WO2024199454A1

WO2024199454A1 - Antibodies and variants thereof against human cluster of differentiation 3 protein

Info

Publication number: WO2024199454A1
Application number: PCT/CN2024/084851
Authority: WO
Inventors: Xinpo JIANG
Original assignee: Nanjing Jinsirui Science and Technology Biology Corp
Current assignee: Nanjing Jinsirui Science and Technology Biology Corp
Priority date: 2023-03-29
Filing date: 2024-03-29
Publication date: 2024-10-03
Anticipated expiration: 2025-09-29
Also published as: CN120958019A; EP4688832A1

Abstract

Provided is an antibody or antigen binding fragment thereof capable of binding specifically to a CD3 protein and a multispecific antibody comprising the anti-CD3 antibody or antigen binding fragment thereof. Also provided are the multispecific antibody, the pharmaceutical compositions, methods of making and using the antibody or antigen-binding fragment thereof, or use of the multispecific antibody in the manufacture of a medicament for the treatment of a cancer.

Description

ANTIBODIES AND VARIANTS THEREOF AGAINST HUMAN CLUSTER OF DIFFERENTIATION 3 PROTEIN

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from PCT international application PCT/CN2023/084860 filed March 29, 2023, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present application relates to an antibodies or antigen binding fragment thereof capable of binding specifically to a CD3 protein. The present application also relates to a bispecific antibody comprising the anti-CD3 antibody or antigen binding fragment thereof.

BACKGROUND

The immune system is a host defense system comprising a collection of cells, tissues, and organs that work together to protect against attacks by “foreign” invaders or abnormal cells arose by mutation. The invaders are primarily infection-causing organisms such as bacteria, viruses, parasites, and fungi. The capacity of the immune system to detect and destroy abnormal cells prevents the development of many cancers and helps to fight cancers. The immune system comprised of the central immune organs and the peripheral immune organs work together as one unit to fight infectious disease. The capability to fend off millions of structurally different foreign enemies shows the complexity of the immune system. This complexity is fulfilled by a dynamic communication network of organs, tissues, cells, and molecules. These organs, tissues, cells, and molecules cooperate with each other and keep the immune system in balance to fight foreign invasion and maintain self-tolerance at the same time.

T cells are specialized lymphocytes that are an essential part of the immune system. T cells are often divided into helper, cytotoxic or regulatory T cells during an adaptive immune response. These T cells can also be classified into naive, effector and memory subsets based on their activation status or can be separated into two major subtypes, CD4+ and CD8+ T cells. In addition to the conventional T cells mentioned above, there are innate-like T cells, including the γδ T cells and natural killer T cells that behave different immune response. CD3 is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+naive T cells) and T helper cells (CD4+ naive T cells) . It is composed of four distinct chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with the T-cell receptor (TCR) and the CD3-zeta (ζ-chain) to generate an activation signal in T lymphocytes. The TCR, CD3-zeta, and the other CD3 molecules together constitute the TCR complex.

Human tumors are the consequence of combination of genetic and epigenetic changes. When tumor cells form, some of the antigens on their surface may change. These so-called neo-antigens would be detected by the immune system and are to be destroyed as foreign objects. The abnormal cells are eliminated before they progress to advanced cancer stage. However, tumor cells develop multiple resistance mechanisms to evade and suppress the immune system. A common mechanism applied by tumors is to manipulate immune checkpoint pathways by overexpressing the inhibitory immune checkpoint modulators. Cancer immunotherapy exploits the host’s immune system to treat cancer. The mechanisms ranging from activating effector cells to blocking inhibitory factors boosts the immune system and produces antitumor activities. Drugs blocking inhibitory immune checkpoint pathways have demonstrated promising clinical activities in various solid tumors. T cell activation is a regulatory event of the adaptive immune response. T cell activation is initiated by interaction of TCRs with major histocompatibility complex (MHC) proteins on the surface of other cells. CD3-bispecific antibodies (BsAbs) become a new treatment modality in cancer immunotherapy. CD3-BsAbs can simultaneously bind to its antigen expressed on tumor cells and to CD3 on a T cell. By crosslinking a T cell and a tumor cell by CD3-BsAbs, an immunological synapse is formed, which results in T-cell activation and the secretion of inflammatory cytokines and cytolytic molecules that kill the tumor cells in the process. CD3-BsAbs are a powerful modality that can engage all available T cells in the field of immunotherapy.

SUMMARY

In one aspect, there is provided an anti-CD3 antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain (VH) comprising: 1) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 13 and 23; 2) a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 14, and 24; and 3) a HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, and 25, and a light chain variable domain (VL) comprising: 1) a LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, and 28 ; 2) a LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 19 and 29; and 3) a LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 , 20, and 30.

In some embodiments, 1) the VH comprises the HCDR1, HCDR2, and HCDR3 sequences having the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; 2) the VH comprises the HCDR1, HCDR2, and HCDR3 sequences having the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively; or 3) the VH comprises the HCDR1, HCDR2, and HCDR3 sequences having the amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively.

In some embodiments, the VH comprises an amino acid sequence that is at least 90%identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 12 and 22, and the VL comprises an amino acid sequence that is at least 90%identical to a sequence selected from the group consisting of SEQ ID NOs: 7, 17 and 27, respectively.

In some embodiments, the VH comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12 and 22, or a variant thereof comprising up to about 3 amino acid substitutions in the VH; and the VL comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17 and 27, or a variant thereof comprising up to about 3 amino acid substitutions in the VL.

In some embodiments, 1) the VH comprises an amino acid sequence of SEQ ID NO: 2, and the VL comprises an amino acid sequence of SEQ ID NO: 7; 2) the VH comprises an amino acid sequence of SEQ ID NO: 12, and the VL comprises an amino acid sequence of SEQ ID NO: 17; or 3) the VH comprises an amino acid sequence of SEQ ID NO: 22, and the VL comprises an amino acid sequence of SEQ ID NO: 27.

In some embodiments, the CD3 is human CD3 or cynomolgus CD3.

In some embodiments, the anti-CD3 antibody is a mouse antibody.

In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof is an activator of a Jurkat T cell, with an EC50 value determined through IL-2 promoter activity greater than 0.01 μg/mL; preferably between about 10^-2 μg/mL and 1.0 μg/mL, such as between about 0.018 μg/mL and 0.82 μg/mL.

In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof is an activator of a Jurkat T cell, with an EC50 value determined through IL-2 promoter activity greater than that of OKT3 antibody.

In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof is an activator of primary human T cell, with an EC50 value determined through IFN-γ concentration in the supernatant of the primary human T cell greater than 0.1 μg/mL; preferably between about 0.1 μg/mL and 50.0 μg/mL, such as between about 0.50 μg/mL and 10.14 μg/mL.

In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof is an activator of a primary human T cell, with an EC50 value determined through IFN-γ concentration in the supernatant of the primary human T cell greater than that of OKT3.

In some embodiments, the anti-CD3 antibody is a chimeric antibody comprising: 1) a VH comprises an amino acid sequence of SEQ ID NO: 22, and a VL comprises an amino acid sequence of SEQ ID NO: 34; 2) a VH comprises an amino acid sequence of SEQ ID NO: 36, and a VL comprises an amino acid sequence of SEQ ID NO: 38; or 3) a VH comprises an amino acid sequence of SEQ ID NO: 40, and a VL comprises an amino acid sequence of SEQ ID NO: 42, and, optionally, the VH is fused to a constant region of human IgG, preferably human IgG1.

In some embodiments, the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and a Jurkat T cell is lower than 1 μg/mL; preferably between about 0.1 μg/mL and 1.0 μg/mL, such as between about 0.20 μg/mL and 0.45 μg/mL.

In some embodiments, the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and a Jurkat T cell is lower than that of OKT3 antibody.

In some embodiments, the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and a primary human T cell is lower than 1 μg/mL; preferably between about 0.1 μg/mL and 1.0 μg/mL, such as between about 0.19 μg/mL and 0.30 μg/mL.

In some embodiments, the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and a primary human T cell is lower than that of OKT3 antibody.

In some embodiments, the EC50 value of the binding between the anti-CD3 antibody or antigen-binding fragment thereof and a cynomolgus primary T cell is lower than 10 μg/mL; preferably between about 0.1 μg/mL and 5.0 μg/mL, such as between about 0.14 μg/mL and 1.6 μg/mL.

In some embodiments, the chimeric antibody is an activator of a Jurkat T cell, with an EC50 value determined through IL-2 promoter activity greater than 0.01 μg/mL; preferably between about 0.01 μg/mL and 10.0 μg/mL, such as between about 0.01 μg/mL and 5.0 μg/mL.

In some embodiments, the chimeric antibody is an activator of a Jurkat T cell, with an EC50 value determined through IL-2 promoter activity greater than that of OKT3 antibody.

In some embodiments, the chimeric antibody is an activator of primary human T cell, with an EC50 value determined through IFN-γ concentration in the supernatant of the primary human T cell greater than 0.1 μg/mL; preferably between about 0.1 μg/mL and 50.0 μg/mL, such as between about 0.20 μg/mL and 15.0 μg/mL.

In some embodiments, the chimeric antibody is an activator of a primary human T cell, with an EC50 value determined through IFN-γ concentration in the supernatant of the primary human T cell greater than that of OKT3 antibody

In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof does not bind to a NK cell, a monocyte or a B cell.

In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a Fab’, a (Fab’) ₂, an Fv, a single chain Fv (scFv) , and an sdAb.

In some embodiments, the multispecific antibody is in the format of BiTE.

In some embodiments, the multispecific antibody comprise the amino acid sequence of SEQ ID NO: 45 or 46.

In some embodiments, compared to the OKT3 antibody, the anti-CD3 antibody or antigen-binding fragment thereof displays a decreased propensity of inducing cytokine release syndrome.

In another aspect, there is provided a multispecific antibody comprising a first binding moiety and a second binding moiety, wherein the first binding moiety comprises the anti-CD3 antibody or antigen-binding fragment thereof described above, and the second binding moiety is able to bind to an antigen other than CD3.

In some embodiments, the multispecific antibody is a bispecific antibody.

In some embodiments, the antigen is a cell surface antigen.

