HK1250045B - Cell-based assay for detecting anti-cd3 homodimers - Google Patents

Description

Cell-based assay for the detection of anti-CD3 homodimers

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/167,761, filed May 28, 2015, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Sequence listing submission as ASCII text file

The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of a Sequence Listing (File Name: 146392022940SEQLISTING.txt, Record Date: May 26, 2016; Size: 17 KB).

Field of the Invention

The present invention provides methods for analyzing a preparation of multispecific antibodies, wherein at least one antigen-binding fragment of the multispecific antibody binds CD3. In some embodiments, the present invention provides methods for determining the presence of anti-CD3 homodimers in a composition of one or more multispecific antibodies, wherein at least one antigen-binding fragment of the multispecific antibody binds CD3.

Background of the Invention

T cell-dependent bispecific (TDB) antibodies are designed to bind to target antigens expressed on cells, and are typically bound to T cells by binding to the CD3 e subunit of the T cell receptor. The combination of bispecific antibodies with the extracellular domain of the target antigen and the CD3 of the T cell leads to T cell recruitment to the target cell, thereby causing T cell activation and target cell elimination. In the absence of target cells, a single anti-CD3 arm cannot cross-link the TCR to induce T cell activation and target cell killing. Anti-CD3 homodimers are product-related impurities formed during the manufacturing process of TDB antibodies, and can cross-link the TCR and induce low-level T cell activation in the presence or absence of target cells. If the presence of high levels may cause the TDB biological efficacy in vitro to decrease, then anti-CD3 homodimers may also affect therapeutic efficacy. Anti-CD3 homodimers can have off-target effects by inducing low-level T cell activation and inducing T cells to produce inflammatory cytokines in the absence of target cells. Therefore, there is a need to control the levels of T cell activation product-associated variants present during the TDB manufacturing process, and sensitive, reproducible, and quantitative impurity assays are needed to detect anti-CD3 homodimers that may be present in the purified product to support the development of safe and effective clinical drug candidates.

Impurity determination requires the ability to distinguish between product/process-related impurities and the desired product. Many traditional methods for Chinese Hamster Ovary Cell Protein (CHOP) impurity detection use binding assay formats, in which the presence of process-related CHO proteins can be sensitively detected in the product to assess product purity and safety. These CHOP antibodies are specific for CHOP proteins but cannot recognize the product, so they generally have no effect on sensitive detection of impurities in the presence of the final product. Similar methods can be used for bispecific antibodies, provided that the antibodies can be identified to distinguish between the final product and impurities. Implementation of available anti-CD3 homodimer binding assay formats will require the development of highly specialized antibodies, which may not be possible for this antigen or other bispecific antibodies in general. Alternative physiochemical-based methods (RP-HPLC, mass spectrometry) can also be used to detect product-related impurities, and rely on the ability to fully separate product-related impurities from the product to detect the amount of impurities present. The amount of impurities can be detected relative to other substances present in the material, or by spiking in variant standards and comparing the percentage of the material present to the spiked standard. However, many of these methods may involve additional sample handling and processing steps to separate variants from the desired product material, and these steps may alter the material or limit the sensitivity and accuracy of the method. In addition, it is also desirable to know whether the structural isoforms or other potential T cell activating impurities that may be present in the bispecific test article (purified product, DS, DP, stability samples, stress samples) of any anti-CD3 homodimer product-related impurities are biologically active so that the appropriate risk can be attributed to the impurities. The novel anti-CD3 homodimer assay method described herein uses a cell-based approach to detect biologically active anti-CD3 homodimer impurities, thereby avoiding the challenges and limitations of detecting homodimer impurities in bispecific formulations using binding assays or physiochemical-based formats.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

summary

The present invention provides a method for detecting anti-CD3 homodimers in a composition comprising a T cell-dependent bispecific antibody (TDB), wherein the bispecific antibody comprises a target antigen-binding fragment and a CD3-binding fragment, the method comprising contacting a population of T cells with the composition, wherein the T cells comprise a nucleic acid encoding a reporter gene operably linked to a response element responsive to T cell activation, and wherein the T cell population does not comprise the target antigen, wherein expression of the reporter gene indicates the presence of the anti-CD3 homodimer.

