RU2788127C2 - New bispecific polypeptide complexes against cd3/cd19 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to bispecific polypeptide complexes that specifically bind to CD3 and CD19, and can be used in medicine for the treatment of B-cell lymphoma. CD3/CD19 bispecific polypeptide complexes are obtained recombinantly, which contain a combination of four polypeptide sequences: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 or SEQ ID NO: 30.

EFFECT: invention makes it possible to obtain bispecific polypeptide complexes capable of simultaneously binding to CD3 and CD19, T-cells and B-cells and causing subsequent lysis of a malignant CD19-positive B-cell.

13 cl, 23 dwg, 2 tbl, 2 ex

Description

CROSS REFERENCE

This application claims priority under International Patent Application No. PCT/CN2017/103032, filed September 22, 2017.

FIELD OF THE INVENTION

The present invention relates generally to anti-CD3×CD19 bispecific polypeptide complexes comprising antibody variable regions fused to TCR constant regions.

BACKGROUND OF THE INVENTION

Bispecific antibodies are emerging as a new class of therapeutic antibodies. They can bind two different targets, or two different epitopes on a target, creating an additive or synergistic effect that is superior to that of individual antibodies. Much effort has been directed to construct new bispecific formats such as DVD-Ig, CrossMab, BiTE, etc. (Spiess et al., Molecular Immunology, 67(2), pp. 95-106 (2015)). However, these formats can potentially have various limitations in terms of stability, solubility, short half-life and immunogenicity.

Among these bispecific antibody formats, IgG-like bispecific antibody is a common format: one arm binds to target A and the other arm binds to target B. Structurally, it consists of half antibody A and half antibody B with a size and shape similar to natural IgG . To facilitate subsequent modification, it is desirable that such bispecific molecules can be easily produced, like normal IgGs, from a single host cell with a high level of expression and a properly assembled shape. Unfortunately, the pairing of cognate light and heavy chains, as well as the assembly of two different semi-antibodies, cannot be automatically controlled. All kinds of mismatches can lead to significant product heterogeneity.

The preferential heterodimeric assembly of two different heavy chains has been accomplished through mutations in the Fc region, such as ridge-to-recess (Ridgway et al., Protein Engineering, 9(7), pp. 617-21(1996); Merchant et al. ., Nature Biotechnology, 16(7), pp.677-681(1998)), electrostatic control (Gunasekaran et al., Journal of Biological Chemistry, 285(25), pp.19637-19646 (2010)) or negative design (Kreudenstein et al., mAbs, 5(5), pp.646-654 (2013); Leaver-Fay et al., Structure, 24(4), pp.641-651 (2016)). However, the selective pairing of the light and heavy chains of each individual antibody is still a difficult task. The area of contact between light and heavy chains includes the variable domain (VH-VL) and the constant domain (CH1-CL). Various methods have been used in developing orthogonal contact areas to facilitate cognate mating. Roche changed the CH1 and CL domains and created the CrossMab platform (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187-11192 (2011)), in Medimmmune introduced an alternative disulfide bond (Mazor et al., mAbs, 7(2), pp.377-389 (2015)), Amgen performed additional electrostatic control in the CH1-CL region (Liu et al. , Journal of Biological Chemistry, 290(12), pp.7535-7562 (2015)), and at Lilly (Lewis et al., Nature Biotechnology, 32(2), pp.191-198 (2014)) and Genentech (Dillon et al., mAbs, 9(2), pp.213-230 (2017)) carried out mutations in both variable and constant domains.

Human CD19 is a type I transmembrane protein belonging to the immunoglobulin superfamily (Carter et al., Curr Dir Autoimmun, 7: 4-32 (2004)). It is expressed on most B cells but is not found on plasma cells, stem cells, or normal myeloid lineage (Tedder, Nat Rev Rheumatol, 5 (10): 572-577 (2009)). CD19s are important in setting endogenous thresholds for B cell signaling through modulation of both B cell receptor (BCR) dependent and independent signals (Wang et al., Experimental Hematology & Oncology, 1:36 (2012)). CD19 is more widely expressed than CD20. CD19 expression patterns are conserved in B-cell malignancies, including all subtypes of B-cell lymphoma, from slow-growing to aggressive forms, as well as B-cell chronic lymphocytic leukemia and non-T-cell acute lymphoblastic leukemia, as well as targeting tumors from undifferentiated B cells, such as acute lymphoblastic leukemia (ALL), which are not targets for rituximab. For the treatment of lymphoma, several anti-CD19 monoclonal antibodies have been investigated (US Patent Application Publication No. 20140072587 A1, US Patent No. 8242252 B2 and US Patent No. 8097703 B2).

The CD3 T-cell co-receptor is a protein complex consisting of four different chains: the CD3 gamma chain, the CD3 delta chain, and two CD3 epsilon chains. The four chains associate with a molecule known as the T cell receptor (TCR) and the zeta chain to generate an activation signal in T lymphocytes. TCR, zeta chain, and CD3 molecules form a TCR complex in which TCR, as a subunit, recognizes and binds to an antigen, and CD3, as a subunit, carries and transmits antigenic stimulation along the signaling pathway and, ultimately, regulates T-cell activity. The CD3 protein is present in almost all T cells. The CD3-TCR complex modulates both innate and adaptive immune response T cell functions, as well as cellular and humoral immune functions. These include the destruction of pathogenic organisms and the control of tumor growth through a wide range of cytotoxic effects. Mouse monoclonal antibodies specific for human CD3, such as OKT3 (Kung et al., Science, 206: 347-9 (1979)), were first generation CD3 antibodies developed for treatment. Although OKT3 has a strong immunosuppressive effect, its clinical use has been hampered by serious side effects associated with its immunogenic and mitogenic potential. (Chatenoud, Nature Reviews, 3:123-132 (2003)). OKT3 induces an antiglobulin response, promoting its rapid clearance and neutralization (Chatenoud et al., Eur. J. Immunol., 137:830-8 (1982)). In addition, OKT3 induces T cell proliferation and cytokine production in vitro and results in large scale cytokine release in vivo. (Hirsch et al., J. Immunol, 142: 737-43 (1989)). These serious side effects limit the wider use of OKT3 in transplantation, as well as its application to other clinical areas such as autoimmunity.

A bispecific antibody targeting CD3 and CD19 can bind to T cells and B cells simultaneously. Once the bispecific antibody binds to a CD3 positive T cell and a CD19 positive B cell, a cytolytic synapse is formed. Cytotoxicity is then induced by the release of perforin and granzymes from the granules in the cytotoxic T cell, the latter inducing apoptosis and lysis of the malignant B cell.

There is a great need to design bispecific molecules with the desired level of expression and half-life in vivo for both CD3 and CD19. Such anti-CD3×CD19 bispecific polypeptide complexes are useful in the treatment of conditions associated with CD19, including cancer.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a bispecific polypeptide complex containing a first antigen-binding fragment associated with a second antigen-binding fragment, where:

the first antigen-binding fragment contains:

a first polypeptide comprising, from the N-terminus to the C-terminus, the first heavy chain variable domain (VH1) of the first antibody operably linked to the first constant region (C1) of the T cell receptor (TCR), and

a second polypeptide comprising, from the N-terminus to the C-terminus, the first light chain variable domain (VL1) of the first antibody operably linked to the second TCR constant region (C2),

where:

C1 contains an engineered CBeta containing SEQ ID NO: 1, and C2 contains an engineered SAFA containing SEQ ID NO: 2,

amino acid C48 in SEQ ID NO: 1 and amino acid C41 in SEQ ID NO: 2 are capable of forming a non-native interchain disulfide bond,

C1 and C2 are able to form a dimer, and the non-native interchain disulfide bond is able to stabilize the dimer,

and

the second antigen-binding fragment contains:

a second heavy chain variable domain (VH2) of the second antibody operably linked to the heavy chain CH1 domain of the antibody, and

a second light chain variable domain (VL2) of the second antibody operably linked to the antibody light chain constant domain (CL),

where:

one of the first and second antigen binding fragments is an anti-CD3 binding fragment, and the other is an anti-CD19 binding fragment,

The anti-CD3 binding fragment is derived from an anti-CD3 antibody containing:

a) a heavy chain CDR1 containing SEQ ID NO: 3, b) a heavy chain CDR2 containing SEQ ID NO: 4, c) a heavy chain CDR3 containing SEQ ID NO: 5, d) a kappa light chain CDR1 containing SEQ ID NO: 6, e) a kappa light chain CDR2 comprising SEQ ID NO: 7, and f) a kappa light chain CDR3 comprising SEQ ID NO: 8,

the anti-CD19 binding fragment is derived from an anti-CD19 antibody containing:

a) a heavy chain CDR1 containing SEQ ID NO: 9, b) a heavy chain CDR2 containing SEQ ID NO: 10, c) a heavy chain CDR3 containing SEQ ID NO: 11, d) a kappa light chain CDR1 containing SEQ ID NO: 12, e) a kappa light chain CDR2 comprising SEQ ID NO: 13, and f) a kappa light chain CDR3 comprising SEQ ID NO: 14,

and

the first antigen binding fragment and the second antigen binding fragment are less susceptible to mismatch than if both the first and second antigen binding fragments were natural Fab analogs.

In some embodiments, the anti-CD3 binding fragment of the bispecific polypeptide complex is derived from an anti-CD3 antibody comprising a heavy chain variable domain sequence comprising SEQ ID NO: 15 and a light chain variable domain sequence comprising SEQ ID NO: 17.

In some embodiments, an anti-CD19 binding fragment of a bispecific polypeptide complex is derived from an anti-CD19 antibody comprising a heavy chain variable domain sequence comprising SEQ ID NO: 19 and a light chain variable domain sequence comprising SEQ ID NO: 21.

In some embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.

In some embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 30.

In one aspect, the present invention relates to a conjugate containing a bispecific polypeptide complex provided herein conjugated to a fragment.

In one aspect, the present invention provides an isolated polynucleotide encoding a bispecific polypeptide complex provided herein.

In one aspect, the present invention provides an isolated vector containing a polynucleotide provided herein.

In one aspect, the present invention provides a host cell containing an isolated polynucleotide as provided herein or an isolated vector as provided herein.

In one aspect, the present invention provides a method for expressing a bispecific polypeptide complex provided herein, comprising culturing a host cell provided herein under conditions under which the bispecific polypeptide complex is expressed.

In one aspect, the present invention relates to a composition containing a bispecific polypeptide complex presented herein.

In one aspect, the present invention provides a pharmaceutical composition comprising the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.

In one aspect, the present invention provides a method of treating a disease or condition associated with CD19 in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of the bispecific polypeptide complex provided herein. In some embodiments, the disease or condition can be alleviated, eliminated, treated or prevented when the first antigen and the second antigen are both modulated.

In some embodiments, the first VH is operably linked to CBeta on a first conjugation domain that contains the C-terminal V/C conjugation fragment of an antibody and has the amino acid sequence of SEQ ID NO: 23 (LEDLKNVFPP), and the first VL is operably linked to SApha on the second domain. conjugation, which contains the N-terminal fragment of the V/C conjugation of TCR and has the amino acid sequence of SEQ ID NO: 24 (KPDIQNPDP).

In some embodiments, the first antigen-binding fragment is associated with a first dimerization domain, and the second antigen-binding fragment is associated with a second dimerization domain, where the first and second dimerization domains are associated. In some embodiments, the association occurs via a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or a combination thereof.

In some embodiments, the first and/or second dimerization domain comprises at least a portion of an antibody hinge region, optionally derived from IgG1, IgG2, or IgG4.

In some embodiments, C1 contains an engineered CBeta and the first dimerization domain is operably linked to the engineered CBeta on a third conjugation domain that contains SEQ ID NO: 25 (YGPPCPPCPAPEFLGGP).

In some embodiments, the second dimerization domain is operably linked to the heavy chain variable domain of the second antigen binding fragment.

In some embodiments, the first and second dimerization domains are different and associate in such a way as to prevent homodimerization and/or promote heterodimerization.

In some embodiments, the first and second dimerization domains are capable of associating into heterodimers in a ledge-in-recess, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased ductility.

In another aspect, the present invention relates to a kit containing a polypeptide complex provided herein for the detection, diagnosis, prognosis or treatment of a disease or condition.

The above and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 presents schematic representations of the studied antibody formats. Both an anti-CD3 T3 antibody and an anti-CD19 U4 antibody have been developed. To create a unique light and heavy chain contact region that is orthogonal to a regular antibody, the T3 constant region (CL and CH1) was replaced with TCR constant domains. To construct bispecific antibodies in the E17 and F16 formats, TCR-modified T3 and native U4 were used in combination with protrusion-to-recess mutations in the Fc domain.

Figures 2A-2D show overlapping positions of an antibody Fv model and TCR structures providing a fusion control for an antibody Fv and a TCR constant region. Figure 2A shows a model of the structure of the Fv antibody, which was built based on the sequence of the T3 antibody against CD3, developed within the laboratory. Figure 2B shows the TCR structure from PDB 4L4T. Figure 2C shows a structural model of an Fv antibody spanning the TCR variable region in various directions. Rough chimeric proteins were created by removing the TCR variable domain at overlapping positions as shown in FIG. 2D. The overlapped residues in the conjugation region contributed to the construction of the conjugation region. The VL chain of the antibody and the alpha chain of the TCR are colored white. VH and beta chains are colored black.

3A shows the sequence of the native TCR alpha chain and its corresponding sequence with mutated cysteine residues. TRAC_Human corresponds to the natural sequence of the alpha chain constant region. 4L4T_Alpha_Crystal corresponds to a crystal structure sequence (PDB code 4L4T) with S55C mutations that can form an interchain disulfide bond. The gray area corresponds to the constant region used as the backbone of the chimeric protein in the present invention.

FIG. 3B shows the sequence of the native TCR beta chain and its corresponding sequence with mutated cysteine residues.

On figa-4B shows the sequence and numbering of the constant regions of the TCR Calf and Cbeta with remote N-glycosylation. 4A shows the sequences and numbering of the TCR alpha constant region. 4B shows the sequences and numbering of the TCR beta constant region.

5 shows flow cytometry histograms of the cynomolgus monkey CD19 transfected cell line, WBP701.CHO-K1.cpro1.FL.C9, and the parental CHO-K1 cell line.

Figure 6 shows DNS-PAGE W3438-T3U4.F16-1.uIgG4.SP. M: protein marker; lane 1: W3438-T3U4.F16-1.uIgG4.SP, under non-reducing conditions; Lane 3: W3438-T3U4.F16-1.uIgG4.SP, under reducing conditions.

Figure 7 shows the chromatogram of the size exclusion HPLC W3438-T3U4.F16-1.uIgG4.

Figure 8 shows DNS-PAGE W3438-T3U4.E17-1.uIgG4.SP. M: protein marker; lane 1: W3438-T3U4.E17-1.uIgG4.SP, under non-reducing conditions; lane 2: W3438-T3U4.E17-1.uIgG4.SP, under reducing conditions.

Figure 9 shows the chromatogram of the size exclusion HPLC W3438-T3U4.E17-1.uIgG4.SP.

10A-10B show binding of W3438-T3U4.E17-1.uIgG4.SP to Ramos cells (FIG. 10A) and Jurkat cells (FIG. 10B) by fluorescent sorting (FACS).

11A-11B show binding of W3438-T3U4.F16-1.uIgG4.SP to Ramos cells (FIG. 11A) and Jurkat cells (FIG. 11B) by fluorescent sorting (FACS).

Figure 12 shows the binding of W3438-T3U4.E17-1.uIgG4.SP to a cell expressing cynomolgus monkey CD19 by fluorescence sorting (FACS).

13 shows binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus monkey CD3 by ELISA.

FIGS. 14A-14B show the affinity of W3438-T3U4.E17-1.uIgG4.SP for human CD19 and CD3, measured by binding to Ramos (FIG. 14A) and Jurkat (FIG. 14B) cells.

On figa-15B shows the double binding of W3438-T3U4.E17-1.uIgG4.SP with CD19 and CD3 (figa); and a negative control (FIG. 15B).

16A-16B shows the cytotoxic activity of W3438-T3U4.E17-1.uIgG4.SP on Raji cells (Fig. 16A) and the cytotoxic activity of W3438-T3U4.F16-1.uIgG4.SP on Raji cells (Fig. 16B) .

On figa-17D shows the expression of CD69 and CD25 on T cells in the presence or absence of CD19+ target cells. Percentage expression of CD69 on T cells in a subpopulation of CD4+ T cells (FIG. 17A); percent expression of CD69 on T cells in the CD8+ T cell subpopulation (FIG. 17B); percent expression of CD25 on T cells in the CD4+ T cell subpopulation (FIG. 17C); percent expression of CD25 on T cells in the CD8+ T cell subset (FIG. 17D).

18A-18D show the release of IFN-γ and TNF-α cytokines by T cells in the presence or absence of CD19+ target cells. Release of IFN-γ in the CD4+ T cell subpopulation (FIG. 18A); Release of TNF-α in the CD4+ T cell subset (Figure 18B); Release of IFN-γ in CD8+ T cell subpopulation (Fig. 18C); Release of TNF-α in the CD8+ T cell subpopulation (FIG. 18D).

On figa-19B shows the stability of W3438-T3U4.E17-1.uIgG4.SP in human serum. Binding of serum-incubated W3438-T3U4.E17-1.uIgG4.SP samples to Ramos cells on indicated days (FIG. 19A); binding of serum-incubated W3438-T3U4.E17-1.uIgG4.SP samples to Jurkat cells on indicated days (FIG. 19B).

Figure 20 shows the binding of W3438-T3U4.E17-1.uIgG4.SP to C1Q by ELISA. An IgG1 antibody was used as an analytical control.

Figure 21 shows the tumor volume curve after administration of W3438-T3U4.E17-1.uIgG4.SP at various doses to humanized PBMC mice with tumor xenografts from Raji cells. The experimental points represent the group mean, the whiskers represent the standard error of the mean (SEM). An irrelevant IgG4 antibody was used as a negative control.

22 shows serum concentrations of W3438-T3U4.E17-1.uIgG4.SP in cynomolgus monkey after a single dose of 1 mg/kg. Serum samples from two monkeys were analyzed by ELISA.