In some embodiments, the antigen is a tumor antigen.

In some embodiments, the antigen other than CD3 is selected from the group consisting of CD19, CD20, EGFR, BCMA, GPRC5D, EpCAM, DLL3, and HER2.

In some embodiments, the first binding moiety and/or the second binding moiety is selected from the group consisting of a Fab, a Fab’, a (Fab’) ₂, an Fv, a single chain Fv (scFv) , and an sdAb.

In another aspect, there is provided a pharmaceutical composition comprising the anti-CD3 antibody or antigen-binding fragment thereof or the multispecific antibody; and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition further comprises one or more therapeutic agents other than the anti-CD3 antibody or antigen-binding fragment thereof or the multispecific antibody.

In some embodiments, the therapeutic agent is an antibody specific for CD39, CTLA-4, PD-L1, TIM-3, LAG-3 or A2aR.

In another aspect, there is provided a method of enhancing immune function in a subject comprising administrating to the subject in need thereof a therapeutically effective amount of the anti-CD3 antibody or antigen-binding fragment thereof, the multispecific antibody or the pharmaceutical composition.

In another aspect, there is provided a method of treating a cancer in a subject comprising administrating to the subject in need thereof a therapeutically effective amount of the anti-CD3 antibody or antigen-binding fragment thereof, the multispecific antibody, or the pharmaceutical composition.

In another aspect, there is provided use of the anti-CD3 antibody or antigen-binding fragment thereof or the multispecific antibody in the manufacture of a medicament for the treatment of a cancer.

In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof, the multispecific antibody, or the pharmaceutical composition is for use in treating a cancer in a subject in need thereof.

In some embodiments, the cancer is multiple myeloma.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the binding of Mouse Hybridoma on Human CD3.

Figure 2 shows the binding of Mouse anti-CD3 on Cyno CD3.

Figure 3 shows the activity of anti-CD3 mAbs on reporter T cell activation.

Figure 4 shows the activity of anti-CD3 mAbs on primary T cell Activation.

Figure 5 shows the binding of chimeric anti-CD3 antibodies to CD3 on Jurkat cells.

Figure 6 shows the binding of chimeric anti-CD3 antibodies to human CD3 on human primary T cells.

Figure 7 shows the binding of chimeric anti-CD3 antibodies to cyno CD3 on cyno primary T cells.

Figure 8 shows the activity of anti-CD3 chimeric antibodies on reporter T cell activation.

Figure 9 shows the activity of anti-CD3 chimeric antibodies on primary T cell activation.

Figure 10 shows the binding activity of anti-CD3 chimeric antibodies on human NK cells.

Figure 11 shows the binding activity of anti-CD3 chimeric antibodies on human monocytes.

Figure 12 shows the binding activities of anti-CD3 chimeric on human B cells.

Figure 13 shows the binding of BiTEs antibodies to human CD3 on Jurkat cells. Black: Secondary detection antibody; Red: 64F9G7/BCMA T cell engager; Green: SP34/BCMA T cell engager; Orange: 10A7C8/BCMA T cell engager; Blue: 39B12G6/BCMA T cell engager.

Figure 14 shows the binding of BiTEs antibodies to human BCMA on RPMI 8226 cells. Black: Secondary detection antibody; Red: 64F9G7/BCMA T cell engager; Green: SP34/BCMA T cell engager; Orange: 10A7C8/BCMA T cell engager; Blue: 39B12G6/BCMA T cell engager. Figure 15. shows BiTEs induced T cell specific killing on human BCMA expressed RPMI 8226 cells.

DETAILED DESCRIPTION

The present disclosure provides anti-CD3 monoclonal antibodies and their applications. The present disclosure pertains to the amino acid sequences of the heavy chain variable domains (V_H) and the light chain variable domains (V_L) of the mouse anti-CD3 monoclonal antibodies clones: 39B12G6, 10A7C8, and 64F9G7. The present disclosure also provides chimeric anti-CD3 monoclonal antibodies by fusing variable domains of the heavy and light chains of the disclosed clones with constant regions of a human IgG (e.g., IgG1) . The present disclosure provides humanized forms of the heavy chain variable domains (V_H) and the light chain variable domains (V_L) of the mouse anti-CD3 monoclonal antibodies clones. In some examples, CDRs of mouse anti-CD3 monoclonal antibodies are grafted onto human IgG framework sequences, and the acquired humanized variable domains may be or may not be fused to a constant region of a human IgG.

I. Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms used herein and are not meant to limit the scope of the present disclosure.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element" means one element or more than one element.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having. ”

When used herein “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising” , “containing” , “including” , and “having” whenever used herein in the context of an aspect or embodiment of the application can be replaced with the term “consisting of” or “consisting essentially of” to vary scopes of the disclosure.

As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance where two elements are conjoined by “and/or” , a first option refers to the applicability of the first element without the second, a second option refers to the applicability of the second element without the first, and a third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or. ”

Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about. ” Thus, a numerical value typically includes ± 10%of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

The term “antibody, ” “antibody moiety” or “antibody construct” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.

The basic 4-chain antibody unit is a heterotetrameric protein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen-binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (V_H) followed by three constant domains (C_H) for each of the α and γ chains and four C_H domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (V_L) followed by a constant domain at its other end. The V_L is aligned with the V_H and the C_L is aligned with the first constant domain of the heavy chain (C_H1) . Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a V_H and V_L together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds) , Appleton &Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C_H) , immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated α, δ, ε, γ and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in the C_H sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

The term “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

The term “single-domain antibody” or “sdAb” refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs) . The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred herein as “V_HHs” (Variable domain of the heavy chain of the Heavy chain antibody) . Some V_HHs can also be known as nanobodies. Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363: 446-8 (1993) ; Greenberg et al., Nature 374: 168-73 (1995) ; Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond) , 8: 1013-26 (2013) ) . A basic V_HH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain can be referred to as “V_H” and “V_L” , respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies from the Camelid species have a single heavy chain variable region, which is referred to as “V_HH” . V_HH is thus a special type of V_H.

The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR) . The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991) ) . The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, deamidations) that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes) , each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the application can be made by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, phage-display technologies, and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

The terms “full-length antibody, ” “intact antibody, ” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length 4-chain antibodies include those with heavy and light chains including an Fc region. Full-length heavy-chain only antibodies include the heavy chain (such as V_HH) and an Fc region. The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody can have one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab′, F (ab′) ₂ and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8 (10) : 1057-1062 [1995] ) ; single-chain antibody molecules; single-domain antibodies (such as V_HH) , and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V_H) , and the first constant domain of one heavy chain (C_H1) . Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F (ab′) ₂ fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab′fragments differ from Fab fragments by having a few additional residues at the carboxy-terminus of the C_H1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′in which the cysteine residue (s) of the constant domains bear a free thiol group. F (ab′) ₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

The term “constant domain region” or “constant region” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the C_H1, C_H2 and C_H3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ( “κ” ) and lambda ( “λ” ) , based on the amino acid sequences of their constant domains.

“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and-binding site. This fragment consists of a dimer of one heavy-and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) may have the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the V_H and V_L antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) .

“Functional fragments” of the antibodies described herein comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the V_H and V_L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the V_H and V_L domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Nat’l. Acad. Sci. USA 90: 6444-6448 (1993) .

The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat’l. Acad. Sci. USA, 81: 6851-6855 (1984) ) . “Humanized antibody” is used as a subset of “chimeric antibodies” .

“Humanized” forms of non-human (e.g., llama or camelid) antibodies are antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an CDR (hereinafter defined) of the recipient are replaced by residues from an CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, framework ( “FR” ) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions can include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321: 522-525 (1986) ; Riechmann et al., Nature 332: 323-329 (1988) ; and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992) . See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma &Immunol. 1: 105-115 (1998) ; Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995) ; Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994) ; and U.S. Pat. Nos. 6,982,321 and 7,087,409.

The term “hypervariable region, ” “HVR, ” or “HV, ” when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, single-domain antibodies comprise three HVRs (or CDRs) : HVR1 (or CDR1) , HVR2 (or CDR2) , and HVR3 (or CDR3) . HVR3 (or CDR3) displays the most diversity of the three HVRs, and is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363: 446-448 (1993) ; Sheriff et al., Nature Struct. Biol. 3: 733-736 (1996) .

The term “Complementarity Determining Region” or “CDR” are used to refer to hypervariable regions as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) .

A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ) . Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ) . The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The amino acid residues of a single-domain antibody (such as V_HH) are numbered according to the general numbering for VH domains given by Kabat et al. ( “Sequence of proteins of immunological interest” , US Public Health Services, NIH Bethesda, Md., Publication No. 91) , as applied to V_HH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2) : 185-195. According to this numbering, FR1 of a V_HH comprises the amino acid residues at positions 1-30, CDR1 of a V_HH comprises the amino acid residues at positions 31-35, FR2 of a V_HH comprises the amino acids at positions 36-49, CDR2 of a V_HH comprises the amino acid residues at positions 50-65, FR3 of a V_HH comprises the amino acid residues at positions 66-94, CDR3 of a V_HH comprises the amino acid residues at positions 95-102, and FR4 of a V_HH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that-as is well known in the art for V_H domains and for V_HH domains-the total number of amino acid residues in each of the CDRs can vary and cannot correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering cannot be occupied in the actual sequence, or the actual sequence can contain more amino acid residues than the number allowed for by the Kabat numbering) .

“Framework” or “FR” residues are those variable-domain residues other than the HVR residues as herein defined.

As use herein, the term “specifically binds, ” “specifically recognizes, ” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antigen binding protein (such as a mAb) , which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antigen binding protein (such as a mAb) that specifically binds a target (which can be an epitope) is an antigen binding protein (such as a mAb) that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds other targets. In some embodiments, the extent of binding of an antigen binding protein (such as a mAb) to an unrelated target is less than about 10%of the binding of the antigen binding protein (such as a mAb) to the target. In some embodiments, an antigen binding protein specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require, exclusive binding.