In some embodiments of the above embodiments, reporter gene is luciferase, fluorescent protein, alkaline phosphatase, beta lactamase or beta galactosidase. In other embodiments, luciferase is firefly luciferase, Renilla luciferase or nano luciferase. In some embodiments, in response to the response element of T cell activation is NFAT promoter, AP-1 promoter, NFκB promoter, FOXO promoter, STAT3 promoter, STAT5 promoter or IRF promoter. In some embodiments, in response to the response element of T cell activation include any one or more T cell activation response elements from NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.

In some embodiments of the above embodiments, the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. In some embodiments, the T cell population is a population of Jurkat T cells or CTLL-2 T cells.

In some embodiments of the above embodiments, the T cell population is contacted with a composition comprising a bispecific antibody at a concentration ranging from 0.01 ng/mL to 50 ng/mL. In some embodiments, the reporter gene is detected after any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours after contacting the cells with the composition.

In some aspects, the present invention provides a method for quantifying the amount of an anti-CD3 homodimeric antibody in a composition comprising a TDB, wherein the TDB comprises a target antigen-binding fragment and a CD3-binding fragment, the method comprising contacting a population of T cells with the composition at one or more TDB concentrations, wherein the T cells comprise a nucleic acid encoding a reporter gene operably linked to a promoter responsive to T cell activation, and wherein the population of T cells does not comprise the target antigen; and correlating expression of the reporter gene as a function of antibody concentration to a standard curve generated by contacting the T cells with varying concentrations of purified anti-CD3 homodimer.

In some embodiments of the above quantification of the amount of anti-CD3 homodimer antibody in the composition, the reporter gene is luciferase, fluorescent protein, alkaline phosphatase, beta lactamase or beta galactosidase. In other embodiments, the luciferase is firefly luciferase, Renilla luciferase or nanoluciferase.

In some embodiments of the amount of anti-CD3 homodimer antibodies in the above quantitative composition, reporter gene is luciferase, fluorescent protein, alkaline phosphatase, beta lactamase or beta galactosidase. In other embodiments, luciferase is firefly luciferase, Renilla luciferase or nano luciferase. In some embodiments, the response element in response to T cell activation is NFAT promoter, AP-1 promoter, NFκB promoter, FOXO promoter, STAT3 promoter, STAT5 promoter or IRF promoter. In some embodiments, the response element in response to T cell activation includes any one or more T cell activation response elements from NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.

In some embodiments of the above embodiments, the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. In some embodiments, the T cell population is a population of Jurkat T cells or CTLL-2 T cells.

In some embodiments of the above embodiments, the T cell population is contacted with a composition comprising a bispecific antibody at a concentration ranging from 0.01 ng/mL to 50 ng/mL. In some embodiments, the reporter gene is detected after any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours after contacting the cells with the composition.

In some aspects, the present invention provides an engineered T cell for detecting anti-CD3 homodimers in a composition comprising a bispecific antibody, wherein the bispecific antibody comprises a target antigen binding fragment and a CD3 binding fragment, wherein the T cell comprises a reporter gene operably linked to a response element responsive to T cell activation.

In some embodiments of the above aspects, the T cell comprises a reporter gene, wherein the reporter gene is luciferase, fluorescent protein, alkaline phosphatase, beta lactamase or beta galactosidase. In other embodiments, the luciferase is firefly luciferase, Renilla luciferase or nanoluciferase.

In some embodiments of the above embodiments, T cells include reporter genes, wherein the reporter genes are luciferase, fluorescent protein, alkaline phosphatase, beta lactamase or beta galactosidase. In other embodiments, luciferase is firefly luciferase, Renilla luciferase or nano luciferase. In some embodiments, the response element in response to T cell activation is NFAT promoter, AP-1 promoter, NFκB promoter, FOXO promoter, STAT3 promoter, STAT5 promoter or IRF promoter. In some embodiments, the response element in response to T cell activation includes any one or more T cell activation response elements from NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.

In some embodiments of the above embodiments, the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. In some embodiments, the T cell population is a population of Jurkat T cells or CTLL-2 T cells.

In some aspects, the present invention provides a kit for detecting anti-CD3 homodimers in a composition comprising a bispecific antibody, wherein the bispecific antibody comprises a target antigen binding fragment and a CD3 binding fragment, wherein the kit comprises engineered T cells comprising a reporter gene operably linked to a response element responsive to T cell activation. In some embodiments, the kit further comprises an anti-CD3 homodimer assay standard and/or an anti-CD3 homodimer control.