Figures 23A-23B show Anti-Drug Antibody (ADA) in serum samples from Monkey #1 (Figure 23A) and Monkey #2 (Figure 23B), including both before and after administration of W3438-T3U4.E17 -1.uIgG4.SP.

DETAILED DESCRIPTION OF THE INVENTION

The following description of the invention is only intended to illustrate various embodiments of the invention.

Definitions

In this document, singular nouns are used to refer to one or more (i.e., at least one) entity. For example, "polypeptide complex" means one or more polypeptide complexes.

As used herein, the term “about” or “approximately” refers to an amount, level, value, number, frequency, percentage, degree, size, volume, mass, or length that varies by as many as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference amount, level, value, number, frequency, percentage, degree, size, volume, mass, or length. In specific embodiments, the terms "about" or "about" before a numerical value means deviations of the values in the direction of decrease or increase by 15%, 10%, 5% or 1%.

Throughout the present invention, unless the context otherwise requires, the words “comprise”, “comprises” and “comprising” will be understood to mean the inclusion of the specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps. or elements. By "consisting of" is meant to include and limit everything that follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements may be present. The term "consisting essentially of" means the inclusion of any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the invention in relation to the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, however, other elements are optional and may or may not be present depending on whether they affect the activity or operation of the listed elements.

Reference throughout the present invention to “one embodiment”, “an embodiment”, “a specific embodiment”, “a related embodiment”, “a certain embodiment”, “an additional embodiment”, or “the next embodiment”, or combinations thereof means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the occurrences of the above phrases in various places in this specification do not necessarily all refer to the same embodiment. In addition, specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues or a combination of several polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is a synthetic chemical mimetic of the corresponding natural amino acid, as well as to natural amino acid polymers and non-natural amino acid polymer. The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are amino acids encoded by the genetic code, as well as amino acids that are further modified, such as hydroxyproline, gamma-carboxyglutamate and O-phosphoserine. Amino acid analogs refer to compounds that have a basic chemical structure similar to a naturally occurring amino acid, i.e. an alpha carbon that is bonded to hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. Alpha carbon refers to the first carbon atom that attaches to a functional group such as a carbonyl. The beta carbon refers to the second carbon atom bonded to the alpha carbon, and the system continues to designate the carbon atoms in alphabetical order with Greek letters. Amino acid mimetics refer to chemical compounds whose structure differs from the general chemical structure of an amino acid, but which functions similarly to a naturally occurring amino acid. The term "protein" usually refers to long chain polypeptides. The term "peptide" generally refers to short polypeptides. Polypeptide sequences are generally characterized in that the left terminus of the polypeptide sequence is the amino terminus (N-terminus); the right end of the polypeptide sequence is the carboxyl end (C-terminus). "Polypeptide complex" in the context of the present description refers to a complex containing one or more polypeptides that are associated to perform certain functions. In some embodiments, the polypeptides are immunodependent.

As used herein, the term “antibody” includes any immunoglobulin, monoclonal antibody, polyclonal antibody, polyspecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. A native intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region (“HCVR”) and first, second and third constant regions (CH1, CH2 and CH3), while each light chain consists of a variable region (“LCVR”) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, with mammalian light chains being classified as λ or κ. The antibody has a "Y" shape, with the "trunk" of Y consisting of the second and third constant regions of two heavy chains linked together via a disulfide bond. Each "arm" Y includes the variable region and the first constant region of one heavy chain associated with the variable and constant regions of one light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops called complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2 and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3). CDR boundaries for antibodies can be defined or identified according to the definitions of Kabat, Chothia or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al. , J Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987 ); Chothia, C. et al. , Nature. Dec 21-28; 342(6252):877-83 (1989); Kabat EA et al. , National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are inserted between flanking regions known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support hypervariable loops. Each HCVR and LCVR contains four FRs, and the CDRs and FRs are arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit different effector functions. Antibodies are categorized based on the amino acid sequence of their heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major classes of antibodies are further subclassified, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). chain).

As used herein, the term "variable domain", in relation to an antibody, refers to the variable region of an antibody, or fragment thereof, containing one or more CDRs. Although the variable domain may contain an intact variable region (such as HCVR or LCVR), it is also possible to include a smaller than intact variable region, but retain the ability to bind to an antigen or form an antigen binding site.

As used herein, the term “antigen binding fragment” refers to an antibody fragment formed from a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain an intact native antibody structure. Examples of an antigen binding fragment include, without limitation, variable domain, variable region, diabody, Fab, Fab', F(ab') ₂ , Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv '), a disulfide-stabilized diabody (ds diabody), a multispecific antibody, a camel single domain antibody, a nanobody, a domain antibody, and a divalent domain antibody. The antigen-binding fragment is capable of binding to the same antigen that the parent antibody binds to. In some embodiments, the antigen binding portion may comprise one or more CDRs from a particular human antibody grafted onto a framework region from one or more different human antibodies. More detailed antigen binding fragment formats are described in Spiess et al., 2015 (Supra) and Brinkman et al., MAbs, 9(2), pp.182-212 (2017), which are incorporated herein by reference in their entirety.

The term “Fab”, in relation to an antibody, refers to that part of an antibody that consists of one light chain (both variable and constant region) associated with the variable region and the first constant region of one heavy chain via a disulfide bond. In some embodiments, both the light chain and heavy chain constant regions are replaced by TCR constant regions.

"Fab" refers to a Fab fragment that includes part of the hinge region.

“F(ab') ₂ ” refers to the Fab' dimer.

The term "Fd region (Fd)", in relation to an antibody, refers to the amino-terminal half of a heavy chain fragment that can be combined with a light chain to form a Fab.

The term "Fc", in relation to an antibody, refers to that portion of the antibody which consists of the second (CH2) and third (CH3) constant regions of the first heavy chain linked to the second and third constant regions of the second heavy chain via a disulfide bond. The Fc portion of an antibody is responsible for various effector functions such as ADCC and CDC but is not involved in antigen binding.

The term "hinge region", in relation to an antibody, includes the portion of the heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region contains approximately 25 amino acid residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently.

As used herein, the term “CH2 domain” refers to the portion of a heavy chain molecule that extends, for example, from about amino acids 244 to amino acids 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360, the Caybott numbering system; and amino acids 231- 340, EU numbering system, see Kabat, E., et al., U.S. Department of Health and Human Services, (1983)).

The "CH3 domain" extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 amino acids. Some classes of immunoglobulins, such as IgM, further include a CH4 region.

“Fv”, in relation to an antibody, refers to the smallest fragment of an antibody that contains the entire antigen binding site. The Fv fragment consists of a single light chain variable domain linked to a single heavy chain variable domain. A number of Fv constructs have been proposed, including dsFv, in which the bond between the two domains is enhanced by an introduced disulfide bond; and scFv can be made using a peptide linker to link the two domains together as a single polypeptide. Fv constructs were also prepared containing an immunoglobulin heavy or light chain variable domain associated with a corresponding immunoglobulin heavy or light chain variable and constant domain. Fv has also been multimerized to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

“ScFab” refers to a Fd fusion polypeptide linked to a light chain via a polypeptide linker, resulting in a single chain Fab (scFab) fragment.

"TriFab" refers to a trivalent bispecific fusion protein consisting of three units with Fab functional groups. TriFabs contain two regular Fabs fused to an asymmetric Fab-like fragment.

“Fab-Fab” refers to a fusion protein formed by fusing the Fd chain of the first arm of a Fab to the N-terminus of the Fd chain of the second arm of a Fab.

“Fab-Fv” refers to a fusion protein formed by the fusion of HCVR to the C-terminus of an Fd chain and LCVR to the C-terminus of a light chain. The "Fab-dsFv" molecule can be formed by introducing an interdomain disulfide bond between the HCVR domain and the LCVR domain.

“MAb-Fv” or “IgG-Fv” refers to a fusion protein formed by fusion of an HCVR domain to the C-terminus of one Fc chain and an LCVR domain either expressed alone or fused to the C-terminus of the other to produce a bispecific trivalent IgG fusion protein -Fv (mAb-Fv), with Fv being stabilized by an interdomain disulfide bond.

The terms “ScFab-Fc-scFv2” and “ScFab-Fc-scFv” refer to a fusion protein formed by the fusion of a single chain Fab with Fc and disulfide-stabilized Fv domains.

The term “attached IgG” refers to a fusion protein with a Fab arm fused to IgG to form a bispecific (Fab) ₂ -Fc format. It can form "IgG-Fab" or "Fab-IgG", the Fab being fused to the C-terminus or N-terminus of the IgG molecule with or without a linker. In some embodiments, the attached IgG can be further modified to IgG-Fab ₄ format (see Brinkman et al., 2017, supra ).

“DVD-Ig” refers to an antibody with two variable domains, which is formed by the fusion of an additional HCVR domain and an LCVR domain of second specificity with an IgG heavy chain and a light chain. “CODV-Ig” refers to a linked format in which two HCVR domains and two LCVR domains are linked in such a way as to allow cross-pairing of HCVR-LCVR variable domains that are either (N- to C-terminal) in the order HCVRA-HCVRB and LCVRB-LCVRA, or in the order HCVRB-HCVRA and LCVRA-LCVRB.

“CrossMab” refers to the technology of pairing an unmodified light chain with a corresponding unmodified heavy chain and pairing a modified light chain with a corresponding modified heavy chain, resulting in an antibody with reduced mismatch in the light chain.

“BiTE” is a bispecific T cell recruiter molecule comprising a first scFv with a first specificity for an antigen in the LCVR-HCVR orientation linked to a second scFv with a second specificity in the HCVR-LCVR orientation.

"WuXiBody" is a bispecific antibody containing a soluble chimeric protein with antibody variable domains and TCR constant domains, wherein the subunits (such as alpha and beta domains) of the TCR constant domains are linked via an engineered disulfide bond.

“Percent (%) sequence identity”, in relation to an amino acid sequence (or nucleic acid sequence), is defined as the percentage of amino acid (or nucleic acid) residues in the candidate sequence that are identical to the amino acid (or nucleic acid) residues in the reference sequence, after alignment of sequences and, if necessary, the introduction of gaps, to achieve the maximum number of identical amino acids (or nucleic acids). A conservative substitution of amino acid residues may or may not be considered identical residues. Alignment to determine percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available from the US National Center for Biotechnology Information (NCBI) website, see also Altschul S.F. et al ., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the European Bioinformatics Institute website, see See also Higgins D. G. et al., Methods in Enzymology, 266:383-402 (1996), Larkin M. A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)) and the ALIGN software or Megaalign (DNASTAR). Those skilled in the art may use the standard parameters provided by the tool, or may adjust the parameters according to the setting, for example, by selecting an appropriate algorithm.

The term "antigen" or "Ag", as used herein, refers to a compound, composition, peptide, polypeptide, protein, or substance that can stimulate antibody production or a T cell response in a cell culture or animal, including compositions (e.g., those containing a tumor-specific protein), which are added to a cell culture (such as a hybridoma), or administered or absorbed into an animal. The antigen interacts with the products of specific, humoral or cellular, immunity (for example, an antibody), including those induced by heterologous antigens.

An "epitope" or "antigenic determinant" refers to the site of an antigen to which a binding agent (such as an antibody) binds. Epitopes can be formed from either contiguous amino acids (also referred to as a linear or sequential epitope) or from non-contiguous amino acids aligned by the folding of a tertiary protein structure (also referred to as a configurational or conformational epitope). Epitopes formed from contiguous amino acids are usually linear along the primary amino acid residues on the protein, and small segments of contiguous amino acids can be cleaved from the antigen that binds to molecules of the major histocompatibility complex (MHC) or are preserved when exposed to denaturing solvents, while epitopes formed folding of the tertiary structure are usually lost during treatment with denaturing solvents. An epitope typically includes at least 3 or more, typically at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation.

As used herein, the term “specific binding” or “specifically binds” refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In some embodiments, the polypeptide complex and the bispecific polypeptide complex provided herein specifically bind an antigen with a binding affinity (K _D ) ≤ 10 ^-6 M (e.g., ≤ 5×10 ^-7 M, ≤ 2×10 ^-7 M , ≤ 10 ^-7 M, ≤ 5×10 ^-8 M, ≤ 2×10 ^-8 M, ≤ 10 ^-8 M, ≤ 5×10 ^-9 M, ≤ 2×10 ^-9 M, ≤ 10 ^-9 M or ≤ 10 ^-10 M). As used herein, K _D refers to the ratio of dissociation rate to association rate (k _off /k _on ) and can be determined using surface plasmon resonance techniques, for example using an instrument such as Biacore.

The term “operably linked” or “operably linked” refers to the combination, with or without a spacer or linker, of two or more biological sequences of interest such that they are in a relationship that allows them to function in a particular manner. When used in relation to polypeptides, it is understood that the polypeptide sequences are linked in such a way that allows the associated product to perform the intended biological function. For example, the variable region of an antibody can be operably linked to a constant region to provide a stable product with antigen-binding activity. The term can also be used in relation to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (eg, a promoter, enhancer, silencer, etc. sequence), this means that the polynucleotide sequences are linked in such a way that controlled expression of the polypeptide from the polynucleotide is allowed.

The term "fusion" or "fusion", when used in relation to amino acid sequences (e.g., a peptide, polypeptide, or protein), refers to the combination of two or more amino acid sequences, for example, by chemical bonding or recombinant means, into a single amino acid sequence that is not is natural. The fused amino acid sequence can be obtained by genetic recombination of two coding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotides into a host cell.

As used herein, the term "spacer" refers to an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues or a length of 5 to 15, 20, 30, 50 or more amino acid residues linked by peptide bonds and used for binding one or more polypeptides. The spacer may or may not have a secondary structure. Spacer sequences are known in the art, see, for example, Holliger et al., Proc. Natl. Acad. sci. USA 90:6444-6448 (1993); Poljak et al. Structure 2:1121-1123 (1994). Any suitable spacers known in the art may be used.

The term “antigen specificity” refers to a particular antigen or epitope thereof that is selectively recognized by an antigen-binding molecule.

As used herein, the term "substitution", in relation to an amino acid residue, refers to the natural or induced substitution of one or more amino acids for another in a peptide, polypeptide or protein. A substitution in a polypeptide may result in a reduction, enhancement, or elimination of the polypeptide's function.

A substitution can also be a “conservative substitution”, as applied to an amino acid sequence, which refers to the substitution of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties, or the substitution of those amino acids that are not critical to the activity of the polypeptide. For example, conservative substitutions can be made among amino acid residues with nonpolar side chains (e.g., Met, Ala, Val, Leu and Ile, Pro, Phe, Trp), among residues with uncharged polar side chains (e.g., Cys, Ser Thr, Asn , Gly and Gln), among residues with acidic side chains (for example, Asp, Glu), among amino acids with basic side chains (for example, His, Lys and Arg), among amino acids with beta-branched side chains (for example, Thr, Val and Ile), among amino acids with sulfur-containing side chains (eg Cys and Met) or among residues with aromatic side chains (eg Trp, Tyr, His and Phe). In some embodiments, substitutions, deletions, or additions may also be considered "conservative substitutions". The number of amino acids that are inserted or removed can range from 1 to 5. Conservative substitution usually does not cause a significant change in the conformational structure of the protein and, therefore, may preserve the biological activity of the protein.

As used herein, the term "mutation" or "mutated", in relation to an amino acid residue, refers to a substitution, insertion, or addition of an amino acid residue.

As used herein, the term “T cell” refers to a type of lymphocyte that plays a major role in cellular immunity, including T helper cells (e.g., CD4 ⁺ T cells, T helper type 1, T helper type 2, T- helper type 3, T helper type 17), cytotoxic T cells (eg, CD8 ⁺ T cells), memory T cells (eg, central memory T cells (TCM cells), effector memory T cells (TEM- cells and TEMRA cells) and resident memory T cells (TRM) which are either CD8 ⁺ or CD4 ⁺ ), natural killer T cells (NKT) and inhibitory T cells.

A native "T cell receptor" or native "TCR" is a heterodimeric protein on the surface of T cells that is associated with invariant CD3 chains to form a complex capable of mediating signal transduction. TCR belongs to the immunoglobulin superfamily and is similar to a semi-antibody with one heavy chain and one light chain. The native TCR has an extracellular part, a transmembrane part, and an intracellular part. The extracellular domain of the TCR has a membrane-proximal constant region and a membrane-distal variable region.

As used herein, the term "subject" or "individual" or "animal" or "patient" refers to an animal or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, mitigation, prevention and/or treatment of a disease or disorders. Mammalian subjects include humans, pets, farm animals and zoo animals, sports or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and so on.

Bispecific polypeptide complex

In one aspect, the present invention relates to a bispecific polypeptide complex. As used herein, the term "bispecific" means that there are two antigen-binding fragments, each capable of specifically binding to a different antigen or different epitope on the same antigen. The bispecific polypeptide complex provided herein comprises a first antigen-binding fragment associated with a second antigen-binding fragment, one of which specifically binds to CD3 and the other specifically binds to CD19. In other words, the first antigen-binding fragment may specifically bind to CD3, and the second antigen-binding fragment may specifically bind to CD19. Alternatively, the first antigen-binding fragment may specifically bind to CD19 and the second antigen-binding fragment may specifically bind to CD3.