The term “specificity” refers to selective recognition of an antigen binding protein (such as a mAb) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term “multispecific” as used herein denotes that an antigen binding protein has polyepitopic specificity (i.e., is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules) . “Bispecific” as used herein denotes that an antigen binding protein has two different antigen-binding specificities. Unless otherwise indicated, the order in which the antigens bound by a bispecific antibody listed is arbitrary. That is, for example, the terms “anti-CD3/BCMA, ” and “anti-BCMA/CD3” can be used interchangeably to refer to bispecific antibodies that specifically bind to both CD3 and BCMA. The term “monospecific” as used herein denotes an antigen binding protein (such as a mAb) that has one or more binding sites each of which bind the same epitope of the same antigen.

“Antibody effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC) ; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors) ; and B cell activation.

“Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) . Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair. Binding affinity can be indicated by KD, K_off, or K_on. The term “K_off” , as used herein, is intended to refer to the off rate constant for dissociation of an antibody (or antigen-binding domain) from the antibody/antigen complex, as determined from a kinetic selection set up, expressed in units of s^- ¹. The term “K_on” , as used herein, is intended to refer to the on rate constant for association of an antibody (or antigen-binding domain) to the antigen to form the antibody/antigen complex, expressed in units of M^-1s^-1. The term equilibrium dissociation constant “KD” , as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, and describes the concentration of antigen required to occupy one half of all of the antibody-binding domains present in a solution of antibody molecules at equilibrium, and is equal to K_off/K_on, expressed in units of M. The measurement of KD presupposes that all binding agents are in solution. In the case where the antigen is tethered to a cell membrane, the corresponding equilibrium rate constant is expressed as EC₅₀, which gives a good approximation of KD.

Binding specificity of the antibody or antigen-binding fragment can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests, peptide scans or FACS.

“EC₅₀” or “EC₅₀ value” represents a concentration required for obtaining 50%of a maximum effect. EC₅₀ can be measured by bioassays such as FACS analysis, cell based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA) .

“Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN^TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

The term “vector, ” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors. ”

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of the disease. The methods of the invention contemplate any one or more of these aspects of treatment.

The term “effective amount” used herein refers to an amount of an agent or a combination of agents, sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

The terms “host cell, ” “host cell line, ” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells, ” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny cannot be completely identical in nucleic acid content to a parent cell, but can contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

The term “pharmaceutical formulation” of “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. A “sterile” formulation is aseptic or free from all living microorganisms and their spores.

It is understood that embodiments of the invention described herein include “consisting” and/or “consisting essentially of” embodiments.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X. ”

The term “about X-Y” used herein has the same meaning as “about X to about Y. ”

II. mouse anti-CD3 antibody

An isolated mouse anti-CD3 construct described herein comprises a monoclonal antibody (mAb) moiety that specifically recognizes or binds to human CD3 (or “anti-CD3 mAb” ) . In some embodiments of the invention, an isolated mouse anti-CD3 antibody is a full-length IgG. In some embodiments, a mouse anti-CD3 mAb of the present disclosure can cross-react with CD3 from species other than human or other proteins which are structurally related to human CD3. In some embodiments, an anti-CD3 mAb of the application is able to specifically recognize or bind to cynomolgus CD3 (or cyno CD3) .

In some embodiments, there is provided a mouse anti-CD3 mAb comprising a heavy chain variable domain (VH) with a heavy chain CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 3 (TYAMN) , SEQ ID NO: 13 (SQYLH) , and SEQ ID NO: 23 (TSYIH) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a heavy chain CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 4 (RIRSKIYNYATFYDDSVKD) , SEQ ID NO: 14 (WINPGDDTTKYNEKFKV) , and SEQ ID NO: 24 (WISPGDVNTKYSEKFKG) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a heavy chain CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 5 (YYGNDWIAK) , SEQ ID NO: 15 (DYGYYFDY) , and SEQ ID NO: 25 (DYGYYFDY) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a light chain variable domain (VL) with a light chain CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 8 (RSSTGAVTTSNYAN) , SEQ ID NO: 18 (KSSQSLLNSRTRKNYLA) , and SEQ ID NO: 28 (KSSQSLLNSRTRKNYLA) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a light chain CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 9 (GTSNRAP) , SEQ ID NO: 19 (WASTRES) , and SEQ ID NO: 29 (WASTRES) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a light chain CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 10 (ALWYSTHYV) , SEQ ID NO: 20 (KQSFILRT) , and SEQ ID NO: 30 (KQSFILRT) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions.

In some embodiments, there is provided a mouse anti-CD3 mAb comprising a heavy chain variable domain (VH) with a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 13, and 23; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 14, and 24; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 5, 15, and 25; and a light chain variable domain (VL) with a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 18, and 28; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 9, 19, and 29; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 10, 20, and 30.

In some embodiments, there is provided a mouse anti-CD3 mAb comprising: 1) a VH comprising the heavy chain CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively, and a VL comprises the light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; 2) a VH comprising the heavy chain CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and a VL comprising the light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively; or 3) a VH comprising the heavy chain CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively, and a VL comprising the light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively.

In some embodiments, there is provided a mouse anti-CD3 mAb comprising: 1) a VH comprising an amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 80%(such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 2, and a VL comprising an amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 7; 2) a VH comprising an amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 12, and a VL comprising an amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 17; or 3) a VH comprising an amino acid sequence of SEQ ID NO: 22 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 22, and a VL comprising an amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 27.

In some embodiments, there is provided a mouse anti-CD3 mAb comprising: 1) a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 1, and a light chain comprising an amino acid sequence of SEQ ID NO: 6 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 6; 2) a heavy chain comprising an amino acid sequence of SEQ ID NO: 11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 11, and a light chain comprising an amino acid sequence of SEQ ID NO: 16 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 16; or 3) a heavy chain comprising an amino acid sequence of SEQ ID NO: 21 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 21, and a light chain comprising an amino acid sequence of SEQ ID NO: 26 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 26.

In some embodiments, there is provided an anti-CD3 antibody, such as an mAb (hereinafter referred to as “competing anti-CD3 antibody or competing anti-CD3 mAb” ) , or an antigen binding fragment thereof, that specifically binds to CD3 competitively with any one of the anti-CD3 mAb described herein. In some embodiments, competitive binding can be determined using an ELISA assay. In some embodiments, the competing anti-CD3 antibody and the anti-CD3 antibody described above bind the same epitope on the CD3.

In some embodiments, the binding specificity of a mouse anti-CD3 mAb of the present disclosure to the CD3 is detected through the binding of a mouse anti-CD3 mAb of the present disclosure to a CD3-expresing cell, such as a Jurkat T cell. In some embodiments, the binding of a mouse anti-CD3 mAb of the present disclosure to a CD3-expresing cell is detected by FACS.

After binding to CD3 on a T cell, a mouse anti-CD3 mAb of the present disclosure is able to induce T cell activation. In some embodiments, the T cell activation is determined through IL-2 or IFN-γ secretion. In other embodiments, the T cell activation is determined through the expression of an IL-2 promoter-driven reporter (e.g., luciferase) .

In some embodiments, EC₅₀ of T cell (e.g., Jurkat T cell) activation by a mouse anti-CD3 mAb of the present disclosure as determined through an IL-2 promoter-driven reporter is greater than 0.01 μg/mL, preferably between about 10^-2 μg/mL and 1.0 μg/mL, such as between about 0.018 μg/mL and 0.82 μg/mL.

In some embodiments, EC₅₀ of T cell activation by a mouse anti-CD3 mAb of the present disclosure as determined through an IL-2 promoter-driven reporter is higher than that of OKT3.

In some embodiments, EC₅₀ of T cell activation by a mouse anti-CD3 mAb of the present disclosure as determined through IFN-γ secretion is greater than 0.1 μg/mL, preferably between about 0.1 μg/mL and 50.0 μg/mL, such as between about 0.50 μg/mL and 10.14 μg/mL.

In some embodiments, EC₅₀ of T cell activation by a mouse anti-CD3 mAb of the present disclosure as determined through IFN-γ secretion is higher than that of OKT3.

III Chimeric anti-CD3 antibody

In some embodiments, the anti-CD3 antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Nat’l. Acad. Sci. USA, 81: 6851-6855 (1984) ) . In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a rodent species, such as mouse) and a human constant region. Chimeric antibodies include antigen-binding fragments thereof.

In some embodiments, the chimeric anti-CD3 antibody provided herein comprises the variable region of the mouse anti-CD3 antibody of the present disclosure and a human IgG (e.g., IgG1) constant region comprising the amino acid sequence of SEQ ID NO: 43.

In some embodiments, the chimeric anti-CD3 antibody provided herein is a humanized antibody comprising the CDRs of the mouse anti-CD3 antibody of the present disclosure, human FRs and a human IgG (e.g., IgG1) constant region comprising the amino acid sequence of SEQ ID NO: 43.

In some embodiments, there is provided a chimeric anti-CD3 mAb comprising: 1) a VH comprising an amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 32, and a VL comprising an amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 34; 2) a VH comprising an amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 36, and a VL comprising an amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least about 80%(such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 38; or 3) a VH comprising an amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 40, and a VL comprising an amino acid sequence of SEQ ID NO: 42 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 42.

In some embodiments, the chimeric anti-CD3 antibody provided herein comprises: 1) a heavy chain comprising an amino acid sequence of SEQ ID NO: 31 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 31, and a light chain comprising an amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 33; 2) a heavy chain comprising an amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 35, and a light chain comprising an amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 37; or 3) a heavy chain comprising an amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 39, and a light chain comprising an amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 41.

In some embodiments, the EC₅₀ value of the binding between a chimeric anti-CD3 antibody or antigen-binding fragment thereof of the present disclosure and a Jurkat T cell is lower than 1 μg/mL; preferably between about 0.1 μg/mL and 1.0 μg/mL, such as between about 0.20 μg/mL and 0.45 μg/mL.

In some embodiments, the EC₅₀ value of the binding between a chimeric antibody or antigen-binding fragment thereof of the present disclosure and a Jurkat T cell is lower than that of OKT3 antibody.

In some embodiments, the EC50 value of the binding between a chimeric antibody or antigen-binding fragment thereof of the present disclosure and a primary human T cell is lower than 1 μg/mL; preferably between about 0.1 μg/mL and 1.0 μg/mL, such as between about 0.19 μg/mL and 0.30 μg/mL.