In some embodiments of the above kits, the reporter gene is luciferase, fluorescent protein, alkaline phosphatase, beta lactamase or beta galactosidase. In other embodiments, the luciferase is firefly luciferase, Renilla luciferase or nanoluciferase.

In some embodiments of the above test kits, reporter gene is luciferase, fluorescent protein, alkaline phosphatase, beta lactamase or beta galactosidase. In other embodiments, luciferase is firefly luciferase, Renilla luciferase or nano luciferase. In some embodiments, in response to the response element of T cell activation is NFAT promoter, AP-1 promoter, NFκB promoter, FOXO promoter, STAT3 promoter, STAT5 promoter or IRF promoter. In some embodiments, in response to the response element of T cell activation include any one or more T cell activation response elements from NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.

In some embodiments of the above kits, the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. In some embodiments, the T cell population is a population of Jurkat T cells or CTLL-2 T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and 1B show that the CD20 TDB (αCD20 (Mab2; VH SEQ ID NO: 31 / VL SEQ ID NO: 32) / αCD3 (Mab1; VH SEQ ID NO: 19 / VL SEQ ID NO: 20)) requires antigen CD20-expressing target cells to induce T cell activation and target cell killing. Figure 1A shows the activation of CD8 ⁺ cells as measured by the expression of CD69 and CD25. Circles represent samples that include target cells, and squares represent samples that contain T cells but no target cells. Figure 1B shows target cell killing in the presence of T cells (circles) or in samples in which CD3+ T cells were depleted from the PBMC pool (squares).

Figure 2 shows that anti-CD3 homodimers can activate human donor T cells. Human PBMCs from two different donors were treated with increasing amounts of purified anti-CD3 homodimers (triangles) or CD20TDB (circles). T cell activation was measured by CD69 ⁺ cell % in the human CD8 ⁺ cell population. The EC ₅₀ for CD20TDB was 5.5 ng/ml for cells from donor 1, and 4.4 ng/ml for cells from donor 2. The EC ₅₀ for anti-CD3 homodimers was 526 ng/ml for cells from donor ₁ , and ₁₆₉ ng/ml for cells from donor 2.

Figure 3A shows that anti-CD3 homodimers can reduce the efficacy of CD20TDB. CD20TDB was spiked with different amounts of anti-CD3 homodimers and the responses of target cells (squares) and T cells (diamonds) were measured. Figure 3B shows that low levels of anti-CD3 homodimers spiked into CD20TDB did not significantly reduce CD8 ⁺ T cell activation (left panel) or CD4 ⁺ T cell activation (right panel). CHOTDB is represented by circles, CHO TBD+2.5% HD by squares, and CHO TBD+5% HD by triangles.

Figure 4A shows that anti-CD3 homodimers can weakly activate human ^CD8- T cells from various human donors in the absence of target cells. Figures 4B-4E show that anti-CD3 homodimer activation of human T cells shows a dose-dependent trend of increases in several representative cytokines. The mean cytokine level responses are plotted.

Fig. 5 A illustrates the T cell activation that can monitor anti-CD3 homodimer using reporter gene assay.Human Jurkat CD4 ⁺ T cell line is genetically engineered to stably express the firefly luciferase reporter gene driven by various T cell receptor (TCR) responsive transcriptional response elements (AP-1, NFAT and NFκB), selects stable cell pool, and assesses the response of the pool for treating 4 hours with 1010 μg/mL purified anti-CD3 homodimer. Fluorescence response (luciferase reporter gene activity) is mapped, and the highest response is observed from Jurkat/NFκB luciferase stable pool. Fig. 5 B illustrates Jurkat/NFκB luciferase stable clone.

Figures 6A and 6B illustrate that the purified anti-CD3 homodimer can activate T cells in the presence or absence of target cells. Figure 6A illustrates a comparison of the potential of purified CD20TDB and purified anti-CD3 homodimer activated T cells. Jurkat T cells expressing NFκB luciferase reporter gene are activated by CD20TDB dose-dependently in the presence of target antigen-expressing cells. CD20TDB activates Jurkat/NFκB-firefly luciferase cells in the presence of target antigen-expressing cell lines. In the presence of cells expressing co-stimulatory target antigens, the purified CD20TDB is 1000 times more active than the purified anti-CD3 homodimer. Figure 6B illustrates that in the absence of cells expressing target antigens (squares), CD20TDB does not activate Jurkat/NFκB luciferase cells, but the purified anti-CD3 homodimer dose-dependently induces NFκB-dependent luciferase activity (diamonds).