In some embodiments, the present invention relates to a bispecific polypeptide complex comprising a first antigen-binding fragment associated with a second antigen-binding fragment, where:

the first antigen-binding fragment includes:

a first polypeptide comprising, from the N-terminus to the C-terminus, the first heavy chain variable domain (VH1) of the first antibody operably linked to the first T cell receptor (TCR) constant region (C1), and

a second polypeptide comprising, from the N-terminus to the C-terminus, the first light chain variable domain (VL1) of the first antibody operably linked to the second TCR constant region (C2),

where:

C1 contains an engineered CBeta containing SEQ ID NO: 1, and C2 contains an engineered SAFA containing SEQ ID NO: 2,

amino acid C48 in SEQ ID NO: 1 and amino acid C41 in SEQ ID NO: 2 are capable of forming a non-native interchain disulfide bond,

C1 and C2 are able to form a dimer, and a non-native interchain disulfide bond is able to stabilize the dimer,

and

the second antigen-binding fragment includes:

a second heavy chain variable domain (VH2) of the second antibody operably linked to the heavy chain CH1 domain of the antibody, and

the second light chain variable domain (VL2) of the second antibody operably linked to the antibody light chain constant domain (CL),

where:

one of the first and second antigen binding fragments is an anti-CD3 binding fragment, and the other is an anti-CD19 binding fragment,

the anti-CD3 binding fragment is derived from an anti-CD3 antibody containing:

a) Heavy chain CDR1 containing SEQ ID NO: 3, b) Heavy chain CDR2 containing SEQ ID NO: 4, c) Heavy chain CDR3 containing SEQ ID NO: 5, d) Kappa light chain CDR1 containing SEQ ID NO: : 6, e) a kappa light chain CDR2 comprising SEQ ID NO: 7, and f) a kappa light chain CDR3 comprising SEQ ID NO: 8,

the anti-CD19 binding fragment is derived from an anti-CD19 antibody containing:

a) a heavy chain CDR1 containing SEQ ID NO: 9, b) a heavy chain CDR2 containing SEQ ID NO: 10, c) a heavy chain CDR3 containing SEQ ID NO: 11, d) a kappa light chain CDR1 containing SEQ ID NO: 12, e) a kappa light chain CDR2 comprising SEQ ID NO: 13 and f) a kappa light chain CDR3 comprising SEQ ID NO: 14,

and

the first antigen binding fragment and the second antigen binding fragment are less susceptible to mismatch than if both the first and second antigen binding fragments were natural Fab analogs.

In some embodiments, the bispecific polypeptide complex provided herein comprises a first antigen-binding fragment containing a sequence derived from a TCR constant region, but the second antigen-binding fragment does not contain a sequence derived from a TCR constant region.

The bispecific polypeptide complex provided herein is significantly less prone to mismatch between heavy chain and light chain variable domains. Without wishing to be bound by any theory, it is believed that the stabilized TCR constant regions in the first antigen-binding fragment may specifically associate with each other and therefore contribute to highly specific pairing of putative VH1 and VL1, preventing unwanted mismatches of VH1 or VL1 with other variable regions. , which do not include putative antigen-binding sites.

In some embodiments, the second antigen-binding fragment further comprises an antibody CH1 constant domain operably linked to VH2 and an antibody light chain constant domain operably linked to VL2. Thus, the second antigen binding fragment contains a Fab.

When the first, second, third, and fourth variable domains (e.g., VH1, VH2, VL1, and VL2) are expressed in the same cell, it is highly desirable that VH1 specifically bind to VL1 and VH2 specifically bind to VL2, so that, as a result, the bispecific protein product had the correct antigen-binding specificity. However, in existing technologies, such as interspecific hybridoma (or quadroma), random mating of VH1, VH2, VL1 and VL2 occurs, which, respectively, leads to the formation of up to ten different species, of which only one is a functional bispecific antigen-binding molecule. This not only reduces the yield of the product, but also complicates the purification of the target product.

The bispecific polypeptide complexes presented herein are exceptional in that the variable domains are less mismatched than the first and second antigen-binding fragments that are natural Fab analogs. In an illustrative example, the first antigen-binding domain contains VH1-C1 paired with VL1-C2, and the second antigen-binding domain contains VH2-CH1 paired with VL2-CL. Surprisingly, it has been found that C1 and C2 preferentially associate with each other and are less likely to associate with CL or CH1, whereby the formation of undesirable pairs such as C1-CH, C1-CL, C2-CH and C2-CL is undesirable and significantly abbreviated. As a result of C1-C2 specific association, VH1 binds specifically to VL1, thereby providing a first antigen-binding site, and CH1 specifically binds to CL, thereby allowing VH2-VL2 specific pairing, which provides a second antigen-binding site. Accordingly, the first antigen-binding fragment and the second antigen-binding fragment are less prone to mismatch, and mismatch between, for example, VH1-VL2, VH2-VL1, VH1-VH2, VL1-VL2 is significantly reduced compared to what would be if and the first and second antigen binding fragments were analogues of natural Fabs, for example, in the form of VH1-CH1, VL1-CL, VH2-CH1 and VL2-CL.

In some embodiments, the bispecific polypeptide complex provided herein, when expressed from a cell, has significantly fewer mismatches (e.g., at least 1, 2, 3, 4, 5 or more fewer mismatches) and /or significantly higher product yield (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or higher yield) than a reference molecule expressed under comparable conditions, where the reference molecule otherwise identical to the bispecific polypeptide complex except for the presence of native CH1 instead of C1 and native CL instead of C2.

Antigen-binding fragment including engineered SApha and SBeta

The first antigen-binding fragment provided herein comprises a first antibody heavy chain variable domain operably linked to a first T cell receptor (TCR) constant region and a first antibody light chain variable domain operably linked to a second TCR constant region, wherein the first constant the TCR region and the second TCR constant region are linked through at least one non-native interchain disulfide bond. The first antigen-binding fragment contains at least two polypeptide chains, each of which contains a variable domain derived from an antibody and a constant region derived from a TCR. Thus, the first antigen-binding fragment contains a heavy chain variable domain and a light chain variable domain that are operably linked to a pair of TCR constant regions, respectively. In some embodiments, the pair of TCR constant regions in the first antigen binding fragment are alpha/beta TCR constant regions. The TCR constant regions in the polypeptide complexes provided herein are able to bind to each other to form a dimer through at least one non-native disulfide bond.

Surprisingly, it has been found that the first antigen-binding fragment provided herein with at least one non-native disulfide bond can be recombinantly expressed and assembled into a desired conformation that stabilizes the TCR constant region dimer while providing good antigen-binding activity of the antibody variable regions. In addition, the first antigen-binding fragment has been found to allow for routine antibody engineering, such as modification of glycosylation sites and deletion of some natural sequences. In addition, the polypeptide complexes provided herein can be incorporated into a bispecific format that can be easily expressed and assembled with little or no antigen-binding mismatch due to the presence of TCR constant regions in the first antigen-binding fragment. Additional advantages of the first antigen binding fragment and constructs presented herein will become more apparent in the following description below.

Thus, the first antigen-binding fragment provided herein comprises a first polypeptide comprising, from the N-terminus to the C-terminus, the first heavy chain variable domain (VH) of the first antibody operably linked to the first constant region (C1) of a T cell receptor (TCR), and a second polypeptide containing, from the N-terminus to the C-terminus, the first light chain variable domain (VL) of the first antibody, operably linked to the second constant region of the TCR (C2), where: C1 contains an engineered CBeta containing SEQ ID NO: 1 and C2 contains engineered SApha comprising SEQ ID NO: 2, amino acid C48 of SEQ ID NO: 1 and amino acid C41 of SEQ ID NO: 2 are capable of forming a non-native interchain disulfide bond, C1 and C2 are capable of forming a dimer, and a non-native interchain disulfide bond between C1 and C2 is capable of stabilizing the dimer.

TCR constant region

The first antigen-binding fragment provided herein contains an alpha or beta constant region derived from a TCR.

Figures 3A and 3B show the amino acid sequences of the native TCR constant regions of the TCR alpha and beta chains. For clarity and consistency, each of the amino acid residues in these sequences is numbered in FIGS. 4A and 4B, and such numbering is used herein to refer to a particular amino acid residue in a particular TCR constant region.

The constant region of the human TCR alpha chain is known as TRAC with NCBI accession number P01848 or the amino acid sequence of SEQ ID NO: 31.

The human TCR beta chain constant region has two distinct variants known as TRBC1 and TRBC2 (IMGT nomenclature) (see also Toyonaga B, et al ., PNAs, Vol. 82, pp. 8624-8628, Immunology (1985)). The TRBC1 sequence (SEQ ID NO: 33) was chosen as the backbone for constructing the polypeptide complexes described herein.

In particular, the native TCR beta chain contains a native cysteine residue at position 74 (see Fig. 4B), which is unpaired and therefore does not form a disulfide bond in native TCR alpha/beta. In the polypeptide complexes presented herein, this native cysteine residue at position 74 of the TCR beta chain is mutated to an alanine residue. This can be used to eliminate intrastrand or interstrand mismatches. In some embodiments, the replacement, in some embodiments, may improve the efficiency of TCR refolding in vitro.

As used herein, the first and second TCR constant regions of the first antigen-binding fragment provided herein are capable of forming a dimer having at least one non-native interchain disulfide bond between the TCR constant regions that is capable of stabilizing the dimer.

As used herein, the term “dimer” refers to a bonded structure formed by two molecules, such as polypeptides or proteins, through covalent or non-covalent interactions. Homodimer or homodimerization occurs through two identical molecules, and heterodimer or heterodimerization occurs through two different molecules. The dimer formed by the first and second TCR constant regions is a heterodimer.

A "mutated" amino acid residue refers to a residue that is substituted, inserted, or added and differs from its native counterpart in the corresponding native TCR constant region. For example, if an amino acid residue at a particular position in the wild-type TCR constant region is referred to as a "native" residue, then its mutated counterpart is any residue that differs from the native residue but is at the same position in the TCR constant region. The mutated residue may be another residue that replaces the native residue in the same position, or that is inserted before the native residue and, accordingly, takes its original position.

In the polypeptide complexes provided herein, the first and/or second TCR constant regions have been designed to contain one or more mutated amino acid residues that are responsible for the formation of a non-native interchain disulfide bond. To introduce such a mutated residue into the TCR constant region, the TCR region coding sequence can be manipulated, for example, to change the codon encoding the native residue to the codon encoding the mutated residue, or to insert the codon encoding the mutated residue before the codon of the native residue.

In the polypeptide complexes provided herein, the first and/or second TCR constant regions were designed to contain one or more mutated cysteine residues such that, upon replacement with cysteine residues, a non-native interchain disulfide bond could be formed between the two TCR constant regions.

The non-native interchain disulfide bond is capable of stabilizing the first antigen-binding moiety. Such stabilization effects can be implemented in various ways. For example, the presence of a mutated amino acid residue or a non-native interchain disulfide bond may allow a polypeptide complex to be stably expressed and/or highly expressed and/or to be combined into a stable complex having the desired biological activity (e.g., antigen-binding activity) and/or to be expressed and assembled into a highly efficient the desired stable complex having the desired biological activity. The ability of an interchain disulfide bond to stabilize the first and second TCR constant regions can be assessed using appropriate methods known in the art, such as molecular weight determination by DNS-PAGE, or thermal stability determination by differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF) . In an illustrative example, the formation of a stable first antigen-binding fragment presented herein can be confirmed by DNS-PAGE if the product exhibits a molecular weight comparable to the combined molecular weight of the first and second polypeptides. In some embodiments, the first antigen-binding fragment provided herein is stable in the sense that its thermal stability is at least 50%, 60%, 70%, 80%, or 90% of that of the native Fab. In some embodiments, the first antigen-binding fragment provided herein is stable in the sense that its thermal stability is comparable to that of a natural Fab.

Without wishing to be bound by any theory, it is believed that the non-native interchain disulfide bond formed between the first and second TCR constant regions in the first antigen-binding fragment is able to stabilize the heterodimer of the TCR constant regions, thereby increasing the level of correct folding, structural stability and/or level expression of the heterodimer and the first antigen-binding fragment. In contrast to the native TCR, “anchored” on the T-cell surface membrane, the heterodimers of the extracellular domains of the native TCR turned out to be significantly less stable, despite their similarity to the Fab antibody in three-dimensional structure. In addition, the instability of native TCR in the soluble state was a significant obstacle to the determination of its crystal structure. (See Wang, Protein Cell, 5(9), pp. 649-652 (2014)). By introducing a pair of cysteine (Cys) mutations into the constant regions of the TCR and thereby allowing the formation of an interchain non-native disulfide bond, the first antigen-binding fragment can be stably expressed while maintaining the antigen-binding capabilities of the antibody variable region.

A TCR constant region containing a mutated residue is also referred to herein as a "constructed" TCR constant region. In some embodiments, the first TCR constant region (C1) of the polypeptide complex contains a TCR engineered alpha chain (CApha) and the second TCR constant region (C2) contains a TCR engineered beta chain (CBeta). In the polypeptide complexes provided herein, C1 includes engineered CBeta and C2 contains engineered SApha.

In the polypeptide complexes provided herein, the engineered TCR constant region contains one or more mutated cysteine residues within the contact region of the first and/or second engineered TCR constant regions.

As used herein, the term “contact area” refers to the specific area(s) on polypeptides where the polypeptides interact/bind with each other. The contact area includes one or more amino acid residues that are capable of interacting with the corresponding amino acid residue(s) that comes into contact or association when the interaction occurs. The amino acid residues in the contact region may or may not be in a continuous sequence. For example, when the contact area is three-dimensional, the amino acid residues within the contact area can be separated at different positions in the linear sequence.

In some embodiments, one or more disulfide bonds may be formed between engineered SApha and engineered SBeta. In some embodiments, the mutated cysteine residue in CBeta is S56C (corresponding to amino acid C48 in SEQ ID NO: 1) and the mutated cysteine residue in SApha is T49C (corresponding to amino acid C41 in SEQ ID NO: 2), and where the cysteine pair residues is capable of forming a non-native interchain disulfide bond.

As used throughout this document, the term "XnY", in relation to the TCR constant region, is intended to mean that the nth amino acid residue X in the TCR constant region (based on the numbering in FIGS. 4A and 4B provided herein) is replaced by the amino acid residue Y, where X and Y are, respectively, the one-letter abbreviation for the specific amino acid residue. It should be noted that the number n is based only on the numbering shown in FIGS. 4A and 4B and may differ from its actual position. By way of illustration, the CBeth sequence (S56C) (N69Q) shown in SEQ ID NO: 1 is used as an example. Although the change from S to C occurs at the 48th residue in SEQ ID NO: 1, the residue itself is designated as the 56th residue based on the numbering system in FIG. 4B, and hence this S to C substitution is designated S56C and not S48C. Similarly, replacing N with Q is also referred to as N69Q based on the numbering system in FIGS. 4A and 4B. This convention for naming amino acid residue substitutions applies to all TCR constant regions in this specification unless otherwise noted.

In the polypeptide complexes provided herein, engineered CBeta contains or is SEQ ID NO: 1 and engineered SApha contains or is SEQ ID NO: 2.

The amino acid sequences of SEQ ID NOs: 1 and 2 are shown below.

SEQ ID NO: 1

EVAVFEPSEAISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA

SEQ ID NO: 2

AVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDK C VLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS

In the peptide complexes provided herein, one or more of the native glycosylation sites present in the native TCR constant regions have been modified (eg, deleted) in the first antigen-binding fragment provided herein. As used herein, the term "glycosylation site", in relation to a polypeptide sequence, refers to an amino acid residue with a side chain to which a carbohydrate moiety (eg, an oligosaccharide structure) can be attached. Glycosylation of antibody-like polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an aspartic residue, such as an aspartic residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. O-linked glycosylation refers to the addition of one of the sugars (N-acetylgalactosamine, galactose, or xylose) to a hydroxyamino acid, usually serine or threonine. Removal of native glycosylation sites can conveniently be accomplished by altering the amino acid sequence such that one or more of the tripeptide sequences described above (for N-linked glycosylation sites) or one or more serine or threonine residues (for O-linked glycosylation sites) are replaced .

In the first antigen binding fragment provided herein, at least one native glycosylation site is absent from the engineered TCR constant regions, eg, the first and/or second TCR constant regions. Without wishing to be bound by any theory, it is believed that the first antigen-binding fragment provided herein may allow the removal of all or part of the glycosylation sites without affecting protein expression and stability, in contrast to current beliefs that the presence of N- associated glycosylation in the TCR constant region, e.g., SApha (i.e., N34, N68, and N79) and CBeta (i.e., N69), is required for protein expression and stability (see Wu et al., Mabs, 7:2, 364-376, 2015).

In the first antigen-binding fragment presented herein, the N-glycosylation sites in the engineered SApha at N34, N68 and N79 are absent. The engineered SApha sequence lacking a glycosylation site contains or corresponds to SEQ ID NO: 2. In the first antigen-binding fragment provided herein, the N-glycosylation site in engineered CBeta on N69 is missing. The constructed CBeta (TRBC1) sequence, which lacks a glycosylation site, contains or corresponds to SEQ ID NO: 1.

In the first antigen-binding fragment provided herein, TCR-derived constant regions are operably linked to antibody-derived variable regions.

In some embodiments, the first antibody variable domain (VH) is fused to the first TCR constant region (C1) at the first conjugation domain, and the first antibody variable domain (VL) is fused to the second TCR constant region (C2) at the second conjugation domain.

The term “conjugation domain” as used herein refers to a boundary or boundary region where two amino acid sequences are fused or combined. In some embodiments, the first conjugation domain contains at least a portion of the C-terminal fragment of the V/C conjugation of the antibody, and the second conjugation domain contains at least a portion of the N-terminal fragment of the V/C conjugation of the TCR.

As used herein, the term “V/C antibody conjugation” refers to the boundary between the variable domain and the constant domain of an antibody, such as the boundary between a heavy chain variable domain and a CH1 domain, or between a light chain variable domain and a light chain constant domain. Similarly, the term "V/C TCR conjugation" refers to the boundary between a TCR variable domain and a constant domain, for example, the boundary between a TCR alpha variable domain and a constant domain, or between a TCR beta variable domain and a constant domain.

In some embodiments, the first polypeptide contains a sequence containing domains operably linked such as, for example, in formula (I): VH-HCJ-C1, and the second polypeptide contains a sequence containing domains operably linked, such as, for example, in formula (II ): VL-LCJ-C2, where:

VH is the variable domain of an antibody heavy chain;

HCJ is the first conjugation domain as defined above;

C1 is the first TCR constant domain as defined above;

VL is an antibody light chain variable domain;

LCJ is the second conjugation domain as defined above;

C2 is the second constant domain of the TCR as defined above.

In such embodiments, C1 is engineered CBeta that contains or matches SEQ ID NO: 1 and C2 is engineered SApha that contains or matches SEQ ID NO: 2, HCJ contains or matches SEQ ID NO: 23, and LCJ contains or matches SEQ ID NO: 24.