In some embodiments, the EC50 value of the binding between a chimeric antibody or antigen-binding fragment thereof of the present disclosure and a primary human T cell is lower than that of OKT3 antibody.

In some embodiments, the EC50 value of the binding between a chimeric antibody or antigen-binding fragment thereof of the present disclosure and a cynomolgus primary T cell is lower than 10 μg/mL; preferably between about 0.1 μg/mL and 5.0 μg/mL, such as between about 0.14 μg/mL and 1.6 μg/mL.

After binding to CD3 on a T cell, a chimeric anti-CD3 mAb of the present disclosure is able to induce T cell activation. In some embodiments, the T cell activation is determined through IL-2 or IFN-γ secretion. In other embodiments, the T cell activation is determined through the expression of an IL-2 promoter-driven reporter (e.g., luciferase) .

In some embodiments, EC₅₀ of T cell (e.g., Jurkat T cell) activation by a chimeric anti-CD3 mAb of the present disclosure as determined through an IL-2 promoter-driven reporter is greater than 0.01 μg/mL, preferably between about 10^-2 μg/mL and 10 μg/mL, such as between about 0.01 μg/mL and 5.0 μg/mL.

In some embodiments, EC₅₀ of T cell activation by a chimeric anti-CD3 mAb of the present disclosure as determined through an IL-2 promoter-driven reporter is higher than that of OKT3.

In some embodiments, EC₅₀ of T cell activation by a chimeric anti-CD3 mAb of the present disclosure as determined through IFN-γ secretion is greater than 0.1 μg/mL, preferably between about 0.1 μg/mL and 50.0 μg/mL, such as between about 0.20 μg/mL and 15.0 μg/mL.

In some embodiments, EC₅₀ of T cell activation by a chimeric anti-CD3 mAb of the present disclosure as determined through IFN-γ secretion is higher than that of OKT3.

In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) , e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008) , and are further described, e.g., in Riechmann et al., Nature 332: 323-329 (1988) ; Queen et al., Proc. Nat’l Acad. Sci. USA 86: 10029-10033 (1989) ; US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (describing SDR (a-CDR) grafting) ; Padlan, Mol. Immunol. 28: 489-498 (1991) (describing “resurfacing” ) ; Dall’Acqua et al., Methods 36: 43-60 (2005) (describing “FR shuffling” ) ; and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) (describing the “guided selection” approach to FR shuffling) .

In some embodiments, the mAbs are modified, such as humanized, without diminishing the native affinity of the domain for antigen and while reducing its immunogenicity with respect to a heterologous species. For example, the amino acid residues of the antibody heavy chain and light chain variable domains (VH and VL) can be determined, and one or more of the mouse amino acids, for example, in the framework regions, are replaced by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e., the humanization does not significantly affect the antigen binding capacity of the resulting polypeptide. Humanization of mouse monoclonal antibodies requires the introduction and mutagenesis of a limited amount of amino acids in two chains, the light and the heavy chain and the preservation of the assembly of both chains.

IV Construct comprising the anti-CD3 mAb

The anti-CD3 construct comprising the anti-CD3 mAb can be of any possible format.

In some embodiments, the anti-CD3 construct comprising the anti-CD3 mAb can further comprise additional polypeptide sequences, such as one or more antibody moieties. Such additional polypeptide sequences can or cannot change or otherwise influence the (biological) properties of the anti-CD3 mAb, and can or cannot add further functionality to the anti-CD3 mAb described herein. In some embodiments, the additional polypeptide sequences confer one or more desired properties or functionalities to the anti-CD3 mAb of the application.

In some embodiments, the additional polypeptide sequences can be a second antibody moiety (such as sdAb, scFv) that specifically recognizes a second antigen. In some embodiments, the second antigen is not CD3. In some embodiments, the second antibody moiety specifically recognizes the same epitope on CD3 as the anti-CD3 mAb described herein. In some embodiments, the second antibody moiety specifically recognizes a different epitope on CD3 as the anti-CD3 mAb described herein.

In some embodiments, the additional polypeptide sequences can increase the molecule’s stability, solubility, or absorption, reduce immunogenicity or toxicity, eliminate or attenuate undesirable side effects, and/or confer other advantageous properties to and/or reduce undesired properties of the anti-CD3 construct of the invention, compared to the anti-CD3 mAb described herein per se.

In some embodiments, an anti-CD3 mAb is a full-length IgG. In some embodiments, the anti-CD3 mAb comprises the constant regions of IgG, such as any of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the constant region is human constant region. In some embodiments, the constant region is human IgG1 constant region.

In some embodiments, the full-length anti-CD3 IgG is rodent, chimeric, human, partially humanized, or fully humanized.

In some embodiments, the anti-CD3 mAb is not a full-length anti-CD3 IgG. In some embodiments, the anti-CD3 mAb does not comprise an Fc domain, and do not have the ability to induce ADCC in vivo.

In some embodiments, the anti-CD3 construct comprises an anti-CD3 mAb described herein fused to one or more other antibody moiety (such as an antibody moiety that specifically recognizes another antigen) . The one or more other antibody moiety can be of any antibody or antibody fragment format, such as an sdAb, a full-length antibody, a Fab, a Fab’, a (Fab’) ₂, an Fv, a single chain Fv (scFv) , an scFv-scFv, a minibody, or a diabody. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003) . For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York) , pp. 269-315 (1994) ; see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F (ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. For a review of multispecific antibodies, see Weidle et al., Cancer Genomics Proteomics, 10 (1) : 1-18, 2013; Geering and Fussenegger, Trends Biotechnol., 33 (2) : 65-79, 2015; Stamova et al., Antibodies, 1 (2) : 172-198, 2012. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404, 097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003) ; and Hollinger et al., Proc. Nat’l. Acad. Sci. USA 90: 6444-6448 (1993) . Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003) . Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage) , as described herein. In some embodiments, the one or more other antibody moiety is antibody mimetics, which are small engineered proteins, comprising antigen-binding domains reminiscent of antibodies (Geering and Fussenegger, Trends Biotechnol., 33 (2) : 65-79, 2015) . These molecules are derived from existing human scaffold proteins and comprise a single polypeptide. Exemplary antibody mimetics that can be comprised within the anti-CD3 construct described herein can be, but are not limited to, a designed ankyrin repeat protein (DARPin; comprising 3-5 fully synthetic ankyrin repeats flanked by N-and C-terminal Cap domains) , an avidity multimer (avimer; a high-affinity protein comprising multiple A domains, each domain with low affinity for a target) , or an Anticalin (based on the scaffold of lipocalins, with four accessible loops, the sequence of each can be randomized) .

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983) ) , WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991) ) , and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168) . Multi-specific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1) ; cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985) ) ; using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148 (5) : 1547-1553 (1992) ) ; using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Nat’l. Acad. Sci. USA, 90: 6444-6448 (1993) ) ; and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152: 5368 (1994) ) ; and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991) ; and creating polypeptides comprising tandem single-domain antibodies (see, e.g., U.S. Patent Application No. 20110028695; and Conrath et al. J. Biol. Chem., 2001; 276 (10) : 7346-50) . Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies, ” are also included herein (see, e.g., US2006/0025576A1) .

In some embodiments, the two or more antibody moieties within the anti-CD3 construct can be optionally connected by a peptide linker. The length, the degree of flexibility and/or other properties of the peptide linker (s) used in the anti-CD3 construct can have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers can be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiment, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

In some embodiments, an isolated antibody or antigen binding fragment of the application is a bispecific or multispecific antibody that comprises an anti-CD3 IgG described herein fused to a second antibody moiety, wherein the second antibody moiety binds specifically to another antigen, preferably a tumor-associated antigen or a tumor specific antigen, such as CD19, CD20, EGFR, BCMA, GPRC5D, EpCAM, DLL3, and HER2.

In some embodiments, the bispecific antibody is in the format of a Bispecific T Cell Engager (BiTE) . A “BiTE” generally refers to a single polypeptide chain molecule that has two antigen binding domains, one of which binds to an immune effector cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell (e.g., a tumor cell) . In some embodiments, a BiTE is a fusion protein consisting of two single-chain variable fragments (scFvs) joined by a linker. In some embodiments, one of the antigen binding domain (a first scFv) is specific for an immune cell, such as a T cell antigen, such as the CD3 receptor, expressed on the surface of T cells. In some embodiments, the second antigen binding domain (a second scFv) binds to a tumor cell via a tumor-specific molecule. Accordingly, BiTEs are able to form a link between T cells and tumor cells by virtue of their specificities for an antigen on the T cell and an antigen on the tumor cell. This leads to activation of the T-cells and may trigger the T cells to exert their cytotoxic effects on tumor cells, independently of MHC I or co-stimulatory molecules. In some embodiments, the second antigen is selected from the group consisting of CD19, EpCAM, CD20, CD123, BCMA, B7-H3, and PSMA.

In some embodiments, the BiTE comprises a first scFv from an anti-CD3 antibody of the present invention, and a second scFv from an anti-BCMA antibody, such as an anti-human BCMA antibody described in WO 2014/140248A1. In some embodiments, the BiTE comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the BiTE comprises the amino acid sequence of SEQ ID NO: 45 without the C terminal His tag. In some embodiments, the BiTE comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the BiTE comprises the amino acid sequence of SEQ ID NO: 46 without the C terminal His tag. In some embodiments, the BiTE is able to activate T cells and/or promotes the killing of target cells expressing antigen BCMA by T cells. In some embodiments, the target cell is a tumor cell of Multiple myeloma (MM) .

Human monoclonal antibodies specific for CD3, or antigen binding fragments thereof, can be conjugated to an agent, such as an effector molecule or detectable marker, using any number of means known to those of skill in the art. Both covalent and non-covalent attachment means may be used. Conjugates include, but are not limited to, molecules in which there is a covalent linkage of an effector molecule or a detectable marker to an antibody or antigen binding fragment that specifically binds CD3. One of skill in the art will appreciate that various effector molecules and detectable markers can be used, including (but not limited to) chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents such as ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S and other labels, target moieties and ligands, etc. The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect.