7 shows the calculation of the anti-CD3 homodimers present in the CD20TDB sample. The luciferase activity observed from the cells treated with the Jurkat/NFκBLuc sample as measured by a fluorescence plate reader (RLU) was compared with the T cell activation response produced by a known amount of anti-CD3 homodimers. The concentration of the anti-CD3 homodimers present in the sample was solved using an equation derived from a curve fitted to the homodimer standard response. The percentage of the anti-CD3 homodimers present in the sample was then determined based on the ratio of the total amount of CD20TDB present in the sample divided by the homodimer detected in the sample.

Figure 8 shows that the anti-CD3 T cell activation assay was accurate for 0.25% to 35% of the CD20 TDB test samples. The spike recovery of the anti-CD3 homodimer indicated that the analytical method was linear over the method range with ^{an R²} of 0.99, a slope of 1.05, and a y-int of 0.078, and showed minimal bias.

Figure 9 shows that the T cell activation assay is able to detect additional product-related impurities. The level of HMWS present in the CD20 TDB material affects T cell activation in the homodimer assay.

Detailed Description of the Invention

The present invention provides a method for detecting anti-CD3 homodimers in a composition comprising a T cell-dependent bispecific antibody (TDB), wherein the bispecific antibody comprises a target antigen-binding fragment and a CD3-binding fragment, the method comprising contacting a population of T cells with the composition, wherein the T cells comprise a nucleic acid encoding a reporter gene operably linked to a promoter responsive to T cell activation, and wherein the T cell population does not comprise the target antigen, wherein expression of the reporter gene indicates the presence of the anti-CD3 homodimer.

In some aspects, the present invention provides a method for quantifying the amount of an anti-CD3 homodimeric antibody in a composition comprising a TDB, wherein the TDB comprises a target antigen-binding fragment and a CD3-binding fragment, the method comprising contacting a population of T cells with the composition at one or more TDB concentrations, wherein the T cells comprise a nucleic acid encoding a reporter gene operably linked to a promoter responsive to T cell activation, and wherein the population of T cells does not comprise the target antigen; and correlating expression of the reporter gene as a function of antibody concentration to a standard curve generated by contacting the T cells with varying concentrations of purified anti-CD3 homodimer.

In other aspects, the invention provides engineered T cells for detecting anti-CD3 homodimers in a composition comprising a TDB, wherein the TDB comprises a target antigen binding fragment and a CD3 binding fragment, wherein the T cell comprises a reporter gene operably linked to a promoter responsive to T cell activation.

In other aspects, the present invention provides a kit for detecting anti-CD3 homodimers in a composition comprising a TDB, wherein the TDB comprises a target antigen binding fragment and a CD3 binding fragment, wherein the kit includes an engineered T cell comprising a reporter gene operably linked to a promoter responsive to T cell activation.

I. Definition

The terms "polypeptide" or "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component or toxin. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically include antibodies.

A "purified" polypeptide (e.g., an antibody or immunoadhesin) means that the purity of the polypeptide has been increased so that it exists in a form that is more pure than it exists in its natural environment and/or when initially synthesized and/or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of a native polypeptide. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of a native polypeptide. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments, or amino acid sequence variants of the native polypeptide, etc. Methods for identifying agonists or antagonists of a polypeptide can include contacting the polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide.

A polypeptide that "binds" an antigen of interest (e.g., a tumor-associated polypeptide antigen target) is one that binds the antigen with sufficient affinity so that the polypeptide can be used as a diagnostic and/or therapeutic agent targeting cells or tissues expressing the antigen, and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the polypeptide to "non-target" polypeptides will be less than about 10% of the binding of the polypeptide to its specific target polypeptide, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

With respect to binding of a polypeptide to a target molecule, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to the binding of a control molecule, which is generally a structurally similar molecule that has no binding activity. For example, specific binding can be measured by competition with a control molecule that is similar to the target (e.g., an excess of unlabeled target). In this regard, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies including TDBs), and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

Antibodies are naturally occurring immunoglobulin molecules that have different structures, all based on the immunoglobulin fold. For example, IgG antibodies have two "heavy" chains and two "light" chains that undergo disulfide bonding to form functional antibodies. Each heavy and light chain itself comprises a "constant" (C) region and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and function in the non-antigen-specific interactions with immune effectors. The antigen-binding specificity of an antibody or an antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a specific antigen.