Variable region of an antibody

The bispecific polypeptide complex provided herein contains a first antigen-binding fragment associated with a second antigen-binding fragment, and one of them specifically binds to CD3, while the other specifically binds to CD19. In the polypeptide complex presented herein, the first antigen-binding fragment contains the first heavy chain variable domain (VH1) and the first light chain variable domain (VL1) of the first antibody, and the second antigen-binding fragment contains the second heavy chain variable domain (VH2) and the second light chain variable domain. a chain (VL2) of a second antibody, wherein the first antibody and the second antibody are different and are selected from the group consisting of an anti-CD3 antibody and an anti-CD19 antibody. In some embodiments, the first antibody is an anti-CD3 antibody and the second antibody is an anti-CD19 antibody. In some other embodiments, the first antibody is an anti-CD19 antibody and the second antibody is an anti-CD3 antibody.

In a typical native antibody, the variable region contains three CDRs located between flanking framework regions (FRs), for example, as indicated in the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, from N-terminus to C-terminus .

a) Anti-CD3 binding fragment

In the polypeptide complex provided herein, the first antigen-binding fragment or the second antigen-binding fragment is an anti-CD3 binding fragment. In some embodiments, the anti-CD3 binding fragment is derived from the anti-CD3 antibody WBP3311_2.306.4-z1 shown in Table A below. The CDR sequences of the WBP3311_2.306.4-z1 antibody are shown below.

Table A Antibody ID: CDR1 CDR2 CDR3 WBP3311_2.306.4-z1 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 vh GFAFTDYYIH WISPGNVNTKYNENFKG DGYSLYYFDY WBP3311_2.306.4-z1 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 VK KSSQSLLNSRTRKNYLA WASTRQS TQSHTLRT

The variable region sequences of the heavy and kappa light chains of the WBP3311_2.306.4-z1 antibody are shown below.

WBP3311_2.306.4-z1-VH

Amino acid sequence (SEQ ID NO: 15):

Последовательность нуклеиновой кислоты (SEQ ID NO: 16):

Nucleic acid sequence (SEQ ID NO: 16):

CAGGTGCAGCTTGTGCAGTCTGGGGCAGAAGTGAAGAAGCCTGGGTCTAGTGTCAAGGTGTCATGCAAGGCTAGCGGGTTCGCCTTTACTGACTACTACATCCACTGGGTGCGGCAGGCTCCCGGACAAGGGTTGGAGTGGATGGGATGGATCTCCCCAGGCAATGTCAACACAAAGTACAACGAGAACTTCAAAGGCCGCGTCACCATTACCGCCGACAAGAGCACCTCCACAGCCTACATGGAGCTGTCCAGCCTCAGAAGCGAGGACACTGCCGTCTACTACTGTGCCAGGGATGGGTACTCCCTGTATTACTTTGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC

WBP3311_2.306.4-z1-VK

Amino acid sequence (SEQ ID NO: 17):

Nucleic acid sequence (SEQ ID NO: 18):

GATATCGTGATGACCCAGAGCCCAGACTCCCTTGCTGTCTCCCTCGGCGAAAGAGCAACCATCAACTGCAAGAGCTCCCAAAGCCTGCTGAACTCCAGGACCAGGAAGAATTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTCATCTACTGGGCCTCCACCCGGCAGTCTGGGGTGCCCGATCGGTTTAGTGGATCTGGGAGCGGGACAGACTTCACATTGACAATTAGCTCACTGCAGGCCGAGGACGTGGCCGTCTACTACTGTACTCAGAGCCACACTCTCCGCACATTCGGCGGAGGGACTAAAGTGGAGATTAAG

CDRs are known to be responsible for antigen binding.

In some embodiments, the anti-CD3 binding fragment provided herein contains the heavy chain CDR3 sequence of WBP3311_2.306.4-z1. In some embodiments, the anti-CD3 binding fragment provided herein comprises a heavy chain CDR3 comprising SEQ ID NO: 5. contact with the antigen and provide the greatest free energy for the affinity of the antibody for the antigen. It is also believed that the heavy chain CDR3 is by far the most diverse antigen binding site CDR in terms of length, amino acid composition and conformation through various diversification mechanisms. (Tonegawa S., Nature. 302:575-81 (1983)). The diversity in the heavy chain CDR3 is sufficient to obtain greater antibody specificity (Xu JL, Davis MM., Immunity. 13: 37-45 (2000)), as well as the desired antigen binding affinity. (Schier R, et al., J Mol Biol. 263:551-67 (1996)).

The anti-CD3 binding fragment provided herein further comprises suitable framework region (FR) sequences, provided that the anti-CD3 binding fragment can specifically bind to CD3.

The humanized anti-CD3 antibody WBP3311_2.306.4-z1 has a specific binding affinity for a CD3 expressing cell (eg, a CD4 T cell) and can activate human T cells and trigger the release of the cytokines TNF-alpha and IFN-gamma.

In some embodiments, the anti-CD3 binding fragment provided herein comprises a heavy chain variable domain sequence of SEQ ID NO: 15 and a light chain variable domain sequence of SEQ ID NO: 17.

The binding affinity of an anti-CD3 binding moiety provided herein can be represented by a K _D value, which is the ratio of dissociation rate to association rate (k _off /k _on ) when binding between an antigen and an antigen-binding molecule reaches equilibrium. Antigen binding affinity (eg, K _D ) may be appropriately determined using suitable methods known in the art, including, for example, flow cytometry. In some embodiments, the binding of an antibody to an antigen at various concentrations can be determined by flow cytometry, the determined mean fluorescence intensity (MFI) can first be plotted against the concentration of the antibody, then the K _D value can be calculated by fitting the intensity dependence of the specific binding fluorescence (Y) and antibody concentration (X) in a single site saturation equation: Y=B _max *X/(KD+X) using Prism software version 5 (GraphPad Software, San Diego, CA) where B _max refers to the maximum specific binding of the test antibody to the antigen.

In some embodiments, the anti-CD3 binding fragment provided herein is capable of specifically binding to cell surface expressed human CD3 or recombinant human CD3. CD3 is a receptor expressed on the cell. Recombinant CD3 is soluble CD3 that is expressed recombinantly and is not bound to the cell membrane. Recombinant CD3 can be obtained through various recombinant technologies. In one example, a CD3 DNA sequence encoding the extracellular domain of human CD3 (NP_000724.1) (Met1-Asp126) can be fused to a C-terminal polyhistidine tag in an expression vector, and then transfected and expressed in 293E cells and purified with using Ni-affinity chromatography.

In some embodiments, the anti-CD3 binding fragment provided herein is capable of specifically binding to human CD3 expressed on the cell surface with a binding affinity (K _D ) of no more than 5×10 ^-9 M, no more than 4×10 ^-9 M, no more than 3×10 ^-9 M, no more than 2×10 ^-9 M, no more than 10 ^-9 M, no more than 5×10 ^-10 M, no more than 4×10 ^-10 M, no more than 3 ×10 ^-10 M, not more than 2×10 ^-10 M, not more than 10 ^-10 M, not more than 5×10 ^-11 M or not more than 4×10 ^-11 M, not more than 3×10 ^-11 M or not more than 2×10 ^-11 M or not more than 10 ^-11 M, as determined by flow cytometry.

In some embodiments, the anti-CD3 binding fragment provided herein cross-reacts with cynomolgus monkey CD3, for example cell surface expressed cynomolgus monkey CD3 or with soluble recombinant cynomolgus monkey CD3.

The binding of an anti-CD3 binding fragment to recombinant CD3 or CD3 expressed on the cell surface can be measured by methods known in the art, for example, by sandwich assays such as ELISA, Western blotting, flow cytometric assays, and other binding assays. . In some embodiments, an anti-CD3 binding fragment provided herein specifically binds to recombinant human CD3 at an EC ₅₀ value (i.e., 50% binding concentration) of not more than 0.01 nM, not more than 0.02 nM , not more than 0.03 nM, not more than 0.04 nM, not more than 0.05 nM, not more than 0.06 nM, not more than 0.07 nM and not more than 0.08 nM, determined by ELISA. In some embodiments, the anti-CD3 binding fragment provided herein specifically binds to human CD3 expressed on the cell surface at an EC ₅₀ value of not more than 0.5 nM, not more than 0.6 nM, not more than 0.7 nM, max 0.8 nM, max 0.9 nM, max 1 nM, max 2 nM, max 3 nM, max 4 nM, max 5 nM, max 6 nM, max 7 nM not more than 8 nM, not more than 9 nM, or not more than 10 nM, determined by flow cytometry.

In some embodiments, the anti-CD3 binding fragment binds to cynomolgus monkey CD3 with a binding affinity similar to that of human CD3. For example, typical antibody WBP3311_2.306.4 binds to cynomolgus monkey CD3 at an affinity or EC ₅₀ value similar to that of human CD3.

In some embodiments, an anti-CD3 binding fragment provided herein specifically binds to recombinant cynomolgus monkey CD3 at an EC ₅₀ value of not more than 0.001 nM, not more than 0.005 nM, not more than 0.01 nM, not more than 0.02 nM, not more than 0.03 nM, not more than 0.04 nM, or not more than 0.05 nM, determined by ELISA.

In some embodiments, the anti-CD3 binding fragment provided herein has a specific binding affinity for human CD3 that is sufficient to provide diagnostic and/or therapeutic utility. A number of therapeutic strategies modulate T-cell immunity by targeting TCR signaling, in particular anti-human CD3 monoclonal antibodies that are in clinical use.

b) Anti-CD19 antibody

In the polypeptide complex provided herein, the first antigen-binding fragment or the second antigen-binding fragment is an anti-CD19-binding fragment. In some embodiments, the anti-CD19 binding fragment is derived from the anti-CD19 antibody W7011-4.155.8-z1-P15 shown in Table B below. The CDR sequences of an anti-CD19 antibody are shown below.

Table B CDR1 CDR2 CDR3 W7011-4.155.8-z1-P15 vh SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 11 GYAFTSYNMY YIDPYN A DTTYNQKFKG TAYAMDY W7011-4.155.8-z1-P15 VK SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 SASSTVNYMH STSNLAS HQWSSYPYT

The variable region sequences of the heavy and kappa light chains of the WBP7011_4.155.8-z1-P15 antibody are shown below.

W7011-4.155.8-z1-P15-VH

Amino acid sequence (SEQ ID NO: 19):

Nucleic acid sequence (SEQ ID NO: 20):

CAAATGCAGCTCGTCCAGTCTGGACCTGAAGTGAAGAAGCCCGGGACATCCGTCAAGGTCTCATGTAAGGCTAGCGGGTACGCATTCACTTCCTACAACATGTACTGGGTGCGCCAGGCCAGAGGACAGAGGTTGGAGTGGATCGGCTACATCGACCCATACAACGCCGATACTACCTACAATCAGAAGTTTAAAGGGCGGGTGACCATTACCCGGGATATGTCCACCTCCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACAGCCGTGTACTACTGCCTGACAACAGCCTATGCCATGGACTATTGGGGCCAGGGCACACTTGTGACTGTGAGCAGT

W7011-4.155.8-z1-P15-VK

Amino acid sequence (SEQ ID NO: 21):

Nucleic acid sequence (SEQ ID NO: 22):

GACATCCAGCTCACCCAATCCCCTTCTTTCCTCTCCGCAAGTGTCGGAGATAGGGTGACTATCACCTGCTCAGCTTCTTCAACCGTGAACTACATGCATTGGTACCAGCAGAAGCCCGGGAAAGCCCCAAAGCTGCTGATCTACAGCACCTCCAATCTGGCCAGTGGAGTGCCAAGCCGGTTTAGCGGGAGCGGCTCCGGCACTGAATTCACTTTGACAATTAGCAGCCTTCAGCCTGAGGACTTTGCCACATATTACTGTCACCAGTGGTCCAGCTACCCCTACACATTCGGGCAGGGCACAAAGCTGGAGATTAAG

CDRs are known to be responsible for antigen binding.

In some embodiments, the anti-CD19 binding fragment described herein comprises the heavy chain CDR3 sequence of the anti-CD19 antibody W7011-4.155.8-z1-P15. In some embodiments, the anti-CD19 binding fragment provided herein comprises a heavy chain CDR3 sequence comprising SEQ ID NO: 11. degrees are in contact with the antigen and provide the highest free energy for the affinity of the antibody for the antigen. It is also believed that the heavy chain CDR3 is by far the most diverse antigen binding site CDR in terms of length, amino acid composition and conformation through various diversification mechanisms. (Tonegawa S., Nature. 302:575-81 (1983)). Diversity in the heavy chain CDR3 is sufficient to obtain greater antibody specificity (Xu JL, Davis MM. Immunity. 13: 37-45 (2000)), as well as the desired antigen-binding affinity (Schier R, et al., J Mol Biol. 263: 551-67 (1996)).

The anti-CD19 binding fragment provided herein further comprises suitable framework region (FR) sequences, provided that the anti-CD19 binding fragment can specifically bind to CD19.

In some embodiments, the anti-CD19 binding fragment provided herein comprises a heavy chain variable domain sequence of SEQ ID NO: 19 and a light chain variable domain sequence of SEQ ID NO: 21.

In some embodiments, an anti-CD19 binding fragment provided herein is capable of specifically binding to human CD19 expressed on cell surfaces with a binding affinity (KD) of no more than 5x10 ^-9 M, no more than ^{1x10 -9} M, no. more than 9×10 ^-10 M, not more than 8×10 ^-10 M, not more than 7×10 ^-10 M, not more than 6×10 ^-10 M, not more than 5×10 ^-10 M, not more than 4×10 ^{- 10} M, not more than 3×10 ^-10 M, not more than 2×10 ^-10 M or not more than 1×10 ^-10 M, determined by flow cytometry.

In some embodiments, the anti-CD19 binding fragment provided herein cross-reacts with cynomolgus monkey CD19, for example cell surface expressed cynomolgus monkey CD19 or soluble recombinant cynomolgus monkey CD19.

Binding of an anti-CD19 binding fragment to cell-expressed CD19 can be measured by methods known in the art, for example, by sandwich assay such as ELISA, Western blotting, flow cytometry analysis, and other binding assays. In some embodiments, an anti-CD19 binding fragment provided herein specifically binds to human CD19 expressed in a cell at an EC ₅₀ value of not more than 0.01 nM, not more than 0.02 nM, not more than 0.03 nM , not more than 0.04 nM, not more than 0.05 nM, not more than 0.1 nM, not more than 0.2 nM, not more than 0.3 nM, not more than 0.4 nM, not more than 0.5 nM, not more than 0.5 nM, not more than 0.6 nM, not more than 0.7 nM, not more than 0.8 nM, not more than 0.9 nM, or not more than 1 nM, determined by flow cytometry.

In some embodiments, the anti-CD19 binding fragment binds to cynomolgus monkey CD19 with a binding affinity similar to that of human CD19. In some embodiments, an anti-CD19 binding fragment provided herein specifically binds to cynomolgus monkey CD19 expressed on a cell at an EC ₅₀ value of not more than 0.2 nM, not more than 0.5 nM, not more than 0, 8 nM, 1 nM max, 2 nM max, or 3 nM max, determined by flow cytometry.

In some embodiments, an anti-CD19 binding fragment provided herein is internalized by a CD19 expressing cell at an EC ₅₀ value of not more than 1 pM, not more than 2 pM, not more than 3 pM, not more than 4 pM. no more than 5 pm, no more than 6 pm, no more than 7 pm, no more than 8 pm, no more than 9 pm, no more than 10 pm, no more than 11 pm, no more than 12 pm, no more than 13 pm, no more than 14 pm, no more than 15 pm, no more than 16 pm, no more than 17 pm, no more than 18 pm, no more than 19 pm, no more than 20 pm, no more than 21 pm, no more than 22 pm, no more than 23 pm, no more than 24 pm, 25 pm max, 30 pm max, 35 pm max, 40 pm max, 45 pm max, or 50 pm max, as determined by Fab-Zap analysis.

Bispecific polypeptide complex

In some embodiments, the first and/or second antigen binding fragment is multivalent, eg divalent, trivalent, tetravalent. As used herein, the term "valence" refers to the presence of a certain number of antigen binding sites in a given molecule. As such, the terms "bivalent", "tetravalent" and "hexavalent" denote the presence of two antigen-binding molecules, respectively, two binding sites, four binding sites and six binding sites, respectively. A divalent molecule may be monospecific if both binding sites are designed to specifically bind the same antigen or the same epitope. Similarly, a trivalent molecule can be bispecific, for example, when two binding sites are monospecific for the first antigen (or epitope) and the third binding site is specific for the second antigen (or epitope). In some embodiments, the first and/or second antigen-binding fragment in the bispecific polypeptide complex provided herein may be divalent, trivalent, or tetravalent, with at least two binding sites specific for the same antigen or epitope. This, in some embodiments, provides stronger binding to the antigen or epitope than the monovalent counterpart. In some embodiments, in a bivalent antigen-binding fragment, the first binding site and the second binding site are structurally identical (i.e., have the same sequences) or structurally different (i.e., have different sequences, albeit with the same specificity).

In some embodiments, the first and/or second antigen-binding fragment is multivalent and contains two or more antigen-binding sites operably linked together, with or without a spacer.

In some embodiments, the second antigen binding fragment contains two or more Fabs of the second antibody. The two Fabs may be operably linked to each other, for example, the first Fab may be covalently attached to the second Fab via a heavy chain with or without a spacer in between.

In some embodiments, the first antigen-binding fragment is associated with the first dimerization domain and the second antigen-binding fragment is associated with the second dimerization domain. As used herein, the term “dimerization domain” refers to a peptide domain that is capable of associating with another peptide domain to form a dimer or, in some examples, allows two peptides to spontaneously dimerize.

In some embodiments, the first dimerization domain may be associated with a second dimerization domain. The association can occur through any suitable interaction or bond or connection, for example, through a linker, disulfide bond, hydrogen bond, electrostatic interaction, salt bridge or hydrophobic-hydrophilic interaction, or a combination thereof. Exemplary dimerization domains include, without limitation, an antibody hinge region, an antibody CH2 domain, an antibody CH3 domain, and other suitable protein monomers capable of dimerizing and binding to each other. The hinge region, CH2 and/or CH3 domain can be derived from any antibody isotype, such as IgG1, IgG2 and IgG4.