Effector molecules and detectable markers can be linked to an antibody or antigen binding fragment of interest using any number of means known to those of skill in the art. Both covalent and non-covalent attachment means may be used. The procedure for attaching an effector molecule or detectable marker to an antibody or antigen binding fragment varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH) , free amine (-NH₂) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule or detectable marker.

The antibodies or antigen binding fragments disclosed herein can be derivatized, for example, by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies) . Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate) . Such linkers are commercially available.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules) , toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or antigen binding fragment or other polypeptide. For example, the antibody or antigen binding fragment can be conjugated with small molecular weight drugs such as Monomethyl Auristatin E (MMAE) , Monomethyl Auristatin F (MMAF) , maytansine, maytansine derivatives, including the derivative of maytansine known as DM1 (also known as mertansine) , or other chemotherapeutic agents to make an antibody drug conjugate (ADC) . In several embodiments, various chemotherapeutic agents described herein can be conjugated to the provided antibodies to generate a conjugate.

V Anti-CD3 antibody variants

In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it can be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of “Preferred substitutions. ” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions, ” and as further described below in reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, or decreased immunogenicity.

Table 1. Amino acid substitutions

Amino acids can be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody) . Generally, the resulting variant (s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity) .

Alterations (e.g., substitutions) can be made in HVRs, e.g., to improve antibody affinity. Such alterations can be made in HVR “hotspots, ” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) ) , and/or SDRs (a-CDRs) , with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001) ) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) . A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In some embodiments, substitutions, insertions, or deletions can occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs. Such alterations can be outside of HVR “hotspots” or CDRs. In some embodiments of the variant V_HH sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

In some embodiments, an anti-CD3 construct provided herein is altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the anti-CD3 construct comprises an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15: 26-32 (1997) . The oligosaccharide can include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc) , galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-CD3 construct of the present application can be made in order to create antibody variants with certain improved properties.

In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the anti-CD3 construct provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In some embodiments, an anti-CD3 construct provided herein can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG) , copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers) , and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol) , polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight, and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

VI. Pharmaceutical compositions

Further provided by the present application are pharmaceutical compositions comprising any one of the anti-CD3 constructs (such as anti-CD3 IgG, anti-CD3 fragment, full-length anti-CD3 IgG, anti-CD3 bispecific antibody) , and optionally a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing an anti-CD3 construct described herein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) , in the form of lyophilized formulations or aqueous solutions.

The pharmaceutical composition is preferably to be stable, in which the anti-CD3 construct comprising anti-CD3 mAb described here essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993) . Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation can be kept at 40℃ for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8℃, generally the formulation should be stable at 30℃ or 40℃ for at least 1 month, and/or stable at 2-8℃ for at least 2 years. Where the formulation is to be stored at 30℃, generally the formulation should be stable for at least 2 years at 30℃, and/or stable at 40℃ for at least 6 months. For example, the extent of aggregation during storage can be used as an indicator of protein stability. In some embodiments, the stable formulation of anti-CD3 construct described herein can comprise less than about 10% (preferably less than about 5%) of the anti-CD3 construct present as an aggregate in the formulation.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers (e.g., sodium chloride) , stabilizers, metal complexes (e.g., Zn-protein complexes) ; chelating agents such as EDTA and/or non-ionic surfactants.

Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes) ; and/or nonionic surfactants such as TWEEN^TM, polyethylene glycol (PEG) , and PLURONICS^TM or polyethylene glycol (PEG) .

Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use in the present application include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers can comprise histidine and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typically present in a range from 0.2%-1.0% (w/v) . The addition of a preservative can, for example, facilitate the production of a multi-use (multiple-dose) formulation. Suitable preservatives for use in the present application include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide) , benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

Tonicity agents, sometimes known as “stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1%to 25%by weight, preferably 1%to 5%, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated above) ; amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol) , polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose) ; trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition can be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means. In some embodiments, the pharmaceutical composition is administered locally, such as intratumorally.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate) , or poly (vinylalcohol) ) , polylactides (U.S. Pat. No. 3,773,919) , copolymers of L-glutamic acid and. Ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT^TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) , and poly-D- (-) -3-hydroxybutyric acid.

The pharmaceutical compositions herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. In some embodiments, the therapeutic agent or immunosuppressive agent other than an antibody provided herein is an antibody or an antigen-binding fragment thereof specific for CD39, CTLA-4, PD-L1, TIM-3, LAG-3 or A2aR.

The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 18^th edition.

In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.

VII. Methods of uses or applications

The anti-CD3 construct comprising mAb specifically recognizing CD3 as described herein (such as such as anti-CD3 IgG, anti-CD3 fragment, full-length anti-CD3 IgG, anti-CD3 bispecific antibody) , and the compositions (such as pharmaceutical compositions) thereof are useful for a variety of applications, such as in diagnosis, molecular assays, and therapy.

One aspect of the invention provides a method of treating a CD3 related disease or a condition in an individual in need thereof, comprising administering to the individual an effective amount of a pharmaceutical composition comprising the anti-CD3 construct described herein. Another aspect of the invention provides a method of treating a disease or a condition associated with expression of specific antigen in an individual in need thereof, where T cell can be crosslinked with the target cell by a CD3-BsAb comprising the anti-CD3 antibody of the present invention and an antibody targeting the specific antigen. In some embodiments, the disease or the condition is tumor or cancer. In some embodiments, the tumor or cancer is a solid tumor or cancer.

The application contemplates, in part, protein constructs (such as anti-CD3 full-length IgG, anti-CD3 bispecific antibody) , nucleic acid molecules and/or vectors encoding thereof, host cells comprising nucleic acid molecules and/or vectors encoding thereof, that can be administered either alone or in any combination with another therapy, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In some embodiments, prior to administration of the anti-CD3 construct, they can be combined with suitable pharmaceutical carriers and excipients that are well known in the art. The compositions prepared according to the disclosure can be used for the treatment or delaying of worsening of cancer.

In some embodiments, there is provided a method of treating cancer comprising administering to the individual an effective amount of a pharmaceutical composition comprising an isolated anti-CD3 construct comprising a mAb specifically recognizing CD3 (such as anti-CD3 full-length IgG, anti-CD3 bispecific antibody) . In some embodiments, the cancer is a solid tumor (such as lung cancer) . In some embodiments, the pharmaceutical composition is administered systemically (such as intravenously) . In some embodiments, the pharmaceutical composition is administered locally (such as intratumorally) . In some embodiments, the method further comprises administering to the individual an additional cancer therapy (such as surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof) . In some embodiments, the individual is a human. In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing) ; (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) prolonging survival; (8) prolonging time to cancer progression; and (9) preventing, inhibiting, or reducing the likelihood of the recurrence of a cancer. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (uninfected by the oncolytic VV) death rate of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the tumor size. In some embodiments, the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10%(including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis. In some embodiments, the method of prolonging survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging time to cancer progression mediated by the pharmaceutical composition described herein can prolongs the time to cancer progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

The methods described herein are suitable for treating a variety of cancers, including both solid cancer and liquid cancer. The methods are applicable to cancers of all stages, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The methods described herein can be used as a first therapy, second therapy, third therapy, or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (such as T-cell therapy) , bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting (i.e., the method can be carried out before the primary/definitive therapy) . In some embodiments, the method is used to treat an individual who has previously been treated. In some embodiments, the cancer has been refractory to prior therapy. In some embodiments, the method is used to treat an individual who has not previously been treated.

In some embodiments, the method is suitable for treating tumors or cancers with aberrant CD3 expression, activity and/or signaling. In some embodiments, the method is suitable for treating tumors or cancers associated with expression of a tumor-associated antigen or a tumor specific antigen, such as CD19, CD20, EGFR, BCMA, GPRC5D, EpCAM, DLL3, and HER2. In some embodiments, the tumor or cancer includes, by way of non-limiting example, a bladder cancer, a cervical cancer, a colon cancer, a colorectal cancer, a gastric cancer, a liver cancer, a lung cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a kidney cancer, a breast cancer, a head and neck cancer, a skin cancer, a sarcoma, a brain tumor, a brain and spinal cord cancer, an adrenal cancer, a uterine cancer, a neuroblastoma, a small round cell tumor, a peripheral nerve sheath tumor, a bone cancer, a rhabdoid tumor, a lymphoma, a multiple myeloma, a leukemia, a neuroendocrine tumor, and a melanoma.

In some embodiments, the pharmaceutical composition is administered systemically (such as intravenously) . In some embodiments, the pharmaceutical composition is administered locally (such as intratumorally) . In some embodiments, the method further comprises administering to the individual an additional cancer therapy (such as surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof) . In some embodiments, the individual is a human. In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing) ; (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) prolonging survival; (8) prolonging time to cancer progression; and (9) preventing, inhibiting, or reducing the likelihood of the recurrence of a cancer. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (uninfected by the oncolytic VV) death rate of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the tumor size. In some embodiments, the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis. In some embodiments, the method of prolonging survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging time to cancer progression mediated by the pharmaceutical composition described herein can prolongs the time to cancer progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

Dosages and desired drug concentrations of pharmaceutical compositions of the present application can vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics, ” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.

When in vivo administration of the anti-CD3 construct comprising an anti-CD3 mAb described herein are used, normal dosage amounts can vary from about 10 ng/kg up to about 100 mg/kg of mammal body weight or more per day, preferably about 1 mg/kg/day to 10 mg/kg/day, such as about 1-3 mg/kg/day, about 2-4 mg/kg/day, about 3-5 mg/kg/day, about 4-6 mg/kg/day, about 5-7 mg/kg/day, about 6-8 mg/kg/day, about 6-6.5 mg/kg/day, about 6.5-7 mg/kg/day, about 7-9 mg/kg/day, or about 8-10 mg/kg/day, depending upon the route of administration. It is within the scope of the present application that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue can necessitate delivery in a manner different from that to another organ or tissue. Moreover, dosages can be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens can be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

In some embodiments, the pharmaceutical composition is administered for a single time (e.g. bolus injection) . In some embodiments, the pharmaceutical composition is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times) . If multiple administrations, they can be performed by the same or different routes and can take place at the same site or at alternative sites. The pharmaceutical composition can be administered twice per week, 3 times per week, 4 times per week, 5 times per week, daily, daily without break, once per week, weekly without break, once per 2 weeks, once per 3 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, or once per year. The interval between administrations can be about any one of 24h to 48h, 2 days to 3 days, 3 days to 5 days, 5 days to 1 week, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 months to 3 months, 3 months to 6 months, or 6 months to a year. Intervals can also be irregular (e.g., following tumor progression) . In some embodiments, there is no break in the dosing schedule. In some embodiments, the pharmaceutical composition is administered every 4 days for 4 times. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

VIII. Methods of preparation

The anti-CD3 construct (such as anti-CD3 monoclonal antibody) described herein can be prepared using any methods known in the art or as described herein.

