. + Mediating T cell-dependent cytotoxicity of target cells.

[0337] Fluorescently labeled CD19 + MEC-1 cells (CD19) + Human chronic B-cell leukemia cell lines and isolated PBMCs or CB15 T cells (standardized T cell lines) as random donors of effector cells were co-incubated in the presence of CD19-binding multispecific binding protein. After incubation at 37°C in a humidified incubator for 4 hours, the release of fluorescent dye from target cells to the supernatant was measured using a spectrophotometer. Target cells incubated without CD19-binding multispecific binding protein and target cells completely lysed by the addition of saponins at the end of incubation were used as negative and positive controls, respectively. Based on the measured number of remaining viable target cells, the percentage of specific cell lysis was calculated using the following formula: [1 - (number of viable target cells)] (样品) / Number of live targets (自发) ×100%. The S-type dose-response curve and EC were calculated using GraphPad software via nonlinear regression / 4-parameter logistic fitting. 50 The lysis values ​​obtained for a given concentration of multispecific binding protein were used to calculate an S-type dose-response curve using a 4-parameter logistic fit analysis with Prism software. It is expected that the target cell lysis rate induced by the CD19-binding multispecific binding protein will be higher than that induced by similar constructs lacking the CD19-binding domain or the CD3-binding domain.

[0338] Alternatively, a human T-cell-dependent cytotoxicity (TDCC) assay was used to measure the ability of multispecific binding proteins to guide T cells in killing tumor cells (Nazarian et al. 2015, J. Biomol. Screen, 20:519-27). In this assay, T cells and target cancer cell lines were mixed together in 384-well plates at a ratio of 10:1, and varying amounts of multispecific binding proteins were added. After 48 hours, the T cells were washed away, leaving target cells that had not been killed by the T cells attached to the plates. To quantify the remaining viable cells, a... The Luminescent Cell Viability Assay (Promega) anticipates that the killing rate of cancer cells expressing B-cell antigens induced by a multispecific binding protein that binds CD19 will be higher than that induced by similar constructs lacking the CD19-binding domain or CD3-binding domain and / or other negative control molecules.

[0339] Example 5. Pharmacokinetics of a multispecific binding protein with an HSA binding domain

[0340] This embodiment aims to determine the pharmacokinetics of multispecific binding proteins.

[0341] In the context of pharmacokinetic (PK) studies, multispecific binding proteins containing domains binding CD19, CD3, and serum albumin were tested in cynomolgus monkeys to assess the serum elimination time of multispecific binding proteins.

[0342] The multispecific binding protein was administered via intravenous bolus or intravenous infusion. The multispecific binding protein was administered at doses ranging from 0.5 μg / kg to 3 μg / kg, 6 μg / kg, 12 μg / kg, and 15 μg / kg, respectively, within linear pharmacokinetic ranges. For comparability purposes, serum concentrations of the multispecific binding protein were dose-normalized and molecular weight-normalized (in nmol).

[0343] For each multispecific binding protein, a group of at least two to three animals was used. Blood samples were collected and serum was prepared to determine the serum concentration of the multispecific binding protein. Serum multispecific binding protein levels were measured using an immunoassay. This assay was performed by capturing the multispecific binding protein via the CD19 binding domain, while detection was performed using an antibody targeting the CD3 binding domain of the multispecific binding protein. Serum concentration-time curves were used to determine PK parameters using known analytical methods, such as those described in Ritschel WA and Kearns GL, 1999, IN: Handbook of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Association, Washington, DC, and software such as WinNonlin software. Professional V.3.1WinNonlin TM Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.).

[0344] Alternatively, the serum half-life of various multispecific binding proteins containing a serum albumin-binding domain was compared with the serum half-life of a control construct that could bind CD19 and CD3 but lacked a serum albumin-binding domain by including another group of cynomolgus monkeys receiving the control construct in the experiment. Additional domains may be included to make the control construct similar in size to the multispecific binding proteins.

[0345] It is expected that the multispecific binding protein that binds CD19 will have a significantly longer serum half-life compared to similar constructs and / or other negative control molecules that can bind CD19 and CD3 but lack a serum albumin-binding domain.