The term "disulfide bond" refers to a covalent bond with an R-S-S-R' structure. The amino acid cysteine contains a thiol group which can form a disulfide bond with a second thiol group, for example from another cysteine residue. A disulfide bond can be formed between the thiol groups of two cysteine residues located respectively on two polypeptide chains, thereby forming an interchain bridge or an interchain bond.

A hydrogen bond is formed by electrostatic attraction between two polar groups when a hydrogen atom is covalently bonded to a highly electronegative atom such as nitrogen, oxygen, or fluorine. A hydrogen bond can form in a polypeptide between backbone oxygens (e.g., chalcogen groups) and amide hydrogens (nitrogen groups) of two residues, respectively, such as a nitrogen group in Asn and an oxygen group in His, or an oxygen group in Asn and a nitrogen group. in Lys. The hydrogen bond is stronger than the van der Waals force but weaker than covalent or ionic bonds and is critical for maintaining secondary structure and tertiary structure. For example, an alpha helix is formed with a regular spacing between amino acid residues at positions i and i+4, and a beta sheet is a 3-10 amino acid stretch of peptide chain formed when two peptides are joined by at least two or three backbones. hydrogen bonds to form a twisted folded sheet.

Electrostatic interaction is a non-covalent interaction and is important for protein folding, stability, plasticity, and function, including ionic interactions, hydrogen bonding, and halogen bonding. Electrostatic interactions can form within a polypeptide, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp in the first strand and Arg, Val or Thr in the second strand.

Salt bridges are short-range electrostatic interactions that mainly arise from the anionic carboxylate Asp or Glu, and the cationic ammonium from Lys or guanidinium Arg, which are spatially close pairs of oppositely charged residues in native protein structures. Charged and polar residues at mostly hydrophobic interfaces can act as "hot spots" for binding. Among others, residues with ionizable side chains, such as His, Tyr and Ser, can also participate in the formation of a salt bridge.

A hydrophobic interaction can occur between one or more Val, Tyr and Ala in the first strand and one or more Val, Leu and Trp in the second strand or His and Ala in the first strand and Thr and Phe in the second strand (see Brinkmann, et al, 2017, supra ).

In some embodiments, the first and/or second dimerization domain comprises at least a portion of an antibody hinge region. In some embodiments, the first and/or second dimerization domain may further comprise an antibody CH2 domain and/or an antibody CH3 domain. In some embodiments, the first and/or second dimerization domain contains at least a portion of the Hinge-Fc region, i.e. Hinge-CH2-CH3 domain. In some embodiments, the first dimerization domain may be operably linked to the C-terminus of the first TCR constant region. In some embodiments, the second dimerization domain may be operably linked to the C-terminus of the CH1 constant region of the antibody of the second antigen-binding fragment.

In the polypeptide complex provided herein, C1 contains engineered CBeta and the first dimerization domain is operably linked to engineered CBeta on a third conjugation domain that contains SEQ ID NO: 25.

In the polypeptide complex provided herein, the first dimerization domain is operably linked to the C-terminus of the engineered TCR constant region and together forms the chimeric constant region. In other words, the chimeric constant region contains a first dimerization domain operably linked to the engineered TCR constant region.

In some embodiments, the chimeric constant region comprises an engineered CBeta fused to a first hinge-Fc region derived from IgG1, IgG2, or IgG4. Exemplary sequences of such a chimeric constant region are shown in Example 2 .

In some embodiments, the chimeric constant region further comprises an antibody first CH2 domain and/or an antibody first CH3 domain. For example, the chimeric constant region further comprises a first antibody CH2-CH3 domain fused to the C-terminus of a third conjugation domain. Illustrative sequences for such a chimeric constant region are shown in Example 2 .

These pairs of chimeric constant regions and second TCR constant domains are practical in that they can be manipulated to fuse with the desired variable region of an antibody to provide a polypeptide complex as described herein. For example, an antibody heavy chain variable region can be fused to a chimeric constant region (containing C1), thus providing the first polypeptide chain of the polypeptide complex provided herein; and similarly, the light chain variable region of an antibody can be fused to a second TCR constant domain (containing C2), thus providing a second polypeptide chain of the polypeptide complex provided herein.

In some embodiments, the second dimerization domain comprises a hinge region. The hinge region can be derived from an antibody such as IgG1, IgG2, or IgG4. In some embodiments, the second dimerization domain may optionally further comprise an antibody CH2 domain and/or an antibody CH3 domain, such as, for example, a hinge-Fc region. The hinge region can be attached to the heavy chain of an antibody of a second antigen binding site (eg, Fab).

In a bispecific polypeptide complex, the first and second dimerization domains are capable of associating into a dimer. In some embodiments, the first and second dimerization domains are different and associate and associate in a manner that prevents homodimerization and/or promotes heterodimerization. For example, the first and second dimerization domains can be chosen such that they are not identical and that they preferentially form heterodimers with each other rather than form homodimers within their type. In some embodiments, the first and second dimerization domains are capable of associating into heterodimers via ridge-to-recess formation, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased ductility.

In some embodiments, the first and second dimerization domains contain CH2 and/or CH3 domains that are respectively mutated to allow for the formation of a ridge-in-recess. An overhang can be obtained by replacing a small amino acid residue with a larger one in the first CH2/CH3 polypeptide, and a recess can be obtained by replacing a large residue with a smaller one. See Ridgway et al., 1996 supra for more details on the pit mutation sites., Spiess et al., 2015 supra and Brinkmann et al., 2017 higher.

Bispecific format

In the polypeptide complex provided herein, the first antigen-binding fragment and the second binding fragment are associated in an Ig-like structure. The Ig-like structure is similar to a natural antibody having a Y-shaped design, with two "arms" for antigen binding and one "stem" for association and stabilization. Similarity to a natural antibody can provide various advantages such as good in vivo pharmacokinetics, desired immunological response, stability, and so on. An Ig-like structure comprising a first antigen-binding fragment provided herein, coupled to a second antigen-binding fragment provided herein, has been found to have thermal stability comparable to that of an Ig (eg, IgG). In some embodiments, the Ig-like structure provided herein is at least 70%, 80%, 90%, 95%, or 100% of the natural IgG structure.

The bispecific polypeptide complex provided herein comprises four polypeptide chains: i) VH1-C1-hinge-CH2-CH3; ii) VL1-C2; iii) VH2-CH1-hinge-CH2-CH3 and iv) VL2-CL, wherein C1 and C2 are capable of forming a dimer containing at least one non-native interchain bond and two hinge regions and/or two CH3 domains are capable of forming one or more interchain bonds that can promote dimerization.

The bispecific polypeptide complexes described herein have a longer in vivo half-life and are relatively easier to manufacture when compared to bispecific polypeptide complexes in other formats.

Sequences of Bispecific Complexes

In some embodiments, the first antigen-binding fragment of the bispecific complex is capable of specifically binding to CD3 and the second antigen-binding fragment is capable of specifically binding to CD19. In other embodiments, the first antigen-binding fragment of the bispecific complex is capable of specifically binding to CD19 and the second antigen-binding fragment is capable of specifically binding to CD3.

In some embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29 (E17) as shown in Example 2 . In some embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 30 (F16) as shown in Example 2 . In such embodiments, the first antigen-binding fragment binds to CD3 and the second antigen-binding fragment binds to CD19. Construct E17 is a bispecific bivalent antibody, and construct F16 is a bispecific and trivalent antigen binding complex with two Fab repeats of an anti-CD19 antibody.

In some embodiments, the bispecific polypeptide complex comprises four polypeptide chains, including: i) VH1 operably linked to the first chimeric constant region; ii) VL1 operably linked to the second chimeric constant region; iii) VH2 operably linked to a light chain constant region of a conventional antibody, and iv) VL2 operably linked to a light chain constant region of a standard antibody. In some embodiments, the first chimeric constant region may comprise a C1-hinge-CH2-CH3, where each is as defined above. In some embodiments, the implementation, the second chimeric constant region may contain C2, as defined above. In some embodiments, the heavy chain constant region of a reference antibody may comprise CH1-hinge-CH2-CH3, where each is as defined above. In some embodiments, the light chain constant region of a standard antibody may comprise CL as defined above.

How to obtain

The present invention relates to isolated nucleic acids or polynucleotides that encode a polypeptide complex and to a bispecific anti-CD3×CD19 polypeptide complex provided herein.

As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single or double stranded form. Unless specifically limited, the term encompasses polynucleotides, including known analogs of naturally occurring nucleotides, that have similar binding properties as a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly includes its conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly stated sequence. In particular, degenerate codon substitutions can be generated by generating sequences in which the third position at one or more selected (or all) codons has been replaced with a mixed base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

Nucleic acids or polynucleotides encoding a polypeptide complex and a bispecific polypeptide complex provided herein can be constructed using recombination techniques. To this end, DNA encoding an antigen-binding fragment of a parent antibody (such as a CDR or variable region) can be isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody). ). Similarly, DNA encoding the TCR constant region can be obtained. By way of example, a polynucleotide sequence encoding a variable domain (VH) and a polynucleotide sequence encoding a first TCR constant region (C1) are generated and operably linked to allow transcription and expression in a host cell to produce the first polypeptide. Similarly, the polynucleotide sequence encoding VL is operably linked to the polynucleotide sequence encoding C1 to allow expression of the second polypeptide in the host cell. Optionally, the polynucleotide coding sequences for one or more spacers are also operably linked to other coding sequences to allow expression of the desired product.

The coding polynucleotide sequences may further be operably linked to one or more regulatory sequences, optionally in an expression vector, such that expression or production of the first and second polypeptides is feasible and under appropriate control.

The coding polynucleotide sequence(s) can be inserted into a vector for further cloning (DNA amplification) or for expression using recombination techniques known in the art. In another embodiment, the polypeptide complex and the bispecific polypeptide complex provided herein can be obtained by homologous recombination known in the art. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (eg, SV40, CMV, EF-1α), and a transcription termination sequence.

As used herein, the term “vector” refers to a carrier into which a polynucleotide encoding a protein can be operably inserted to cause expression of that protein. Typically, the construct also includes appropriate regulatory sequences. For example, a polynucleotide molecule may include regulatory sequences located in the 5' flanking region of a nucleotide sequence encoding a guide RNA and/or a nucleotide sequence encoding a site-directed modifying polypeptide operably linked to the coding sequences in a manner that allows expression of the desired transcript/gene in host cell. The vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic element it carries in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1 bacteriophage artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and viruses. animals. Categories of animal viruses used as vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (eg, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (eg, SV40). The vector may contain a variety of expression control elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. The vector may also include substances that facilitate its entry into the cell, including, but not limited to, a viral particle, a liposome, or a protein coat.

In some embodiments, the vector system includes mammalian systems, bacterial systems, yeast systems, etc., and contains plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2 and etc. and commercially available vectors. Suitable vectors may include plasmid or viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

Vectors containing the polynucleotide sequence(s) provided herein can be introduced into a host cell for gene cloning or expression. As used herein, the phrase “host cell” refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.

Suitable host cells for cloning or expression of DNA in the vectors of the present invention are prokaryotic, yeast or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria such as gram negative or gram positive organisms e.g. Enterobacteriaceae such as Escherichia e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella e.g. Salmonella typhimurium, Serratia e.g. Serratia marcescans and Shigella as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa and Streptomyces.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding a polypeptide complex and a bispecific polypeptide complex. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and strains are generally available and applicable in this document, for example, host organisms Schizosaccharomyces pombe; Kluyveromyces such as, for example, K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906) , K. Thermotolerans and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium and Aspergillus such as A. nidulans and A. niger.

Suitable host cells for the expression of the glycosylated polypeptide complex and the bispecific polypeptide complex provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants and corresponding permissive insect host cells have been identified from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. A variety of virus strains for transfection are commonly available, such as Autographa californica NPV variant L-1 and Bombyx mori NPV strain Bm-5, and such viruses can be used as a virus in accordance with the present invention, in particular for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

However, vertebrate cells are of the greatest interest, and propagation of vertebrate cells in culture (tissue culture) has become a common procedure. Examples of useful mammalian host cell lines are monkey kidney line CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), eg Expi293; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2).

Host cells transformed with the expression or cloning vectors described above can be cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify cloning vectors.

To produce the polypeptide complex and the bispecific polypeptide complex provided herein, host cells transformed with an expression vector can be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimal Culture Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing host cells. In addition, any of the carriers described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980) US Pat. No. 4,767,704; 4657866; 4927762; 4560655; or 5122469; W090/03430; WO 87/00195; or US patent RE 30985, can be used as a culture medium for host cells. Any of these media may, if necessary, be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the preparation GENTAMYCIN ^TM ), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary additives may also be included at appropriate concentrations that are known to those skilled in the art. Culture conditions such as temperature, pH, and the like are those previously used with the host cell selected for expression and will be apparent to one skilled in the art.

In one aspect, the present invention relates to a method for expressing a polypeptide complex and a bispecific polypeptide complex provided herein, comprising culturing a host cell provided herein under conditions under which the polypeptide complex or the bispecific polypeptide complex is expressed.

In some embodiments, the present invention provides a method for producing a bispecific polypeptide complex provided herein, comprising a) introducing into a host cell: one or more polynucleotides encoding a first antigen-binding fragment, comprising a first polynucleotide encoding a first polypeptide containing from N- C-terminally, the first heavy chain variable domain (VH) of the first antibody operably linked to the first TCR constant region (C1), the second polynucleotide encoding the second polypeptide, N-terminally to C-terminally, the first light chain variable domain (VL ) a first antibody operably linked to a second TCR constant region (C2), and one or more additional polynucleotides encoding a second antigen-binding fragment, where: C1 contains an engineered CBeta containing SEQ ID NO: 1 and C2 contains an engineered SApha containing SEQ ID NO: 2, amino acid C48 in SEQ ID NO: 1 and amino acid C 41 in SEQ ID NO: 2 are capable of forming a non-native interchain disulfide bond, C1 and C2 are capable of forming a dimer, and the non-native interchain disulfide bond is capable of stabilizing the C1 and C2 dimer, the first antigen-binding fragment and the second antigen-binding fragment are less likely to be mismatched than if and the first and second antigen binding fragments would be natural Fab analogs and the first antibody has a first antigenic specificity and the second antibody has a second antigenic specificity, b) allowing the host cell to express the bispecific polypeptide complex.

In some embodiments, the method further comprises isolating the bispecific polypeptide complex.

Using recombination techniques, the bispecific polypeptide complex provided herein can be produced intracellularly, in the periplasmic space, or directly secreted into the environment. If the product is produced intracellularly, in the first step, particulate matter, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describes a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cellular debris can be removed by centrifugation. When the product is secreted into the medium, supernatants from such expression systems are typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of incidental contaminants.

The bispecific polypeptide complex provided herein, obtained from cells, can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, wherein the affinity chromatography is the preferred purification method.

When the bispecific polypeptide complex provided herein contains an immunoglobulin Fc domain, then Protein A can be used as an affinity ligand depending on the species and isotype of the Fc domain that is present in the polypeptide complex. Protein A can be used to purify polypeptide complexes based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as pore size glass or polystyrenedivinylbenzene provide higher flow rates and shorter processing times than can be achieved with agarose.

When the bispecific polypeptide complex provided herein contains a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) can be used for purification. Other protein purification methods such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin chromatography, SEPHAROSE ^TM anion or cation exchange resin chromatography (e.g. polyaspartic acid column), chromatofocusing, DNS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

After any pre-purification step(s), the mixture containing the polypeptide complex of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably performed at low salt concentrations (for example, from 0 to 0.25 M salt).

In some embodiments, the bispecific polypeptide complex provided herein can be readily purified in high yields using conventional methods. One of the advantages of a bispecific polypeptide complex is a significant reduction in mismatch between heavy chain and light chain variable domain sequences. This reduces the production of undesirable by-products and allows a high purity product to be obtained in high yield using relatively simple purification methods.

Derivatives

In some embodiments, a bispecific polypeptide complex can be used as a basis for conjugation with the desired conjugates.

It is contemplated that various conjugates may be associated with the polypeptide complex or bispecific polypeptide complex provided herein (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates can be linked to a polypeptide complex or a bispecific polypeptide complex by, among other means, covalent linkage, affinity linkage, intercalation, coordinate linkage, complexation, association, mixing, or addition.

In some embodiments, the bispecific polypeptide complex provided herein can be designed to contain specific sites outside of the epitope-binding portion that can be used to bind to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as, for example, cysteine residues or histidine residues, to facilitate covalent bonding with the conjugate.

In some embodiments, the bispecific polypeptide complex may be linked to the conjugate directly or indirectly, such as through another conjugate or through a linker.

For example, a bispecific polypeptide complex having a reactive residue such as a cysteine can be linked to a thiol-reactive agent in which the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide, or another thiol-reactive conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem 1:2 Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

In another example, the bispecific polypeptide complex can be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin. In yet another example, a polypeptide complex or a bispecific polypeptide complex can be linked to a linker that further binds to the conjugate. Examples of linkers include bifunctional linkers such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-( p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and hys-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 (1978)) and N-succinimidyl-4-(2 -pyridylthio)pentanoate (SPP) to provide a disulfide bond.

The conjugate may be a detectable label, a pharmacokinetically modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of a detectable label may include fluorescent labels (eg, fluorescein, rhodamine, dansil, phycoerythrin, or Texas red), enzyme-substrate labels (eg, horseradish peroxidase, alkaline phosphatase, luceriferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes (for example, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, ¹¹¹ In, ¹¹² In, ¹⁴ C, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ²¹¹ At, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi and ³² P, other lanthanides, luminescent labels), chromophore fragment, digoxigenin, biotin/avidin, DNA molecule or gold for detection. In some embodiments, the conjugate may be a pharmacokinetically modifying moiety, such as PEG, that enhances the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol-propylene glycol copolymers, and the like. In some embodiments, the conjugate may be a cleansing moiety, such as a magnetic microcarrier. A “cytotoxic moiety” can be any agent that is detrimental to cells or that can damage or cause cell death. Examples of a cytotoxic moiety include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromannite, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum(II) (DDP), cisplatin, anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg. , dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agents (eg vincristine and vinblastine).