Rodent monoclonal antibodies can be obtained using methods known in the art such as by immunizing a rodent species (such as mouse or rat) and obtaining hybridomas therefrom, or by cloning a library of Fab fragment or single chain Fc (scFv) using molecular biology techniques known in the art and subsequent selection by ELISA or FACS with individual clones of unselected libraries or by using phage display.

For recombinant production of the monoclonal antibodies, the nucleic acids encoding the monoclonal antibodies are isolated or synthesized and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.

Developing an anti-human CD3 antibody is very important in the field of immunotherapy. The anti-human CD3 antibody which only binds to CD3 is not enough. The anti-human CD3 antibody has to further activate primary T cells. The antigen, human CD3, is composed of four distinct chains, a CD3γ chain, a CD3δ chain, two CD3ε chains, and CD3ζ-chain. It is impossible to express and purify human CD3 protein in vitro with all four distinct chains. Primary human T cells which have endogenous membrane CD3 expression are isolated from peripheral blood mononuclear cells (PBMC) . They are a good choice for the immunogen to immune the animal to get anti-human CD3 antibody. However, using primary human T cells is not as good as using the purified human CD3 protein as the immunogen. The magnitude of all proteins besides human CD3 on the membrane of primary human T cells induces adaptive immune responses in B cells upon exposure to an immunized animal. The B cells of the immunized animal will generate various antibodies targeting these proteins respectively. To distinguish anti-human CD3 antibody among the antibody pool is not an easy task. Based on this, it is very difficult to get anti-human CD3 antibody. Globally, the number of anti-human CD3 antibody is limited. The most famous anti-human CD3 antibody, OKT3, does not bind to non-human primate CD3 protein. Safety issues are also arisen as OKT3 initiates the cytokine releasing syndrome due to its high T cell activation activity. Our anti-human CD3 not only bind to human CD3 protein, it also induces T cell activations after binding to CD3 proteins in T cells. Comparing to OKT3, the higher binding affinity of our anti-human CD3 on CD3, but lower T cell activation provides a unique feature on its potential application on immunotherapy. Holding T cell tightly with higher affinity binding to CD3 expressed on T cells, its lower T cell activation activity makes T cells extremely effective at their natural killing function on tumor cells with lower side effects comparing to OKT3. It could establish a proper therapeutic window between balancing its efficacy and safety windows. Furthermore, it shows cross-reactivity against non-human primate CD3 protein.

Example 1. Preparation of mouse anti-human CD3 monoclonal antibody

For animal immunization, the immunogen is primary T cells isolated from human PBMC. Five female Balb/c mice were injected intraperitoneally with 5x10⁶ primary huma T cells. Then, mice were boost intraperitoneally with 5x10⁶ primary huma T cells every two weeks up to 2 times.

Four mice were chosen for hybridoma fusion and screening. The isolated spleens were made into homogenized single cell suspension, and the single cell suspensions were also made for the myeloma cells (SP 2/0 cells) . 8.9×10⁷ spleen cell and 4.1×10⁷ SP 2/0 cells were fusion through electrofusion method. The fused cells from each hybridoma fusion were re-suspend in 100 ml DMEM/10%FBS medium containing thymus nucleoside pyrimidine, hypoxanthine and aminopterin hybridoma selective reagent. The cell suspension was distributed 100 μl each into fifty 96 wells. The 96 well plates were cultured in 37℃ incubator with 6%CO2 concentration for 7 days. Then the hybridoma supernatants were tested by FACS binding to detect the binding of the anti-human CD3 antibody to CD3 expressed in Jurkat cells. The hybridoma supernatants from 39B12G6, 10A7C8, and 64F9G7 together with positive control, commercialized anti-human CD3 antibody (Clone UCHT1) were confirmed with solid human CD3 binding in a single dose (Figure 1) . Negative controls, PBS or mouse IgG isotype control, did not have any binding activity. Mouse anti-human CD3 monoclonal antibodies, 39B12G6, 10A7C8, and 64F9G7, were purified from hybridoma supernatants. It was confirmed that mouse anti-human CD3 monoclonal antibodies, 39B12G6, 10A7C8, and 64F9G7, bound to cynomolgus CD3 in a single dose FACS study (Figure 2) by using primary T cells isolated from cynomolgus. Negative controls, PBS or mouse IgG isotype control, did not have any binding activity on primary T cells isolated from cynomolgus.

Example 2. Activation of Jurkat cell with mouse anti-human CD3 monoclonal antibody

Jurkat cells are immortalized cell lines of human T lymphocytes, which are widely used to study the signal transduction of T cell activation. After T cells are activated, they secrete interleukin 2 (IL-2) . A T activation reporter cell line was generated by constructing an IL-2 promoter-driven luciferase reporter gene into the Jurkat cells (Jurkat/IL 2 promotor-Luciferase) . Measuring T cell activation by luciferase intensity is a very reliable method to evaluate T cell activation. 39B12G6, 10A7C8, and 64F9G7 were tested in the T activation reporter study. 8,000 CHO-K1 overexpressed with human CD80 (CHO-K1/CD80) cells were seeded into each well of 384-well plate. After incubating overnight, 40,000 Jurkat/IL 2 promotor-Luciferase cells together with tested antibody samples were added into the well. The luciferase intensity in each well was read by a plater-reader after 5 hours. All mouse anti-human CD3 monoclonal antibodies initiated a concentration-dependent luciferase signals in the T activation reporter cell line (Figure 3) . The EC50 values for reporter T cell activations were range from 0.018 μg/ml to 0.82 μg/ml (Table 2) .

Table 2 Anti-human CD3 hybridomas on reporter T cell activation

Example 3. Activation of primary T cell with mouse anti-human CD3 monoclonal antibody 39B12G6, 10A7C8, 64F9G7, and OKT3 were coated on 96 well plate and incubated overnight at 4 ℃. 2 x 105 Primary human pan-T cells derived from peripheral blood mononuclear cells by using human Pan T Cell Isolation Kit (Miltenyi: Cat. #: 130-096-535) were added into the coated wells and incubated 72 hours at 37 ℃ in a humidified atmosphere with 5%CO2. IFN-γ in the supernatants of each well were measured by using Human IFN gamma kit from Cisbio. 39B12G6, 10A7C8, 64F9G7, and OKT3 initiated a concentration-dependent IFN-γ releasing in the primary T cells (Figure 4) . The EC₅₀ values for primary T cell activations were range from 0.50 μg/ml to 10.14 μg/ml (Table 3) .

Table 3 Anti-human CD3 hybridomas on primary T cell activation

Example 4. Isotype analysis

The express mouse isotype ELISA kit (Clonotyping System-HRP, SouthernBiotech) was used to identify the isotype of the monoclonal antibodies. TRIzol (Ambion) was then used to extract total RNA of the monoclones from 3 × 10⁶ -5 × 10⁶ of the hybridoma cells. Isotype specific primer and universal primer (PrimeScriptTM 1stStrand cDNA Synthesis Kit, Takara) were used to reverse transcript the RNA into cDNA, then RACE PCR (GenScript) was applied to amplify the variable region of the antibody heavy chain and light chain, and PCR products were subcloned into the pMD18-T vector system (Takara) . Vector specific primers were used to validate and sequence the inserted segment. Finally, variable region DNA/protein sequence of 39B12G6, 10A7C8, and 64F9G7, were obtained. 39B12G6 is mouse IgG2b kappa isotype. 10A7C8 is mouse IgG1 kappa isotype. 64F9G7 is mouse IgG1 lambda isotype. The chimeric anti-human CD3 monoclonal antibodies (39B12G6, 10A7C8, or 64F9G7 chimeric) were made by fusing variable domains of the heavy and light chains of 39B12G6, 10A7C8, or 64F9G7 with the constant region of human IgG1.

Example 5. Binding affinity of chimeric anti-human CD3 monoclonal antibody

Binding affinities of the chimeric anti-human CD3 monoclonal antibodies human or cynomolgus CD3 were determined using a fluorescence-activated cell sorting (FACS) -based assay. The chimeric anti-human CD3 monoclonal antibodies and OKT3 were prepared (starting at 7.5 μg/ml, 2-fold serial dilution with 10 concentrations) as primary antibodies for FACS analysis. Jurkat cells were dissociated from adherent culture flasks. Human primary T cells were purified from human PBMC by using human Pan T Cell Isolation Kit (Miltenyi: Cat. #: 130-096-535) . Dissociated Jurkat cells, human primary T cells, or cynomolgus PBMC were mixed with varying concentrations of the mouse anti-human CD3 monoclonal antibodies (both in a 96-well plate) . The mixture was equilibrated for 30 minutes at room temperature, washed three times with FACS buffer (PBS containing 1%BSA) . F (ab’) 2 goat anti-human IgG Fc PE (Thermo: Cat. #: H10104) as the secondary antibody for the chimeric anti-human CD3 monoclonal antibodies or goat anti-mouse IgG PE (Biolegend: Cat. #: 405307) was added to incubate at room temperature for 45 min. Finally, the cells were washed 3 time with PBS, and the signal was read out by FACS BD Calibur. Data was analyzed with PRISMTM (GraphPad Software, San Diego, CA) using non-linear regression and EC50 values were calculated. As shown in Figure 5 and Table 4, the FACS study demonstrated that the chimeric anti-human CD3 monoclonal antibodies bound to human CD3 expressed in Jurkat cells with EC₅₀ values range 0.20 μg/ml to 0.44 μg/ml. 64F9G7 had a higher binding affinity to CD3 expressed on Jurkat cells comparing to that of OKT3.