[0346] Example 6. Determination of antigen affinity by flow cytometry

[0347] This embodiment aims to determine the affinity of multispecific binding proteins for antigens.

[0348] Testing the various multispecific binding proteins disclosed in this paper with human CD3 + Cells and corresponding B cell surface antigen-positive cells such as human CD19 + Cell binding affinity. The binding affinity of the multispecific binding protein to cynomolgus monkey CD3 was also tested. + Cells and corresponding B cell surface antigen-positive cells such as CD19 in cynomolgus monkeys +Cell binding affinity.

[0349] CD3 + and CD19 + Cells were co-incubated with 100 μL of serially diluted multispecific binding protein buffer. After washing three times with FACS buffer, cells were incubated on ice for 45 min with 0.1 mL of 10 μg / mL mouse monoclonal anti-idiotype antibody in the same buffer. After the second wash cycle, cells were co-incubated with 0.1 mL of 15 μg / mL FITC-conjugated goat anti-mouse IgG antibody under the same conditions as before. As a control, cells were co-incubated with anti-His IgG, and then co-incubated with FITC-conjugated goat anti-mouse IgG antibody in the absence of multispecific binding protein. Cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to remove dead cells. 1 × 10⁻⁶ cells were measured using a commercial flow cytometer and software. 4 Fluorescence of live cells. The average fluorescence intensity of the cell sample was calculated using software such as CXP (Beckman-Coulter, Krefeld, Germany) or Incyte (Merck Millipore, Schwalbach, Germany). The KB for single-site binding can be calculated using normalized fluorescence intensity values ​​and known equations. D The values ​​are calculated using equations such as those provided in GraphPad Prism software (GraphPad Software, La Jolla Calif, USA). CD3 binding affinity and cross-reactivity are related to CD3. + Jurkat cells and cynomolgus monkey CD3 + The HSC-F cell line was evaluated in titration and flow cytometry experiments. It was targeted at human CD19. + Tumor cell lines were evaluated for CD19 binding and cross-reactivity. K+ can be determined using CHO cell lines expressing recombinant human antigen or recombinant cynomolgus monkey antigen. D K is used to calculate cross-reactivity. D ratio.

[0350] Example 7. Cytokine production induced by multispecific binding proteins

[0351] This embodiment aims to determine the ability of multispecific binding proteins to induce immune cells to produce cytokines.

[0352] The AlphaLISA assay (Perkin Elmer) for TNFα and interferon-γ was used to obtain the results in target cells such as CD19. +Evidence of T cell activation by the multispecific binding protein of the present invention (such as the CD19-binding multispecific binding protein) in the presence of B cells. For this assay, as described in the cytotoxicity assay, primary human T cells and human tumor cells expressing B cell surface antigens were incubated in the presence of the CD19-binding multispecific binding protein. After 48 hours of incubation, 2 μL aliquots of sample supernatant were analyzed according to the manufacturer's instructions. TNFα or interferon-γ levels induced by the CD19-binding multispecific binding protein are expected to be higher than those induced by similar constructs lacking the CD19-binding domain or CD3-binding domain and / or other negative control molecules.

[0353] Incorporation

[0354] All publications and patents (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.) cited throughout this specification, whether above or below, are incorporated herein by reference in their entirety for all purposes. If any material incorporated by reference contradicts or is inconsistent with this specification, this specification shall supersede any such material.

[0355] equivalent

[0356] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the foregoing embodiments are to be considered illustrative in all respects and not limiting of the invention described herein. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all variations falling within the equivalent meaning and scope of the claims are intended to be included therein.

Claims

1. An antigen binding domain that binds human serum albumin, comprising a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 consist of the amino acid sequences of SEQ ID NOs: 123, 124, and 126, respectively.

2. The antigen binding domain of claim 1, wherein the VH comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO:

121.

3. The antigen binding domain of claim 1, wherein the antigen binding domain binds human serum albumin with a K D dissociation constant of less than or equal to 2 nM. D Protein A with a melting temperature of greater than or equal to 60 °C, or a combination thereof.