Methods for conjugating conjugates to proteins such as antibodies, immunoglobulins or fragments thereof are described, for example, in US Pat. No. 5,208,020; US Pat. No. 6,441,163; WO2005037992; WO2005081711; and WO2006/034488, which are incorporated herein by reference in their entirety.

pharmaceutical composition

The present invention also relates to a pharmaceutical composition containing the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable” indicates that said carrier, excipient, diluent, excipient(s) and/or salt are generally chemically and/or physically compatible with the other ingredients making up the composition and physiologically compatible with its recipient.

The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is biologically active and non-toxic to the subject. Pharmaceutically acceptable carriers for use in the pharmaceutical compositions described herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, isolating agents. or chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavors, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylhydroxytoluene, and/or propyl gallate. As described herein, the inclusion of one or more antioxidants, such as methionine, in a pharmaceutical composition provided herein reduces the oxidation of a polypeptide complex or a bispecific polypeptide complex. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving protein stability and maximizing shelf life. Therefore, in certain embodiments, compositions are provided that include the polypeptide complex or bispecific polypeptide complex described herein and one or more antioxidants, such as methionine.

To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as saline for injection, Ringer's solution for injection, isotonic dextrose for injection, sterile water for injection, or dextrose and lactate Ringer's solution for injection, non-aqueous vehicles such as fixed vegetable oils, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as bisulfate sodium, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycoltetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers may be added to pharmaceutical compositions in multiple dose packs that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, p-hydroxybenzoic acid methyl and propyl esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic excipients may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolaminoleate, or cyclodextrin.

Pharmaceutical compositions may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral preparations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

In some embodiments, the pharmaceutical compositions are provided as an injectable composition. Injectable pharmaceutical compositions may be prepared in any conventional form, such as, for example, liquid solution, suspension, emulsion, or solid forms suitable for preparing a liquid solution, suspension, or emulsion. Formulations for injection may include sterile and/or pyrogen-free solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a carrier immediately before use; and sterile and/or pyrogen-free emulsions. Solutions may be aqueous or non-aqueous.

In some embodiments, unit dose parenteral preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile and non-pyrogenic, as is known and practiced in the art.

In some embodiments, a sterile lyophilized powder is prepared by dissolving a polypeptide complex or a bispecific polypeptide complex, as described herein, in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agent. The solvent may contain a buffer such as citrate, sodium or potassium phosphate, or other similar buffer known to those skilled in the art, in one embodiment, at approximately neutral pH. Subsequent sterilizing filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, provides the desired composition. In one embodiment, the resulting solution will be dispensed into lyophilization vials. Each vessel may contain a single dose or more doses of a polypeptide complex, a bispecific polypeptide complex provided herein, or a composition thereof. Overfilling the vessels by a small amount in excess of what is needed for one dose or more doses (eg, about 10%) is acceptable to ensure accurate sampling and accurate dosing. The lyophilized powder can be stored under suitable conditions, for example at 4° C. to room temperature.

Reconstitution of the lyophilized powder with water for injection provides a dosage form for parenteral use. In one embodiment, sterile and/or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the chosen therapy and can be determined empirically.

Method of treatment

Also provided are methods of therapy comprising: administering a therapeutically effective amount of a polypeptide complex or a bispecific polypeptide complex provided herein to a subject in need of such treatment for the treatment or prevention of a condition or disorder. In some embodiments, the subject is identified as having a disorder or condition that may respond to the polypeptide complex or bispecific polypeptide complex provided herein.

As used herein, the term “treating” or “treatment” of a condition includes preventing or alleviating a condition, slowing the onset or rate of progression of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, ameliorating or eliminating symptoms associated with a condition, initiating a complete or partial regression of a condition, healing a condition, or a combination of some of these effects.

The therapeutically effective amount of the bispecific polypeptide complex provided herein will depend on various factors known in the art, such as, for example, body weight, age, medical history, current medications, the health of the subject, and the possibility of cross-reactivity, allergies, sensitivities and side effects, as well as the route of administration and the degree of development of the disease. One of ordinary skill in the art (eg, physician or veterinarian) may proportionally reduce or increase the dose, depending on these and other factors or requirements.

In some embodiments, the bispecific polypeptide complex provided herein may be administered at a therapeutically effective dose of about 0.01 to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg). In some of these embodiments, the polypeptide complex or bispecific polypeptide complex provided herein is administered at a dose of about 50 mg/kg or less, and in some of these embodiments, the dose is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In some embodiments, the implementation, the dose of administration may vary during the course of treatment. For example, in some embodiments, the initial administration dose may be higher than the subsequent administration dose. In some embodiments, the dose of administration may vary during the course of treatment depending on the response of the subject.

Dosing regimens may be adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single dose or more divided doses may be administered over time.

The bispecific polypeptide complex provided herein may be administered by any route known in the art, such as, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical).

In some embodiments, the condition or disorder being treated with the bispecific polypeptide complex provided herein is a malignant tumor or cancerous condition, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neuropathies, neuropsychiatric conditions, trauma, inflammation or bleeding disorder.

As used herein, the term “cancer” or “cancer condition” refers to any medical condition mediated by the growth, proliferation, or metastasis of tumor or malignant cells, and includes both solid cancer and non-solid cancer, such as leukemia. As used herein, the term "tumor" refers to a solid mass of tumor and/or malignant cells.

In relation to a malignant tumor, the term "treat" or "treatment" may refer to inhibiting or slowing the growth, proliferation or metastasis of tumor or malignant cells, preventing or delaying the development of growth, proliferation or metastasis of tumor or malignant cells, or any combination of these effects. When applied to a tumor, the term "treat" or "treatment" includes elimination of all or part of the tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay in tumor development, or any combination of these effects.

For example, in relation to the use of the bispecific polypeptide complex described herein for the treatment of cancer, a therapeutically effective amount is a dose or concentration of the polypeptide complex capable of eliminating all or part of the tumor, inhibiting or slowing tumor growth, inhibiting the growth or proliferation of cells that mediate a cancerous condition, inhibit the metastasis of tumor cells, reduce the intensity of any symptom or sign associated with a tumor or cancerous condition, prevent or delay the development of a tumor or cancerous condition, or capable of producing any combination of these effects.

In some embodiments, the conditions and disorders include tumors and malignancies, e.g., non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, cancer esophagus, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphomas, myelomas, fungoid granuloma, Merkel cell cancer and other hematological malignancies such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and EBV-negative post-transplant lymphoproliferative syndrome, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal cancer, and H HV8-associated primary effusion lymphoma, Hodgkin's lymphoma, central nervous system (CNS) neoplasms such as primary CNS lymphoma, spinal cord tumor, brainstem glioma.

In some embodiments, the conditions and disorders include a CD19-related condition such as B-cell lymphoma, optionally Hodgkin's lymphoma, or non-Hodgkin's lymphoma, wherein the non-Hodgkin's lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, B-cell lymphoma marginal zone lymphoma (MZL), mucosal lymphoid tissue (MALT), small cell lymphocytic lymphoma (chronic lymphocytic leukemia, CLL) or mantle cell lymphoma (MCL), acute lymphoblastic leukemia, or Waldenström macroglobulinemia (WM).

The bispecific polypeptide complex can be administered alone or in combination with one or more additional therapeutics or agents.

In some embodiments, when used to treat cancer or a tumor or proliferative disease, the bispecific polypeptide complex provided herein may be administered in combination with chemotherapy, radiation therapy, cancer surgery (e.g., tumorectomy), one or more antiemetics or other agents for the treatment of complications arising from chemotherapy, or any other therapeutic agent for use in the treatment of cancer or any associated medical disorder. As used herein, the term "administered in combination" means simultaneous administration as part of the same pharmaceutical composition, simultaneous administration as separate compositions, or at different points in time as separate compositions. It is believed that a composition administered before or after another agent is administered “in combination” with that agent, as the phrase is used herein even if the composition and the second agent are administered in different ways. Whenever possible, additional therapeutic agents administered in combination with a polypeptide complex or a bispecific polypeptide complex provided herein are administered according to the schedule provided in the additional therapeutic agent information leaflet or in accordance with the drug formulary. (Physicians' Desk Reference, 70th Ed (2016)) or according to protocols well known in the art.

In some embodiments, the therapeutic agents may induce or enhance an immune response against a cancer. For example, a cancer vaccine may be used to elicit an immune response to a specific tumor or cancer. Cytokine therapy can also be used to enhance the presentation of the tumor antigen to the immune system. Examples of cytokine therapy include, without limitation, interferons such as interferon-α, -β and -γ, colony stimulating factors such as macrophage-CSF, granulocyte macrophage CSF and granulocyte-CSF, interleukins such as IL-1, IL-1α IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor necrosis factors such as TNF-α and TNF-β. Agents that inactivate immunosuppressive targets may also be used, for example, TGF-beta inhibitors, IL-10 inhibitors, and Fas ligand inhibitors. Another group of agents includes agents that activate immune susceptibility to tumor or cancer cells, for example, agents that increase T cell activation (for example, an agonist of costimulatory T cell molecules such as CTLA-4, ICOS, and OX-40) , and agents that enhance dendritic cell function and antigen presentation.

Sets

The present invention also relates to kits containing the bispecific polypeptide complex presented herein. In some embodiments, the kits are used to detect the presence or level of, or to capture or enrich, one or more targets of interest in a biological sample. The biological sample may contain a cell or tissue.

In some embodiments, the kit contains a bispecific polypeptide complex provided herein that is conjugated to a detectable label. In some other embodiments, the kit contains the unlabeled bispecific polypeptide complex provided herein and also contains a secondary labeled antibody that is capable of binding to the unlabeled bispecific polypeptide complex provided herein. The kit may further comprise instructions for use and packaging that separates each of the components in the kit.

In some embodiments, the bispecific polypeptide complex provided herein is associated with a support or device. The support or device used can be, for example, magnetic microcarriers, a microtiter pad or a test strip. They can be used for binding assays (such as ELISA), immunographic assays, capture, or enrichment of a target molecule in a biological sample.

The following examples are given to better illustrate the claimed invention and should not be interpreted as limiting the scope of the invention. All specific compositions, substances and methods described below, in whole or in part, are included in the scope of the present invention. These specific compositions, substances and methods are not intended to limit the invention, but merely to illustrate specific embodiments included within the scope of the invention. A person skilled in the art can develop equivalent compositions, substances and methods without the use of inventive abilities and without departing from the scope of the present invention. It is understood that various changes can be made to the procedures described herein, while still remaining within the scope of the present invention. The inventors assume that such options are included in the scope of the present invention.

EXAMPLES

EXAMPLE 1 Design and construction of antibody and TCR chimeric proteins

TCR sequences

TCRs are heterodimeric proteins consisting of two chains. Approximately 95% of human T cells have TCRs composed of alpha and beta chains. Given that more crystal structures are available for the TRBC1 beta chain, TRBC1 sequences were chosen as the main backbone for constructing the polypeptide complex described herein (“WuXiBody”). A typical TRBC1 amino acid sequence can be found in the Protein Structure Database (PDB), structure 4L4T.

Interchain disulfide bond TCR

TCR crystal structures were used to form the described WuXiBody design. Unlike native TCR anchored in the T-cell surface membrane, soluble TCR molecules are less stable, although their three-dimensional structure is largely similar to that of the Fab antibody. In fact, the instability of TCR in the soluble state has been a significant hurdle in elucidating its crystal structure (Wang 2014, supra). The present inventors adopted the strategy of introducing a pair of Cys mutations into the TCR constant region and found that it could significantly improve chain assembly and increase expression.

To create a stable and functional WuXiBody, the bonds binding the antibody variable domains and the TCR constant domains, their relative fusion orientations, as well as the Fc binding bonds were carefully chosen. Because the structure of the TCR is very similar to that of the Fab antibody, the present inventors overlaid an antibody Fv homology model onto the TCR variable region (PDB 4L4T, FIG. 2). The superimposed structure indicates that the Fv antibody is structurally compatible with the TCR constant domain. Based on this structural alignment and the corresponding sequences, all relevant technical parameters have been developed. Suitable conjugation regions are described in Example 2 .

EXAMPLE 2 Bispecific anti-CD13×CD19 WuXiBody

Generation of a CD19-expressing cynomolgus monkey cell line

The full length human or cynomolgus CD19 gene was cloned into the pcDNA3.3 vector. Each expression vector was then transfected into CHO-K1 cells, respectively, using Lipofectamine 2000. Cells were cultured in F12-K with 10% FBS. After transfection, blastidine was added 24-48 hours later. After two to three selection passages, cells were enriched in PE-conjugated anti-CD19 antibody and anti-PE microbeads (Miltenyi-013-048-801). Stable single cell clones were isolated by dilution limit and screened by FACS using an anti-CD19 antibody.

Target-expressing tumor cell lines

Raji and Jurkat cell lines were obtained from the American Type Culture Collection (ATCC). Ramos cells were obtained from the European Collection of Cell Cultures (ECACC). All tumor cells were cultured in RPMI1640/10% PBS.

Generation of WuXiBody W3438-T3U4.E17-1.uIgG4.SP and W3438-T3U4.F16-1.uIgG4.SP

The VL, VH, Ck, CH1 genes were amplified by PCR from existing intralaboratory DNA templates. The SALF and CBeta genes were synthesized by Genewiz Inc. The light chain genes of an anti-CD19 native or anti-CD3 chimeric antibody were inserted into a linearized vector containing the CMV promoter and the kappa signal peptide. DNA fragments of VH-CBeta anti-CD3 were inserted into a linearized vector containing the constant region of human IgG4S228P CH2-CH3 with the “protrusion” mutation. Anti-CD19 VH-CH1 DNA fragments were inserted into a linearized vector containing a human IgG4S228P CH2-CH3 constant region with a recess mutation. The vector contains a CMV promoter and a human antibody heavy chain signal peptide.

Expression and purification of W3438-T3U4.E17-1.uIgG4.SP and W3438-T3U4.F16-1.uIgG4.SP

Heavy chain and light chain expression plasmids were co-transfected into Expi293 cells using the Expi293 expression system kit (ThermoFisher-A14635) according to the manufacturer's instructions. Five days after transfection, supernatants were collected and the protein was purified using a Protein A column (GE Healthcare-17543802) and an additional size exclusion column (GE Healthcare-17104301). The antibody concentration was measured using a Nano Drop instrument. Protein purity was assessed by DNS-PAGE and HPLC-SEC.

Target binding according to FACS

Binding of bi-specific antibodies to CD3- and CD19-expressing cells was assessed using Jurkat and Ramos cells, respectively. An irrelevant antibody was used as an isotype control. Cells were distributed into 96-well plates (Corning-3799) at a density of 10 ⁵ cells/well and washed with PBS/1% BSA. Antibodies were serially diluted and incubated with cells at 4°C for 1 hour. PE-conjugated goat anti-human IgG Fc (Jackson-109-115-098) was used for detection. After washing and resuspension, cells were analyzed by flow cytometry (Canto II, BD Biosciences). Data was analyzed using FlowJo software. Four-parameter non-linear regression analysis was used to calculate EC ₅₀ values using Prism GraphPad software.

CD3 binding of the cynomolgus macaque

Binding of the CD3×CD19 bispecific antibody to cynomolgus CD3 was examined by protein binding ELISA. 96-well high protein binding ELISA assay plates (Nunc MaxiSorp, ThermoFisher, Thermo-442404) were coated overnight at 4°C with 100 µl of 1 µg/ml cynomolgus CD3-epsilon protein (Acro, # CDE-C5226 ) in carbonate-bicarbonate buffer (20 mM Na ₂ CO ₃ , 180 mM NaHCO ₃ , PH9.2). All wells were washed once with 300 μl per well of PBS/0.5% Tween-20 (v/v). The wells were then blocked for one hour at room temperature with 200 μl/well PBS/2% BSA (BOVOGEN, #BSAS) and washed three times with 300 μl/well PBS/0.5% Tween-20 (v/v). For primary antibody binding, the CD3xCD19 bispecific antibody, serially diluted in PBS/2% BSA, was added to the appropriate wells and incubated at room temperature for two hours. The plates were washed three times as before before adding 100 μl of 100 ng/ml goat anti-human Fc-HRP IgG secondary antibody (Bethyl, # A80-304P). The plates were incubated at room temperature for one hour and then washed six times as described above. To determine binding, 100 µl of tetramethylbenzidine (TMB) substrate solution (Sigma-860336) was added to all wells for 10 minutes at room temperature in the dark until the reaction was stopped with 100 µl of 2M HCl. The degree of binding of the bispecific antibody to cynomolgus CD3 was determined by measuring the absorbance of OD450 using a SpectraMax® M5e microplate reader. Where appropriate, EC50 binding values were obtained by four parameter non-linear regression analysis using GraphPad Prism5 software.

CD19 binding of cynomolgus monkey

Binding of the CD3×CD19 bispecific antibody to the cynomolgus monkey CD19 target protein expressed on CHOK1 cells was determined by flow cytometry. Briefly, an overexpressed stable cynomolgus monkey CD19 cell line (WBP701.CHOK1.cPro1.C9, WuXi Biologics) was harvested with trypsin and diluted to a concentration of 1×10 ⁶ cells/ml in 1% BSA/1×PBS. 1×10 ⁵ cells/well (100 μl) was added to each well of a 96-well U-shaped plate (Corning, #3799) and centrifuged at 1500 rpm (Eppendorf, #5810R) for 5 minutes before removing the supernatant. Antibodies serially diluted in 1% BSA/1xPBS were added at 100 μl/well to the pelleted cells and incubated at 4° C. for 1 hour. Unbound hIgG4 antibody was used as an isotype control. Cells were washed twice with 180 μl/well of 1% BSA/1×PBS by centrifugation at 1500 rpm for 5 minutes at 4°C. The pelleted cells were resuspended in 100 μl/well of fluorescently labeled anti-human IgG Fc antibody (Jackson, no. 109-115-098) 1:150 diluted in 1% BSA/1×PBS for 30 minutes at 4°C in the dark. The cells were then washed twice as described above. After the final wash, the cells were resuspended in 80 μl of 1% BSA/1×PBS and fluorescence values were measured using a FACS Canto II cytometer (BD Biosciences). The amount of anti-CD19 and CD3 bispecific antibody associated with the cell surface was assessed by measuring the average fluorescence (MFI). Raw FACS data was analyzed using FlowJo software, wells containing no antibody or only secondary antibody were used to establish background fluorescence. Binding EC ₅₀ values were obtained by four parameter non-linear regression analysis using GraphPad Prism 5 software.