The antigen-binding specificity of an antibody is determined by the structural characteristics of the V region. Variability is unevenly distributed over the 110 amino acid spans of the variable domain. In fact, the V region is composed of a relatively constant elongated region called a framework region (FR) with 15-30 amino acids, which is separated by a shorter region called a "hypervariable region" (HVR) that is each 9-12 amino acids long. The variable domains of native heavy and light chains each contain four FRs, mainly using a β-sheet configuration connected by three hypervariable regions connected by a loop, and in some cases, forming a part of a β-sheet structure. The hypervariable region of each chain is closely bound together by FR, and together with the hypervariable regions from other chains, contributes to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Although the constant domains are not directly involved in binding an antibody to an antigen, they exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

Each V region typically comprises three HVRs, e.g., complementarity determining regions ("CDRs," each containing a "hypervariable loop") and four framework regions. Thus, an antibody binding site (the minimum structural unit required to bind a specific desired antigen with substantial affinity) will typically include three CDRs, interspersed with at least three, preferably four, framework regions to maintain and present the CDRs in the proper conformation. Classical four-chain antibodies have an antigen binding site defined by the cooperation of the _VH and _VL domains. Certain antibodies, such as camelid and shark antibodies, lack light chains and rely solely on the binding site formed by the heavy chain. Single-domain engineered immunoglobulins can be prepared in which the binding site is formed solely by the heavy chain or the light chain, without cooperation between _VH and _VL .

The term "variable" refers to the fact that certain parts of the variable domain are widely different in sequence between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed throughout the entire variable domain of an antibody. In the light and heavy chain variable domains, the variability is concentrated in three segments called hypervariable regions. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs, primarily in a β-sheet configuration connected by three hypervariable regions forming loops, and in some cases, forming part of the β-sheet structure. The hypervariable regions of each chain are closely bound together by the FRs and, together with the hypervariable regions from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Although the constant domains do not directly participate in antibody-antigen binding, they perform various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" (HVR) when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region may comprise amino acid residues from a "complementarity determining region" or "CDR" (e.g., approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in _VL , and 31-35B ( _H1 ), 50-65 (H2), and 95-102 (H3) in VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in _VL , and 26-32 ( _H1 ), 52A-55 (H2), and 96-101 (H3) in VH (Chothia and Lesk, 1991)). J. Mol. Biol. 196:901-917(1987)).

"Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

As used herein, a "T cell-dependent bispecific" antibody or "TDB" is a bispecific antibody designed to bind to a target antigen expressed on a cell, and typically binds to a T cell by binding to the CD3e subunit of the T cell receptor.

"Antibody fragments" comprise a portion of an intact antibody, preferably comprising its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV) 4 -Fc, di-scFv, di-scFv or tandem (di, tri)-scFv); and bispecific T cell engagers (BiTEs).

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab') ₂ fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region is composed of a dimer formed by the tight, non-covalent association of a heavy chain variable domain and a light chain variable domain. In this configuration, the three hypervariable regions of each variable domain interact to confine the antigen binding site to the surface of the _VH - _VL dimer. Together, the six hypervariable regions confer antigen-binding specificity to an antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a small number of residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is herein designated as Fab' in which the cysteine residues of the constant domains bear at least one free thiol group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate 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.

The constant domains of the heavy chains of antibodies are classified into different classes depending on the amino acid sequence of the constant domains of their heavy chains. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to different antibody classes are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

"Single-chain Fv" or "scFv" antibody fragments comprise the _VH and _VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that enables the scFv to form the structure required for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabody" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain ( _VH ) connected to a light-chain variable domain ( _VL ) in the same polypeptide chain (VH- _VL ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Mini- _diabodies are more fully described in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies with polyepitopic specificity. Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain ( _VH ) and a light chain variable domain ( _VL ) (wherein the _VHVL unit has _polyepitopic specificity), antibodies with two or more _VL and _VH domains (wherein each _VHVL unit binds to a different epitope), antibodies _with two or more single variable domains (wherein each single variable domain binds to a different epitope), full-length antibodies, antibody fragments (such as Fab, Fv, dsFv, scFv), diabodies, bispecific diabodies, tribodies, trifunctional antibodies, and covalently or non-covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. "Monospecificity" refers to the ability to bind to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

In some examples, the multispecific antibody is a bispecific antibody (eg, a bispecific antibody that binds CD3 and another epitope). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy chain-light chains with 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, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic pulling effects for preparing antibody Fc heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to prepare bispecific antibodies and using single-chain Fv (sFv) 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).