4. A multispecific binding protein, comprising: (a) a first antigen binding domain that binds a first target protein expressed on a target cell; (b) a second antigen binding domain that binds a second target protein expressed on an immune effector cell; and (c) a third antigen binding domain that binds human serum albumin, wherein the third antigen binding domain is the antigen binding domain of claim 1.

5. The multispecific binding protein of claim 4, wherein the first antigen binding domain binds human CD19.

6. The multispecific binding protein of claim 4, wherein the second antigen binding domain binds human CD3.

7. The multispecific binding protein of claim 4, wherein the multispecific binding protein comprises a single polypeptide chain.

8. The multispecific binding protein of claim 7, wherein the third antigen binding domain is not located between the first antigen binding domain and the second antigen binding domain in the polypeptide chain; optionally wherein the third antigen binding domain is located N-terminal to the first antigen binding domain and the second antigen binding domain in the polypeptide chain; optionally wherein the third antigen binding domain is located N-terminal to the first antigen binding domain, and the first antigen binding domain is located N-terminal to the second antigen binding domain in the polypeptide chain; optionally wherein the third antigen binding domain is located N-terminal to the second antigen binding domain, and the second antigen binding domain is located N-terminal to the first antigen binding domain in the polypeptide chain; optionally wherein the third antigen binding domain is located C-terminal to the first antigen binding domain and the second antigen binding domain in the polypeptide chain; optionally wherein the first antigen binding domain is located N-terminal to the second antigen binding domain, and the second antigen binding domain is located N-terminal to the third antigen binding domain in the polypeptide chain; optionally wherein the second antigen binding domain is located N-terminal to the first antigen binding domain, and the first antigen binding domain is located N-terminal to the third antigen binding domain in the polypeptide chain; optionally wherein the first antigen binding domain is located N-terminal to the third antigen binding domain, and the third antigen binding domain is located N-terminal to the second antigen binding domain in the polypeptide chain. ​ ​ ​ ​ ​ ​ ​ the third antigen binding domain is N-terminal to the first antigen binding domain in the polypeptide chain.

9. The multispecific binding protein of claim 4, wherein the first antigen binding domain comprises a single chain variable fragment (scFv); optionally wherein the third antigen binding domain comprises a single domain antibody (sdAb); optionally wherein the second antigen binding domain comprises a scFv; optionally wherein the second antigen binding domain binds human CD3 epsilon; and optionally wherein the second antigen binding domain binds human CD3 epsilon with a K D in the range of 1-100 nM.

9. The multispecific binding protein of claim 4, wherein the first antigen binding domain comprises a single chain variable fragment (scFv); optionally wherein the third antigen binding domain comprises a single domain antibody (sdAb); optionally wherein the second antigen binding domain comprises a scFv; optionally wherein the second antigen binding domain binds human CD3 epsilon; and optionally wherein the second antigen binding domain binds human CD3 epsilon with a K D in the range of 1-100 nM.

10. The multispecific binding protein of claim 4, wherein at least two adjacent antigen binding domains are connected by a peptide linker.

11. The multispecific binding protein of claim 4, wherein the serum half-life of the multispecific binding protein is at least 24, 36, 48, or 60 hours.

12. An antibody comprising the antigen binding domain of claim 1.

13. A pharmaceutical composition comprising: (a) the multispecific binding protein of claim 4 or the antibody of claim 12; and (b) a pharmaceutically acceptable carrier.

14. An isolated polynucleotide encoding the multispecific binding protein of claim 4 or the antibody of claim 12, or a vector comprising a polynucleotide encoding the multispecific binding protein of claim 4 or the antibody of claim 12.

15. A recombinant host cell comprising the polynucleotide or vector of claim 14.

16. A method of producing a multispecific binding protein or antibody, the method comprising culturing the host cell of claim 15 under suitable conditions to allow expression of the multispecific binding protein or the antibody.

17. The method of claim 16, further comprising formulating the isolated multispecific binding protein or antibody with a pharmaceutically acceptable carrier.

18. Use of the multispecific binding protein of claim 4 in the manufacture of a medicament for stimulating an immune response against B cell lymphoma.

19. Use of the multispecific binding protein of claim 4 in the manufacture of a medicament for treating B cell lymphoma in a subject in need thereof.

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