Table 4 Anti-human CD3 monoclonal antibodies binding on Jurkat cells

As shown in Figure 6 and Table 5, the FACS study demonstrated that the chimeric anti-human CD3 monoclonal antibodies bound to human CD3 expressed in human primary T cells with EC₅₀ values range 0.19 μg/ml to 0.30 μg/ml. All the chimeric anti-human CD3 monoclonal antibodies had higher binding affinities to CD3 expressed on human primary T cells comparing to that of OKT3.

Table 5 Anti-human CD3 monoclonal antibodies binding on human primary T cells

The chimeric anti-human CD3 monoclonal antibodies also bound to cynomolgus CD3. As shown in Figure 7 and Table 6, the FACS study demonstrated that the chimeric anti-human CD3 monoclonal antibodies bound to cynomolgus PBMC with EC₅₀ values range 0.14 μg/ml to 1.6 μg/ml. However, OKT3 did not bind to cynomolgus CD3.

Table 6 Anti-human CD3 monoclonal antibodies binding on cynomolgus PBMC

Example 6. Activation of Jurkat cell with chimeric anti-human CD3 monoclonal antibody

The chimeric anti-human CD3 monoclonal antibodies were tested in the T activation reporter study. 8,000 CHO-K1 overexpressed with human CD80 (CHO-K1/CD80) cells were seeded into each well of 384-well plate. After incubating overnight, 40,000 Jurkat/IL-2 promotor-Luciferase cells together with tested antibody samples were added into the well. The luciferase intensity in each well was read by a plater-reader after 5 hours. All chimeric anti-human CD3 monoclonal antibodies initiated a concentration-dependent luciferase signals in the T activation reporter cell line (Figure 8) . The EC₅₀ values for reporter T cell activations were range from 0.047 μg/ml to 3.64 μg/ml (Table 7) . As 39B12G6, 10A7C8, and 64F9G7 were from the class of mouse IgG1 (mIgG1) or mouse IgG2b (mIgG2b) , the nonrelevant mouse IgG1 and mouse IgG2b were used as the negative controls in the study. Mouse IgG1 and mouse IgG2b antibodies did not initiate a concentration-dependent luciferase signals in the T activation reporter cell line.

Table 7 Anti-human CD3 chimeric on reporter T cell activation

Example 7. Activation of primary T cell with chimeric anti-human CD3 monoclonal antibody

The chimeric anti-human CD3 monoclonal antibodies and OKT3 were coated on 96 well plate and incubated overnight at 4 ℃. 2 x 10⁵ Primary human pan-T cells derived from human peripheral blood mononuclear cells by using human Pan T Cell Isolation Kit (Miltenyi: Cat. #: 130-096-535) were added into the coated wells and incubated 72 hours at 37 ℃ in a humidified atmosphere with 5%CO₂. IFN-γ in the supernatants of each well were measured by using Human IFN gamma kit from Cisbio. The chimeric anti-human CD3 monoclonal antibodies, and OKT3 initiated a concentration-dependent INF-γ releasing in the primary T cells (Figure 9) . The EC₅₀ values for primary T cell activations were range from 0.20 μg/ml to 13.73 μg/ml (Table 8) . 64F9G7 had less primary T cell activation than that of OKT3. It will produce less unnecessary side effects in the potential immunotherapy due to overreactive of T cell activation.

Table 8 Anti-human CD3 chimeric on primary T cell activation

Example 8. Target specificity

Target specificity of the chimeric anti-human CD3 monoclonal antibodies were tested on immune cells isolated from human peripheral blood mononuclear cells. 5 μg/ml of the chimeric anti-human CD3 monoclonal antibodies and OKT3 were prepared as primary antibodies for FACS analysis. 2 μg/ml of rat anti-human IgG Fc PE (Biolegend: Cat. #: 410708) as the secondary antibody for the chimeric anti-human CD3 monoclonal antibodies. No significant bindings were detected in 7-ADD (Biolegend: Cat. #: 420403) negative, CD45 (Biolegend: Cat. #: 368540) positive, and CD16 (Biolegend: Cat. #: 302016) /CD56 (Biolegend: Cat. #: 362504) positive NK cells (Figure 10) . 5 μg/ml of the chimeric anti-human CD3 monoclonal antibodies and OKT3 were prepared as primary antibodies for FACS analysis. 2 μg/ml of F (ab’) 2 goat anti-human IgG Fc PE (Thermo: Cat. #: H10104) as the secondary antibody for the chimeric anti-human CD3 monoclonal antibodies. No significant bindings were detected in 7-ADD (Biolegend: Cat. #: 420403) negative, CD45 (Biolegend: Cat. #: 368540) positive, and CD14 (Biolegend: Cat. #: 325620) positive monocytes (Figure 11) . 5 μg/ml of the chimeric anti-human CD3 monoclonal antibodies directly labeled with iFluor 647 and OKT3 directly labeled with APC were prepared as primary antibodies for FACS analysis. No significant bindings were detected in 7-ADD (Biolegend: Cat. #: 420403) negative, CD45 (Biolegend: Cat. #: 368540) positive, and CD19 (Biolegend: Cat. #: 302238) positive B cells (Figure 12) .

Example 9. Bispecific T-cell engagers (BiTEs)

Bispecific T-cell engagers (BiTEs) were expressed to evaluate the strengths of the anti-human CD3 antibodies on T cell mediated cytotoxicity towards tumor cells by simultaneously binding to the target antigen and CD3. Two single-chain variable fragments (scFvs) from an anti-human BCMA monoclonal antibody and an anti-CD3 monoclonal antibody form the canonical BiTE molecule, respectively, with a short GS linker connecting them in tandem. The scFv sequence of anti-human BCMA was from the patent (WO 2014/140248 Al) . The scFv sequence of anti-human CD3, SP34, was also applied for BiTE expression. A total of three BiTEs were expressed for the study, anti-human BCMA-SP34, anti-human BCMA-64F9G7, and anti-human BCMA-10A7C8. The sequences of three BiTEs were shown below.

Sequence of anti-human BCMA-SP34:

Sequence of anti-human BCMA-64F9G7:

Sequence of anti-human BCMA-10A7C8:

The binding affinities of three purified BiTEs on human CD3 were determined using a fluorescence-activated cell sorting (FACS) -based assay. The purified BiTEs were prepared at 45 μg/ml as primary antibodies for FACS analysis. Jurkat cells harvested from culture flasks were mixed with three purified BiTEs. The mixture was equilibrated for 30 minutes at room temperature, washed three times with FACS buffer (PBS containing 1%BSA) . Anti-his antibody (GenScript, Cat. #: A01802-100) as the secondary antibody was added to incubate at room temperature for 45 min. Finally, the cells were washed 3 time with PBS, and the signal was read out by FACS BD Calibur. As shown in Figure 13, the FACS study demonstrated that the three purified BiTEs bound to human CD3 expressed on Jurkat cells.

The binding affinities of three purified BiTEs on human BCMA were determined using a fluorescence-activated cell sorting (FACS) -based assay. The purified BiTEs were prepared at 15 μg/ml as primary antibodies for FACS analysis. Human BCMA expressed cell line, RPMI 8226 cells harvested from culture flasks were mixed with three purified BiTEs. The mixture was equilibrated for 30 minutes at room temperature, washed three times with FACS buffer (PBS containing 1%BSA) . Anti-his antibody (GenScript, Cat. #: A01802-100) ) as the secondary antibody was added to incubate at room temperature for 45 min. Finally, the cells were washed 3 time with PBS, and the signal was read out by FACS BD Calibur. As shown in Figure 14, the FACS study demonstrated that the three purified BiTEs bound to human BCMA expressed on RPMI 8226 cells.

The purified three BiTEs were used in a T cell mediated cytotoxicity study on BCMA expressed tumor cell line, RPMI 8226. RPMI 8226-Luc cell line was generated by stably integration of a constitutive firefly luciferase. The Luciferase activity in RPMI 8226-Luc cells can be measured through luciferase detection reagents, so that surviving RPMI8226-Luc cells in the co-culture system can be detected, thereby indirectly reflecting T cell mediated cell killing activity induced by the BiTEs. RPMI 8226-Luc cells were the target cells in the study. Primary CD8+ T cells derived from peripheral blood mononuclear cells by using human CD8+T cell isolation kit (Miltenyi Cat. #: 130-097-057) were as the effector cells in the study. Gently mixed the RPMI 8226-Luc cells and added them into the wells of 96-well plate. After the addition of RPMI 8226-Luc, covering the 96-well plate with cells and placing it in a horizontal centrifuge, and centrifuging at 300g for 1 minute at room temperature to allow the liquid on the wall of the well to flow into the bottom of the well. The wells with RPMI 8226-Luc cells were labelled with three groups, the sampling group which would add the effector cell and the purified BiTE, the maximum survival group (Max) which would add only completed culture medium, and the minimal survival group (Min) which would add 2%Triton X-100 to kill all RPMI 8226-Luc cells. Adding 0.1 μg/ml anti-human BCMA-SP34, anti-human BCMA-64F9G7, anti-human BCMA-10A7C8, 64F9G7 chimeric, and 10A7C8 chimeric into the wells of the sampling group, respectively. Incubating the sample wells at room temperature for 30 min in cell culture incubator and the adding effector cells. The ratio of effector cell to target cells was 5: 1. After incubating 24 hours, adding Fire-lumi (Fire-lumi Luciferase assay kit, GenScript, Cat. #: L00877C-1000) into the wells and measuring luciferase intensity. The target cell killing percentage = [1- (X-Min) / (Max-Min) ] *100% (X: luciferase intensity of sampling group; Max: luciferase intensity of Max; Min: luciferase intensity of Min) . The BiTE induced primary CD8+ T cells killing on RMI 8226 was shown in Figure 15. Both anti-human BCMA-SP34, anti-human BCMA-64F9G7 induced a significant T cell specific killing on human BCMA expressed RPMI 8226 cells.

Listed below are some amino acid sequences and nucleic acid sequences mentioned herein. The CDR sequences are based on Kabat numbering.