Affinity according to FACS

Binding affinity for CD3 and CD19 was determined by flow cytometry using Jurkat and Ramos cells, respectively. Cells were transferred into 96-well U-bottom (BD) plates at a density of 5×10 ⁴ cells/well. Antibodies to be tested were serially diluted 1:2-fold in 1% 1×PBS/1% BSA and incubated with cells at 4°C for 1 hour. The plates were then centrifuged at 1500 rpm for 4 minutes and the supernatant discarded. A second Alexa647-conjugated goat anti-human IgG Fc, (Jackson, Cat # 109-605-098) or FITC-conjugated anti-goat His (Bethyl, Cat # A190-113F) was added to the resuspended cells and incubated at 4°C for darkness for 30 min. Cells were washed once and resuspended in 100 μl 1×PBS/1% BSA. Fluorescence intensity was measured using flow cytometry (BD Canto II) and analyzed using FlowJo. Fluorescence intensity was converted to bound molecules/cell based on quantitative particles (QuantumTM MESF kits, Bangs Laboratories). K _D was calculated using the Graphpad Prism5 program.

Double binding on target cells

The ability of bispecific antibodies to bind to CD3 T cells and CD19 B cells was examined by FACS. Jurkat cells and Raji cells were prelabeled separately with 20 nM CellTrace Far Red (Invitrogen-C34564) and 50 nM Calcein-AM (Invitrogen-C3099) at 37°C for 30 min at a density of 1*10 ⁶ cells/ml. The prelabeled cell mass was washed twice with PBS/1% BSA, then mixed 1:1 to a final density of 1*10 ⁶ cells/ml. The cell mixture was centrifuged and resuspended with 10 nM antibody followed by incubation for 1 hour. The cell mixture was analyzed by flow cytometry immediately after incubation. Percent bridging conjugation was defined as the percentage of events that are both labeled with Far-Red and Calcein.

Cytotoxicity study

Peripheral blood mononuclear cells (PBMCs) were freshly isolated by Ficoll-Paque PLUS density gradient centrifugation (GE Healthcare-17-1440-03) from heparinized venous blood. The resulting PBMCs were then run through EasySep columns (Stemcell-19053) to enrich CD8+ T cells, which were used as effector cells. The effectiveness of antibodies to mediate the lysis of tumor cells by CD8+ T cells was determined by flow cytometry. In the cytotoxicity assay, Raji CD19 B cells as target cells were prelabeled with 20 nM CellTrace Far Red (Invitrogen-C34564) at 37°C for 30 minutes, followed by double washing of the cell mass with phenol-free RPMI 1640 medium (Invitrogen-11835030) with the addition of 1% FBS. Far Red-stained Raji cells (20,000 cells per well) were incubated in a 96-well round bottom plate (Corning-3799) with isolated CD8+ T cells (5:1 ratio of effector to target cells) and serially diluted antibodies at 37°C in for 4 hours. After incubation, 3 μM propidium iodide (PI, Invitrogen-P3566) was thoroughly mixed to identify dead cells. After 15 min, cells were analyzed by flow cytometry using a FACSCanto II cytometer. Ab-mediated cytotoxicity can be defined as the percentage of PI-positive target cells in Far Red-positive target cells. The EC ₅₀ value of cytotoxicity was determined using the Prism software.

T cell activation study - secreted cytokines TNFα and IFNɣ

T cell activation was determined by the amount of TNFα and IFNɣ secreted into the supernatant. The procedure for isolation of CD4 and CD8-positive T cells is described in the section “T Cell Activation (Intracellular Cytokine TNFα & IFNɣ Staining)”. A mixture of human Raji B cells (2×10 ⁴ cells/well), CD4 or CD8 T cells (1×10 ⁵ cells/well) and antibodies were co-incubated at 37° C. for 24 hours. The supernatant was collected, followed by centrifugation of the reaction mixture at 1500 rpm for 5 minutes. The amount of TNFα and IFNɣ in the supernatant was determined using the human TNF ELISA kit (R&D-DY210) and the human IFNɣ ELISA kit (Capture Ab: Thermo Fisher-M700A, Detection Ab: Thermo Fisher-M701B, Standard substance: PEROTECH-300-02 ) respectively.

The sandwich ELISA procedure was as follows. Overnight at 4°C or room temperature, 96-well high protein binding ELISA plates (ThermoFisher-442404) were coated with 50 µl/well of immobilized antibody in carbonate-bicarbonate buffer (20 mM Na ₂ CO ₃ , 180 mM NaHCO ₃ , pH 9.2) in accordance with the instructions for the kit. All wells were washed three times with 300 μl per well of PBS/0.5% Tween-20 (v/v) and all subsequent washing steps in the assay were performed in a similar manner. The wells were then blocked for one hour with PBS/2% BSA (BovoGen Biologicals-BSAS) for TNFα and 100% casein (Pierce-37528) for IFNɣ, then washed three times, followed by binding of the harvested supernatant or standard substance (50 µl/well ) for 1 hour at room temperature, and three times thereafter. For detection of antibody binding, appropriate antibodies diluted in PBS/2% BSA for TNFα and 50% casein for IFNα were added to appropriate wells and incubated at room temperature for two hours. The plates were washed three times before adding 50 μl of the second SA-HRP antibody. The plates were incubated at room temperature for one hour and then washed six times in the manner described above. To detect binding, 50 µl of tetramethylbenzidine (TMB) substrate solution (Sigma-860336) was added to all wells for 10 minutes before stopping the reaction with 50 µl of 2M HCl. TNFα and IFNɣ were quantified by measuring OD450 absorbance using a SpectraMax® M5e microplate reader.

T cell activation study - expression of surface markers CD25 and CD69

T cell activation was determined by staining the signals of the surface receptors CD25 and CD69. The procedure for isolating CD4 and CD8 positive T cells was described in the section “T Cell Activation (Intracellular Cytokine TNFα & IFNɣ Staining)”. A mixture of human Raji B cells (2×10 ⁴ cells/well), CD4 or CD8 T cells (1×10 5 cells/well) and antibodies were co-incubated at 37° C. for 24 hours. After washing once with 1% BSA, the cell pellet was resuspended with staining buffer, FITC-labeled mouse anti-human CD4 (BD-550628) or PerCpCy5.5-labeled mouse anti-human CD8 (BD-560662), PE-labeled mouse to human CD69 (BD-560968) and APC-labeled mouse anti-human CD25 (BD-555434) followed by a 30-minute incubation at 4°C. After washing the cells twice, the percentage of PE- and APC-positive cells in FITC- or PerCpCy5.5-positive cells was determined by flow cytometry.

Thermal Stability (DSF)

The melting point (Tm) of the antibodies was examined using the QuantStudio® Flex real-time PCR system (Applied Biosystems). 19 µl of antibody solution was mixed with 1 µl of 62.5 X SYPRO Orange solution (Invitrogen) and transferred to a 96-well plate (Biosystems). The plate was heated from 26 to 95° C. at a rate of 0.9° C./min, and the resulting fluorescence data were collected. The negative derivatives of fluorescence changes with respect to various temperatures were determined, and the maximum value was determined as the melting point Tm. If a protein had multiple conformational unfolding transitions, the first two Tm's, named Tm1 and Tm2, were reported. Data collection and determination of Tm were carried out automatically using software.

Serum stability

Freshly drawn human blood from selected donors was placed in polystyrene tubes without anticoagulant. After incubation for 30 minutes at room temperature, human blood was centrifuged at 4000 rpm for 10 minutes to collect the serum layer. The centrifugation step was repeated until the serum was clear. In the detection process, antibodies were mixed with the collected serum in a ratio of 1:9, and aliquots were taken at 37°C for the indicated time: 0 days, 1 day, 4 days, 7 days and 14 days. Samples were flash frozen at various times in liquid nitrogen and stored at -80° C. prior to use. Samples were analyzed by FACS to evaluate the binding capacity of CD3 Jurkat T cells and CD19 Ramos B cells compared to the corresponding antibodies without serum treatment.

Fcγ receptor binding affinity according to SPR (SPR)

The binding affinity of the antibody to FcγR was determined using Biacore T200 (or Biacore 8K). Each receptor was fixed on a CM5 sensor chip with an immobilized anti-histidine (GE) antibody. Antibodies at various concentrations were injected onto the sensor chip at a flow rate of 30 μl/min during an association phase of 60 sec followed by dissociation for 60 sec. The chip was then regenerated with 10 mM glycine (pH 1.5) after each binding cycle.

The empty surface and buffer channel sensorgrams were subtracted from the analytical sensorgrams. Experimental data were fitted to a 1:1 model using Langmuir analysis (for FcγRI) or an equilibrium model (for other receptors). A molecular weight of 150 kDa was used to calculate the molar concentration of antibodies.

C1q binding according to ELISA

ELISA plates (Nunc) were coated with antibody samples at a concentration of 3 μg/ml overnight at 4°C. After blocking and washing, C1q was diluted in a gradient starting at 600 μg/ml and incubated at room temperature for 2 hours. The plates were then washed and further incubated with sheep anti-human C1q Ab-HRP for 1 hour. After washing, TMB substrate was added and the reaction was stopped with 2M HCl. Optical density at 450 nm was read using a microplate reader (Molecular Device).

FcRn binding affinity according to SPR (SPR)

The binding affinity of the antibody to FcRn was determined by Biacore T200 (or Biacore 8K). Each antibody was immobilized on a CM5 sensor chip (GE). FcRn at various concentrations in running buffer (50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ , 150 mM NaCl, 0.05% Tween 20, pH 6.0) were injected onto the sensor chip at a flow rate of 30 μl/min during the phase association for 60 sec followed by dissociation for 60 sec. The chip was then regenerated with 1×PBS (pH 7.4) after each binding cycle.

The empty surface and buffer channel sensorgrams were subtracted from the analytical sensorgrams. The experimental data were approximated by the equilibrium model. A molecular weight of 45 kDa was used to calculate the molar concentration of FcRn.

Efficacy Study in a Raji/PBMC Mouse Model

Raji tumor cells (ATCC® CCL-86™) were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37ºC in a 5% CO ₂ atmosphere in the air. Tumor cells were passaged twice a week in the prescribed manner. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood using Ficoll-Paque Plus according to the manufacturer's instructions.

Each mouse was inoculated subcutaneously in the right flank with a mixture of Raji tumor cells with Matrigel and fresh PBMCs in 0.2 ml of PBS on day 0. Antibody injection was performed starting from day 3 (intravenous biweekly (BIW) × 4 times).

Composition of the study drug Connections Package A drug Conc.
mg/ml Isotype control 9.38 mg/ml 0.031 ml solution + 1.908 ml FSB 1.5 W3438-T3U4.E17-1.uIgG4.SP 2.6 mg/ml B: 0.138 ml solution + 2.254 ml PBS 1.5 W3438-T3U4.E17-1.uIgG4.SP B1: 0.450 ml B+1.800 ml PBS 0.3 W3438-T3U4.E17-1.uIgG4.SP B2: 0.450 ml B1+1.800 ml PBS 0.06

Tumor measurements and expected results

The main expected outcome was to see if the tumor growth could be halted or the mice could be cured. Tumor size was measured twice a week in two dimensions using a caliper and the volume was expressed in mm ³ using the formula: V=0.5 a×b ² where a and b are the larger and smaller tumor diameters, respectively. The T/C value (in percent) is an indicator of antitumor efficacy.

Tumor growth inhibition (TGI) was calculated for each group according to the formula: TGI (%) = [1-(Ti-T0)/(Vi-V0)]×100; Ti - average tumor volume in the treatment group on a given day, T0 - average tumor volume of the treatment group on the day of treatment, Vi - average tumor volume of the control group with the carrier on the same day as Ti, V0 - average tumor volume in the carrier group on the day of the start of treatment.

Analysis of the pharmacokinetics, toxicity and immunogenicity of the drug on the cynomolgus monkey

One male and one female cynomolgus macaque received WBP3438 at a dose of 1 mg/kg once by intravenous bolus injection. The formulation was made in 20 mM NaAc-HAc, 7.0% (w/w) sucrose, 0.02% (w/w) PS80, pH 5.0. PK blood samples were collected before drug administration (day -1), after 0.25 h, 0.5 h, 1 h, 4 h, 8 h, 24 h, day 3, day 7, day 14, day 21 and day 28. Anti-drug antibody (ADA) samples were collected on day 3, day 14 (at 312 h) and day 28 (at 480 h).

The concentrations of WBP3438 and ADA in serum samples were determined by ELISA. WBP3438 serum concentration in monkeys was subjected to non-compartmental pharmacokinetic analysis using Phoenix WinNonlin software (version 6.3, Pharsight, Mountain View, CA). When obtaining the FK parameters, the linear-logarithmic trapezoid rule was used.

The cell was monitored for general health and appearance, in particular for skin irritation. Analysis of whole blood samples for hematology (CBC) and analysis of serum for chemical detection were performed by means of a hematology analyzer (ADVIA2120) and a biochemistry analyzer (HITACHI 7180), respectively.

results

Generation of a cell line expressing CD19 from the cynomolgus monkey

Expression of the cynomolgus monkey CD19 cell line WBP701.CHO-K1.cpro1.FL.C9 was detected using an anti-CD19 antibody by flow cytometry. WBP701.CHO-K1.cpro1.FL.C9 showed high expression of monkey CD19 (FIG. 5).

Generation and optimization of WuXiBody

Figure 1 presents schematic images of the investigated antibodies and their formats. Both an anti-CD3 T3 antibody and an anti-CD19 U4 antibody have been developed. The T3 constant region (CL and CH1) was replaced with a TCR constant region to construct a unique light-heavy chain region that is orthogonal to a regular antibody. TCR-modified T3 and native U4 in combination with protrusion-to-recess mutations in the Fc domain were used to construct bispecific E17 and F16 format antibodies.

The heavy chain and light chain variable region sequences of the anti-CD3 and anti-CD19 binding fragments from W3438-T3U4.E17-1.uIgG4.SP and W3438-T3U4.F16-1.uIgG4.SP are shown below:

The full length sequences of W3438-T3U4.E17-1.uIgG4.SP and W3438-T3U4.F16-1.uIgG4.SP are shown below:

Products W3438-T3U4.F16-1.uIgG4.SP

As a result of transient expression, the expression titer of the W3438-T3U4.F16-1.uIgG4.SP antibody is above 90 mg/L. After two-step purification, the purity of W3438-T3U4.F16-1.uIgG4.SP reaches 97.5% (SEC, Figure 7). W3438-T3U4.F16-1.uIgG4.SP migrates with an average molecular weight of 75 kDa, 55 kDa and 25 kDa in DNS-PAGE under reducing conditions, corresponding to two heavy chains and two light chains. The two light chains may overlap due to similar molecular weights. The antibody migrates at an average molecular weight of 200 kDa under non-reducing conditions, indicating an intact bispecific molecule (FIG. 6).

Products W3438-T3U4.E17-1.uIgG4.SP

As a result of transient expression, the expression titer of W3438-T3U4.E17-1.uIgG4.SP antibody is higher than 100 mg/l. After two-step purification, the purity of W3438-T3U4.E17-1.uIgG4.SP reaches 95% (SEC, Figure 9). W3438-T3U4.E17-1.uIgG4.SP migrates with an average molecular weight of 54 kDa, 56 kDa and 25 kDa in DNS-PAGE under reducing conditions, corresponding to two heavy chains and two light chains. The two light chains may overlap due to similar molecular weights. The antibody migrates at an average molecular weight of 150 kDa under non-reducing conditions, indicating an intact bispecific molecule (FIG. 8).

Linking to a target

Binding of W3438-T3U4.E17-1.uIgG4.SP to CD19 and CD3 was examined in Ramos and Jurkat cells by flow cytometry (Figures 10A-10B). The W3438-T3U4.E17-1.uIgG4.SP antibody showed strong binding activity to Ramos and Jurkat cells with EC ₅₀ values of 15.6 nM and 47 nM, respectively.

Binding of W3438-T3U4.F16-1.uIgG4.SP to CD19 and CD3 was examined in Ramos and Jurkat cells by flow cytometry (Figures 11A-11B). The W3438-T3U4.F16-1.uIgG4.SP antibody showed strong binding activity to Ramos and Jurkat cells, with EC ₅₀ values of 1.8 nM and 19.3 nM, respectively.

Interspecies bonding

Binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus monkey CD19 was examined on WBP701.CHO-K1.cpro1.FL.C9 cell (CD19 expressing cell) by flow cytometry (FIG. 12). The EC ₅₀ value for binding was 26 nM. Binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus CD3 was examined using W331-cynoPro1.ECD.His (cynomolgus monkey CD3 epsilon protein) by ELISA (FIG. 13). The EC ₅₀ value for binding was 0.04 nM.

Affinity for target cells

The binding affinity of W3438-T3U4.E17-1.uIgG4.SP to human CD19 and CD3 was examined on Ramos and Jurkat cells by flow cytometry. The ratio of bound IgG/free IgG relative to bound IgG was plotted as shown in FIG. 14A-14B. Suitable K _D values for CD19 and CD3 binding were 23 nM and 9.0 nM, respectively.

Double binding on target cells

The activity of W3438-T3U4.E17-1.uIgG4.SP to bind CD3 T cells and CD19 B cells was examined using prelabeled Jurkat and Raji cells by flow cytometry (Fig. 15A-15B). Q2 shows the Jurkat and Raji bridge cell population. Compared to the negative control, approximately 18% of the cells were bound via the W3438-T3U4.E17-1.uIgG4.SP bispecific antibody.

Cytotoxicity assay

The cytotoxic activity of W3438-T3U4.E17-1.uIgG4.SP was assessed using CD8+ T cells and Raji cells. W3438-T3U4.E17-1.uIgG4.SP caused rapid and efficient cell lysis after a 4 hour incubation (FIG. 16A) with an EC ₅₀ value of 15 nM. The maximum percentage of cell death was 90%.

The cytotoxic activity of W3438-T3U4.F16-1.uIgG4.SP was assessed using CD8+ T cells and Raji cells. W3438-T3U4.F16-1.uIgG4.SP caused rapid and efficient cell lysis after 4 hours of incubation (FIG. 16B) with an EC ₅₀ value of 3.2 nM. The maximum percentage of cell death was 90%.

Target-specific T cell activation

W3438-T3U4.E17-1.uIgG4.SP was investigated in assays that indicate T cell activation via CD69 and CD25 activation markers in the presence or absence of CD19+ target cells. The results showed that W3438-T3U4.E17-1.uIgG4.SP induces the expression of CD25 and CD69 T cell activation markers in a dose-dependent manner only in the presence of CD19+ target cells (Figures 17A-17D). When the B cell is absent, CD25 and CD69 expression was not observed in both the CD4+ and CD8+ T cell subsets.

W3438-T3U4.E17-1.uIgG4.SP was also investigated in T cell activation assays for cytokine release in the presence or absence of CD19+ target cells. The results showed that W3438-T3U4.E17-1.uIgG4.SP induces the release of IFN-γ and TNF-α in a dose-dependent manner only in the presence of CD19+ target cells (FIGS. 18A-18D). When the B cell is absent, IFN-γ and TNF-α were not detected in both the CD4+ and CD8+ T cell subsets.

thermal stability

The thermal stability of W3438-T3U4.E17-1.uIgG4.SP was examined using real-time PCR. Tm1 and Tm2 for W3438-T3U4.E17-1.uIgG4.SP are 60.2°C and 72.7°C.

Serum stability

W3438-T3U4.E17-1.uIgG4.SP was incubated in serum at 37°C for 14 days. The binding activity of the antibody incubated for 0, 1, 4, 7 and 14 days was determined by flow cytometry. The results showed that W3438-T3U4.E17-1.uIgG4.SP binding activity to both CD3 and CD19 cells did not change after incubation in human serum for 14 days (Figures 19A and 19B).

Fcγ receptor binding

The binding activity of W3438-T3U4.E17-1.uIgG4.SP with FcγRI, FcγRIIa (H167), FcγRIIa (R167), FcγRIIb, FcγRIIIa (F176), FcγRIIIa (V176) and FcγRIIIb was examined by SPR (SPR). Affinities are shown in Table 1. W3438-T3U4.E17-1.uIgG4.SP showed typical human IgG4 binding affinity for all Fcγ receptors.

Table 1. W3438-T3U4.E17-1.uIgG4.SP affinity for Fc receptor according to SPR Fc receptor K _D (M) FcγRI 9.79E-09 FcγRIIa (H167) 2.05E-05 FcγRIIa (R167) 1.58E-05 FcγRIIb 2.41E-05 FcγRIIIa (F176) 2.93E-05 FcγRIIIa (V176) 1.40E-05 FcγRIIIb >4.10E-05

The activity of antibody binding to C1Q was studied by ELISA. W3438-T3U4.E17-1.uIgG4.SP showed no binding signal in the ELISA assay (FIG. 20), with the control human IgG1 antibody showing a normal binding signal.

FcRn binding

The binding of W3438-T3U4.E17-1.uIgG4.SP to FcRn was examined by SPR at pH 6.0. The affinity was 2.58 μM, which is the typical affinity of human IgG4 for FcRn.

Characteristic in vivo

Efficacy Study in PBMC/Raji Cell Xenograft Model

This assay examined the antitumor efficacy of W3438-T3U4.E17-1.uIgG4.SP in a mixed humanized Raji cell-bearing PBMC model in NOG mice. The tumor growth curve is shown in Fig.21.

On day 14, the mean tumor size in the isotype control treatment group reached 342 mm ³ . Treatment with 1.5 mg/kg and 0.5 mg/kg W3438-T3U4.E17-1.uIG4.SP provided significant antitumor activity. The mean tumor size was 78 ^mm3 (T/C=23.0%, TGI=93.9%, p=0.016) and 75 ^mm3 (T/C=22.0%, TGI=95.3%, respectively). p=0.014), with tumor in one animal in the high dose group being completely eliminated. W3438-T3U4.E17-1.uIgG4.SP at a very low dose (0.06 mg/kg) showed no antitumor activity.

Pharmacokinetics of WuXiBody in the cynomolgus monkey

The concentration of W3438-T3U4.E17-1.uIgG4.SP in cynomolgus monkey serum was examined by ELISA (FIG. 22). The calculated pharmacokinetic (PK) parameters are shown in Table 2. The half-life of W3438-T3U4.E17-1.uIgG4.SP for a single intravenous injection at a dose of 1 mg/kg was 152 hours. W3438-T3U4.E17-1.uIgG4.SP showed a much longer half-life in monkeys than blinatumomab with a very short half-life (1.5-2.6 hours) in chimpanzees (European Medicines Agency EMA/CHMP/ expert report). 469312/2015).

Table 2. Pharmacokinetics of W3438-T3U4.E17-1.uIgG4.SP in the cynomolgus monkey FK parameter W3438-T3U4.E17-1.uIgG4.SP C ₀ (µg/ml) 60.4 T _1/2 (h) 152 Vd _ss (l/kg) 0.0513 Cl (ml/min/kg) 0.00462 AUC _0-last (h * mcg / ml) 3552 AUC _0-inf (h*mcg/ml) 3708 MRT _0-last (h) 157 MRT _0-inf (h) 187

Toxicity

All monkeys tolerated the drug well throughout the course of the study. No side effects were observed during the life stage of the study. No obvious changes in feed intake and weight were observed. Hematology and clinical chemistry parameters, including AST (aspartic aminotransferase), ALT (alanine aminotransferase), WBC (leukocytes), HGB (hemoglobin), and HCT (hematocrit), were generally within the reference range.

Immunogenicity

The results of the anti-drug antibody (ADA) W3438-T3U4.E17-1.uIgG4.SP are shown in FIGS. 23A-23B. ADA titers against W3438-T3U4.E17-1.uIgG4.SP in monkey serum 3, 14 and 28 days after administration did not show a significant difference from the values before administration of the drug. Accordingly, a single intravenous injection of W3438-T3U4.E17-1.uIgG4.SP at a dose of 1 mg/kg was not immunogenic in monkeys.

--->

SEQUENCE LIST

<110> WuXi Biologics (Shanghai) CO., LTD.

WUXI BIOLOGICS (CAYMAN) INC.

<120> NEW BISPECIFIC POLYPEPTIDE COMPLEXES AGAINST CD3/CD19

<130> IEC180126PCT

<160> 40

<170>PatentIn version 3.5

<210> 1

<211> 116

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 1

Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln

1 5 10 15

Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val

20 25 30

Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys

35 40 45

Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg

50 55 60

Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn

65 70 75 80

Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu

85 90 95

Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val

100 105 110

Ser Ala Glu Ala

115

<210> 2

<211> 87

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 2

Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys

1 5 10 15

Leu Phe Thr Asp Phe Asp Ser Gln Thr Gln Val Ser Gln Ser Lys Asp

20 25 30

Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met

35 40 45

Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Gln Lys Ser Asp Phe

50 55 60

Ala Cys Ala Asn Ala Phe Gln Asn Ser Ile Ile Pro Glu Asp Thr Phe

65 70 75 80

Phe Pro Ser Pro Glu Ser Ser

85

<210> 3

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 3

Gly Phe Ala Phe Thr Asp Tyr Tyr Ile His

1 5 10

<210> 4

<211> 17

<212> PROTEIN

<213> Mus musculus

<400> 4

Trp Ile Ser Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Asn Phe Lys

1 5 10 15

gly

<210> 5

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 5

Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp Tyr

1 5 10

<210> 6

<211> 17

<212> PROTEIN

<213> Mus musculus

<400> 6

Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 7

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 7

Trp Ala Ser Thr Arg Gln Ser

15

<210> 8

<211> 8

<212> PROTEIN

<213> Mus musculus

<400> 8

Thr Gln Ser His Thr Leu Arg Thr

15

<210> 9

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 9

Gly Tyr Ala Phe Thr Ser Tyr Asn Met Tyr

1 5 10

<210> 10

<211> 17

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 10

Tyr Ile Asp Pro Tyr Asn Ala Asp Thr Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

gly

<210> 11

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 11

Thr Ala Tyr Ala Met Asp Tyr

15

<210> 12

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 12

Ser Ala Ser Ser Thr Val Asn Tyr Met His

1 5 10

<210> 13

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 13

Ser Thr Ser Asn Leu Ala Ser

15

<210> 14

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 14

His Gln Trp Ser Ser Tyr Pro Tyr Thr

15

<210> 15

<211> 119

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 16

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 16

caggtgcagc ttgtgcagtc tggggcagaa gtgaagaagc ctgggtctag tgtcaaggtg 60

tcatgcaagg ctagcgggtt cgcctttact gactactaca tccactgggt gcggcaggct 120

ccgggacaag ggttggagtg gatgggatgg atctccccag gcaatgtcaa cacaaagtac 180

aacgagaact tcaaaggccg cgtcaccatt accgccgaca agagcacctc cacagcctac 240

atggagctgt ccagcctcag aagcgaggac actgccgtct actactgtgc cagggatggg 300

tactccctgt attactttga ttactggggc cagggcacac tggtgacagt gagctcc 357

<210> 17

<211> 112

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 17

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser His Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 18

<211> 336

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 18

gatatcgtga tgacccagag cccagactcc cttgctgtct ccctcggcga aagagcaacc 60

atcaactgca aggctccca aagcctgctg aactccagga cggaagaa ttacctggcc 120

tggtatcagc agaagcccgg ccagcctcct aagctgctca tctactgggc ctccacccgg 180

cagtctgggg tgcccgatcg gtttagtgga tctgggagcg ggacagactt cacattgaca 240

attagctcac tgcaggccga ggacgtggcc gtctactact gtactcagag ccacactctc 300

cgcacattcg gcggagggac taaagtggag attaag 336

<210> 19

<211> 116

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 19

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr

20 25 30

Asn Met Tyr Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Ala Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Leu Thr Thr Ala Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 20

<211> 348

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 20

caaatgcagc tcgtccagtc tggacctgaa gtgaagaagc ccgggacatc cgtcaaggtc 60

tcatgtaagg ctagcgggta cgcattcact tcctacaaca tgtactgggt gcgccaggcc 120

agaggacaga ggttggagtg gatcggctac atcgacccat acaacgccga tactacctac 180

aatcagaagt ttaaagggcg ggtgaccatt acccgggata tgtccacctc caccgcctac 240

atggagctga gcagcctgag gagcgaggac acagccgtgt actactgcct gacaacagcc 300

tatgccatgg actattgggg ccagggcaca cttgtgactg tgagcagt 348

<210> 21

<211> 106

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 21

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Thr Val Asn Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 22

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic

<400> 22

gacatccagc tcacccaatc cccttctttc ctctccgcaa gtgtcggaga tagggtgact 60

atcacctgct cagcttcttc aaccgtgaac tacatgcatt ggtaccagca gaagcccgggg 120

aaagccccaa agctgctgat ctacagcacc tccaatctgg ccagtggagt gccaagccgg 180

tttagcggga gcggctccgg cactgaattc actttgacaa ttagcagcct tcagcctgag 240

gactttgcca catattactg tcaccagtgg tccagctacc cctacacatt cgggcagggc 300

acaaagctgg agattaag 318

<210> 23

<211> 10

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 23

Leu Glu Asp Leu Lys Asn Val Phe Pro Pro

1 5 10

<210> 24

<211> 9

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 24

Lys Pro Asp Ile Gln Asn Pro Asp Pro

15

<210> 25

<211> 17

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 25

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

1 5 10 15

Pro

<210> 26

<211> 207

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 26

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser His Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Gln Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Gln Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

<210> 27

<211> 472

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

115 120 125

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

130 135 140

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

145 150 155 160

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

165 170 175

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg Tyr

180 185 190

Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

195 200 205

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

210 215 220

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

225 230 235 240

Ala Glu Ala Trp Gly Arg Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

245 250 255

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

260 265 270

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

275 280 285

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

290 295 300

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

305 310 315 320

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

325 330 335

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

340 345 350

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

355 360 365

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr

370 375 380

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

385 390 395 400

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

405 410 415

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

420 425 430

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

435 440 445

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

450 455 460

Ser Leu Ser Leu Ser Leu Gly Lys

465 470

<210> 28

<211> 213

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 28

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Thr Val Asn Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 29

<211> 443

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 29

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr

20 25 30

Asn Met Tyr Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Leu Thr Thr Ala Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 30

<211> 667

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 30

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr

20 25 30

Asn Met Tyr Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Leu Thr Thr Ala Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

225 230 235 240

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr

245 250 255

Asn Met Tyr Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

260 265 270

Gly Tyr Ile Asp Pro Tyr Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

275 280 285

Lys Gly Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

290 295 300

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

305 310 315 320

Leu Thr Thr Ala Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

325 330 335

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

340 345 350

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

355 360 365

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

370 375 380

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

385 390 395 400

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

405 410 415

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

420 425 430

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

435 440 445

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

450 455 460

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

465 470 475 480

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

485 490 495

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

500 505 510

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

515 520 525

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

530 535 540

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

545 550 555 560

Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu

565 570 575

Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

580 585 590

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

595 600 605

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

610 615 620

Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

625 630 635 640

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

645 650 655

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

660 665

<210> 31

<211> 142

<212> PROTEIN

<213> Homo sapiens

<400> 31

Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

65 70 75 80

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

85 90 95

Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn

100 105 110

Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val

115 120 125

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 32

<211> 95

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 32

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

65 70 75 80

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

85 90 95

<210> 33

<211> 177

<212> PROTEIN

<213> Homo sapiens

<400> 33

Glu Asp Leu Asn Lys Val Phe Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser

130 135 140

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

145 150 155 160

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

Phe

<210> 34

<211> 130

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 34

Glu Asp Leu Lys Asn Val Phe Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp

130

<210> 35

<211> 95

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 35

Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

65 70 75 80

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

85 90 95

<210> 36

<211> 95

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 36

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

65 70 75 80

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

85 90 95

<210> 37

<211> 95

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 47

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Gln Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Gln Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn

65 70 75 80

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

85 90 95

<210> 38

<211> 129

<212> PROTEIN

<213> Homo sapiens

<400> 38

Glu Asp Leu Asn Lys Val Phe Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala

<210> 39

<211> 130

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 39

Leu Glu Asp Leu Lys Asn Val Phe Pro Glu Val Ala Val Phe Glu

1 5 10 15

Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys

20 25 30

Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val

35 40 45

Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu

50 55 60

Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg

65 70 75 80

Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg

85 90 95

Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln

100 105 110

Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly

115 120 125

Arg Ala

130

<210> 40

<211> 130

<212> PROTEIN

<213> artificial sequence

<220>

<223> synthetic

<400> 40

Leu Glu Asp Leu Lys Asn Val Phe Pro Glu Val Ala Val Phe Glu

1 5 10 15

Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys

20 25 30

Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val

35 40 45

Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu

50 55 60

Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg Tyr Ala Leu Ser Ser Arg

65 70 75 80

Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg

85 90 95

Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln

100 105 110

Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly

115 120 125

Arg Ala

130

<---

Claims (15)

1. A bispecific polypeptide complex that specifically binds to CD3 and CD19, characterized in that the bispecific polypeptide complex contains a combination of four polypeptide sequences: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 . 2. A bispecific polypeptide complex that specifically binds to CD3 and CD19, characterized in that the bispecific polypeptide complex contains a combination of four polypeptide sequences: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 30 . 3. A set of isolated polynucleotide molecules for the expression of a bispecific polypeptide complex that specifically binds to CD3 and CD19, according to claim 1 or 2, where each of said isolated polynucleotide molecules encodes each polypeptide chain of the bispecific polypeptide complex according to claim 1 or 2. 4. A set of isolated vectors for the expression of a bispecific polypeptide complex that specifically binds to CD3 and CD19, according to claim 1 or 2, where each of said isolated vectors contains each of the polynucleotide molecules according to claim 3. 5. A host cell for expressing a bispecific polypeptide complex that specifically binds to CD3 and CD19, according to claim 1 or 2, containing a set of isolated polynucleotide molecules according to claim 3 or a set of isolated vectors according to claim 4. 6. A method for producing a bispecific polypeptide complex that specifically binds to CD3 and CD19 according to claim 1 or 2, comprising culturing a host cell according to claim 5 under conditions under which each polypeptide chain of said bispecific polypeptide complex is expressed and the expressed polypeptide chains are assembled into a bispecific polypeptide complex. 7. A method for obtaining a bispecific polypeptide complex that specifically binds to CD3 and CD19, according to claim 1 or 2, including: a) introducing into the host cell the selected polynucleotide molecules, each of which encodes each polypeptide chain of the bispecific polypeptide complex according to claim 1 or 2; and b) allowing the host cell to express each polypeptide chain of the bispecific polypeptide complex and to assemble the expressed polypeptide chains into the bispecific polypeptide complex. 8. The method of claim 6 or 7, further comprising isolating the bispecific polypeptide complex. 9. A composition for treating a CD19 related disease or condition in a subject, comprising an effective amount of the bispecific polypeptide complex of claim 1 or 2, wherein said CD19 related disease or condition is B-cell lymphoma. 10. A pharmaceutical composition for the treatment of a disease or condition associated with CD19 in a subject, containing an effective amount of a bispecific polypeptide complex according to claim 1 or 2 and a pharmaceutically acceptable carrier, where the specified disease or condition associated with CD19 is a B-cell lymphoma . 11. A method of treating a disease or condition associated with CD19 in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a bispecific polypeptide complex according to claim 1 or 2 or a therapeutically effective amount of a pharmaceutical composition according to claim 10, where the specified disease or the condition associated with CD19 is B-cell lymphoma. 12. The method of claim 11, wherein the B-cell lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma, and wherein the non-Hodgkin's lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL ), mucosal lymphoid tissue (MALT), small cell lymphocytic lymphoma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia, or Waldenström macroglobulinemia (WM). 13. A kit for treating a CD19 related disease or condition in a subject, comprising the bispecific polypeptide complex of claim 1 or 2, wherein said CD19 related disease or condition is B-cell lymphoma.

Images