Hybridoma

64F9G7 (mouse IgG1 Lamda)

Heavy Chain

VH:

CDR1: TYAMN (SEQ ID NO: 3)

CDR2: RIRSKIYNYATFYDDSVKD (SEQ ID NO: 4)

CDR3: YYGNDWIAK (SEQ ID NO: 5)

Light Chain

VL:

CDR1: RSSTGAVTTSNYAN (SEQ ID NO: 8)

CDR2: GTSNRAP (SEQ ID NO: 9)

CDR3: ALWYSTHYV (SEQ ID NO: 10)

10A7C8 (mouse IgG1 Kappa)

Heavy Chain

VH:

CDR1: SQYLH (SEQ ID NO: 13)

CDR2: WINPGDDTTKYNEKFKV (SEQ ID NO: 14)

CDR3: DYGYYFDY (SEQ ID NO: 15)

Light Chain

VL:

FILRTFGGGTRLEIK (SEQ ID NO: 17)

CDR1: KSSQSLLNSRTRKNYLA (SEQ ID NO: 18)

CDR2: WASTRES (SEQ ID NO: 19)

CDR3: KQSFILRT (SEQ ID NO: 20)

39B12G6 (mouse IgG2b Kappa)

Heavy Chain

VH:

CDR1: TSYIH (SEQ ID NO: 23)

CDR2: WISPGDVNTKYSEKFKG (SEQ ID NO: 24)

CDR3: DYGYYFDY (SEQ ID NO: 25)

Light Chain

VL:

CDR1: KSSQSLLNSRTRKNYLA (SEQ ID NO: 28)

CDR2: WASTRES (SEQ ID NO: 29)

CDR3: KQSFILRT (SEQ ID NO: 30)

Chimeric Antibodies

64F9G7

Heavy Chain

VH:

Light Chain

VL:

10A7C8

Heavy Chain

VH:

Light Chain

VL:

39B12G6

Heavy Chain

VH:

Light Chain

VL:

Constant region of Human IgG1

Claims

An anti-CD3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising: 1) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 13 and 23; 2) a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 14, and 24; and 3) a HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, and 25, and a light chain variable domain (VL) comprising: 1) a LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, and 28 ; 2) a LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 19 and 29; and 3) a LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 , 20, and 30.
The anti-CD3 antibody or antigen-binding fragment thereof of claim 1, wherein:

1) the VH comprises the HCDR1, HCDR2, and HCDR3 sequences having the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively;

2) the VH comprises the HCDR1, HCDR2, and HCDR3 sequences having the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively; or

3) the VH comprises the HCDR1, HCDR2, and HCDR3 sequences having the amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claim 1 or 2, wherein the VH comprises an amino acid sequence that is at least 90%identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 12 and 22, and the VL comprises an amino acid sequence that is at least 90%identical to a sequence selected from the group consisting of SEQ ID NOs: 7, 17 and 27, respectively.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the VH comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12 and 22, or a variant thereof comprising up to about 3 amino acid substitutions in the VH; and the VL comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17 and 27, or a variant thereof comprising up to about 3 amino acid substitutions in the VL.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein:

1) the VH comprises an amino acid sequence of SEQ ID NO: 2, and the VL comprises an amino acid sequence of SEQ ID NO: 7;

2) the VH comprises an amino acid sequence of SEQ ID NO: 12, and the VL comprises an amino acid sequence of SEQ ID NO: 17; or

3) the VH comprises an amino acid sequence of SEQ ID NO: 22, and the VL comprises an amino acid sequence of SEQ ID NO: 27.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the CD3 is human CD3 or cynomolgus CD3.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the anti-CD3 antibody is a mouse antibody.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the anti-CD3 antibody or antigen-binding fragment thereof is an activator of a Jurkat T cell, with an EC50 value determined through IL-2 promoter activity greater than 0.01 μg/mL; preferably between about 10^-2 μg/mL and 1.0 μg/mL, such as between about 0.018 μg/mL and 0.82 μg/mL.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the anti-CD3 antibody or antigen-binding fragment thereof is an activator of a Jurkat T cell, with an EC50 value determined through IL-2 promoter activity greater than that of OKT3 antibody.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the anti-CD3 antibody or antigen-binding fragment thereof is an activator of primary human T cell, with an EC50 value determined through IFN-γ concentration in the supernatant of the primary human T cell greater than 0.1 μg/mL; preferably between about 0.1 μg/mL and 50.0 μg/mL, such as between about 0.50 μg/mL and 10.14 μg/mL.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the anti-CD3 antibody or antigen-binding fragment thereof is an activator of a primary human T cell, with an EC50 value determined through IFN-γ concentration in the supernatant of the primary human T cell greater than that of OKT3.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the anti-CD3 antibody is a chimeric antibody comprising:

1) a VH comprises an amino acid sequence of SEQ ID NO: 32, and a VL comprises an amino acid sequence of SEQ ID NO: 34;

2) a VH comprises an amino acid sequence of SEQ ID NO: 36, and a VL comprises an amino acid sequence of SEQ ID NO: 38; or

3) a VH comprises an amino acid sequence of SEQ ID NO: 40, and a VL comprises an amino acid sequence of SEQ ID NO: 42,

and, optionally, the VH is fused to a constant region of human IgG, preferably human IgG1.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and a Jurkat T cell is lower than 1 μg/mL; preferably between about 0.1 μg/mL and 1.0 μg/mL, such as between about 0.20 μg/mL and 0.45 μg/mL.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and a Jurkat T cell is lower than that of OKT3 antibody.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and a primary human T cell is lower than 1 μg/mL; preferably between about 0.1 μg/mL and 1.0 μg/mL, such as between about 0.19 μg/mL and 0.30 μg/mL.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and a primary human T cell is lower than that of OKT3 antibody.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the EC50 value of the binding between the anti-CD3 antibody or antigen-binding fragment thereof and a cynomolgus primary T cell is lower than 10 μg/mL; preferably between about 0.1 μg/mL and 5.0 μg/mL, such as between about 0.14 μg/mL and 1.6 μg/mL.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-17, wherein the chimeric antibody is an activator of a Jurkat T cell, with an EC50 value determined through IL-2 promoter activity greater than 0.01 μg/mL; preferably between about 0.01 μg/mL and 10.0 μg/mL, such as between about 0.01 μg/mL and 5.0 μg/mL.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-18, wherein the chimeric antibody is an activator of a Jurkat T cell, with an EC50 value determined through IL-2 promoter activity greater than that of OKT3 antibody.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-19, wherein the chimeric antibody is an activator of primary human T cell, with an EC50 value determined through IFN-γ concentration in the supernatant of the primary human T cell greater than 0.1 μg/mL; preferably between about 0.1 μg/mL and 50.0 μg/mL, such as between about 0.20 μg/mL and 15.0 μg/mL.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-20, wherein the chimeric antibody is an activator of a primary human T cell, with an EC50 value determined through IFN-γ concentration in the supernatant of the primary human T cell greater than that of OKT3 antibody.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-21, wherein the anti-CD3 antibody or antigen-binding fragment thereof does not bind to a NK cell, a monocyte or a B cell.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the antigen-binding fragment is selected from the group consisting of a Fab, a Fab’, a (Fab’) ₂, an Fv, a single chain Fv (scFv) , and an sdAb.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-23, wherein, compared to the OKT3 antibody, the anti-CD3 antibody or antigen-binding fragment thereof displays a decreased propensity of inducing cytokine release syndrome.
A multispecific antibody comprising a first binding moiety and a second binding moiety, wherein the first binding moiety comprises the anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-24, and the second binding moiety is able to bind to an antigen other than CD3.
The multispecific antibody of claim 25, wherein the multispecific antibody is a bispecific antibody.
The multispecific antibody of claim 25 or 26, wherein the antigen is a cell surface antigen.
The multispecific antibody of any one of claims 25-27, wherein the antigen is a tumor antigen.
The multispecific antibody of any one of claims 25-28, wherein the antigen other than CD3 is selected from the group consisting of CD19, CD20, EGFR, BCMA, GPRC5D, EpCAM, DLL3, and HER2.
The multispecific antibody of any one of claims 25-29, wherein the first binding moiety and/or the second binding moiety is selected from the group consisting of a Fab, a Fab’, a (Fab’) ₂, an Fv, a single chain Fv (scFv) , and an sdAb.
The multispecific antibody of any one of claims 25-29, wherein the multispecific antibody is in the format of BiTE.
The multispecific antibody of any one of claims 25-30, wherein the multispecific antibody comprise the amino acid sequence of SEQ ID NO: 45 or 46.
A pharmaceutical composition comprising an anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-24, or a multispecific antibody of any one of claims 25-32; and a pharmaceutically acceptable carrier.
The pharmaceutical composition of claim 33, further comprising one or more therapeutic agents other than the anti-CD3 antibody or antigen-binding fragment thereof or the multispecific antibody.
The pharmaceutical composition of claim 34, wherein the therapeutic agent is an antibody specific for CD39, CTLA-4, PD-L1, TIM-3, LAG-3 or A2aR.
A method of enhancing immune function in a subject comprising administrating to the subject in need thereof a therapeutically effective amount of the anti-CD3 antibody or antigen-binding fragment thereof of any one of the claims 1-24, the multispecific antibody of any one of claims 25-32, or the pharmaceutical composition of any one of claims 33-35.
A method of treating a cancer in a subject comprising administrating to the subject in need thereof a therapeutically effective amount of the anti-CD3 antibody or antigen-binding fragment thereof of any one of the claims 1-24, the multispecific antibody of any one of the claims 25-32, or the pharmaceutical composition of any one of claims 33-35.
The method of claim 37, wherein the cancer is multiple myeloma.
Use of the anti-CD3 antibody or antigen-binding fragment thereof of any one of the claims 1-24 or the multispecific antibody of any one of claims 25-32 in the manufacture of a medicament for the treatment of a cancer.
The method of claim 39, wherein the cancer is multiple myeloma.
The anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 1-24, the multispecific antibody of any one of claims 25-32 or the pharmaceutical composition of any one of claims 33-35, for use in treating a cancer in a subject in need thereof.
The method of claim 41, wherein the cancer is multiple myeloma.