The term "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) confers antigen binding. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks) and those derived from recombinant methods of human and mouse antibodies (Ward, ES et al., Nature (1989) 341:544-546; Dooley, H. et al., Dev Comp Immunol (2006) 30:43-56; Muyldemans S et al., Trend Biochem Sci (2001) 26:230-235; Holt, LJ et al., Trends Biotechnol (2003):21:484-490; WO 2005/035572; WO 03/035694; Davies, J et al., Febs Lett (1994) 339:285-290; WO 00/29004; WO 02/051870).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variants (described variants generally present in small amounts) that may occur during the production of monoclonal antibodies, the individual antibodies constituting the population are identical and/or bind to the same epitope. Unlike polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, the advantage of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is a characteristic obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the method provided herein can be prepared by the hybridoma method initially described by Kohler et al., Nature 256:495 (1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent number 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chains is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys (such as baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences (U.S. Patent No. 5,693,780).

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Most humanized antibodies are human immunoglobulins (recipient antibodies), in which the residues from the recipient's hypervariable regions are replaced with residues having the desired specificity, affinity, and ability from the hypervariable regions of non-human species (donor antibodies) such as mice, rats, rabbits, or non-human primates. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may be included in residues not seen in the recipient antibody or in the donor antibody. These modifications are carried out to further refine antibody performance. Typically, the humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins and all or substantially all of the FRs are those of human immunoglobulin sequences in addition to the FR replacements described above. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region, typically at least a portion of an immunoglobulin constant region of a human immunoglobulin. For further details, see 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).

For the purposes of this article, a "complete antibody" is an antibody comprising heavy and light chain variable domains and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the complete antibody has one or more effector functions.

"Native antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, although the number of disulfide bridges varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain ( _VH ) at one end, followed by multiple constant domains. Each light chain has a variable domain ( _VL ) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are known to form the interface between the light and heavy chain variable domains.

A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

In some embodiments, antibody "effector functions" refer to those biological activities attributable to the Fc region (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; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause target cell lysis. NK cells, the primary cells used to mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred.

"Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by binding of the first component of the complement system (C1q) to a molecule, such as a polypeptide (e.g., an antibody), complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR. In addition, preferred FcRs are receptors that bind to IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

"Impurity" refers to a substance that is different from the desired polypeptide product. In some embodiments of the present invention, impurities include charge variants of polypeptides. In some embodiments of the present invention, impurities include charge variants of antibodies or antibody fragments. In other embodiments of the present invention, the impurities include, but are not limited to: host cell materials, such as CHOP; leached protein A; nucleic acids; variants, fragments, aggregates or derivatives of the desired polypeptide; another polypeptide; endotoxins; viral contaminants; cell culture medium components, etc. In some examples, impurities can be host cell proteins (HCPs) from, for example, but not limited to, bacterial cells (such as E. coli cells), insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells, etc. In some examples, the impurity is a homodimer (e.g., anti-CD3 homodimer).

As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide with the effector functions of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises an amino acid sequence with a desired binding specificity that is different from the antigen recognition and binding site (i.e., "heterologous") of an antibody and a fusion of an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a continuous amino acid sequence that at least comprises the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin such as IgG-1, IgG-2, IgG-3, or IgG-4 subtype, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

As used in this specification, "reporter molecule" means a molecule that provides an analyzable signal by its chemical properties that allows detection of antigen-bound antibodies. The most commonly used reporter molecules in this type of assay are enzymes, fluorophores or molecules containing radionuclides (i.e., radioisotopes) and chemiluminescent molecules.

As used herein, "substantially the same" means that a value or parameter has not been changed in a significant manner. For example, the ionic strength of the chromatographic mobile phase at the column outlet is substantially the same as the initial ionic strength of the mobile phase if the ionic strength has not changed significantly. For example, the ionic strength at the column outlet is substantially the same as the initial ionic strength if it is within 10%, 5%, or 1% of the initial ionic strength.

References herein to "about" a value or parameter include (and describe) variations to the value or parameter itself. For example, description of "about X" includes description of "X."

As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise. It should be understood that the aspects and variations of the invention described herein include "consisting of" and/or "consisting essentially of" the aspects and variations.

II. Cell-based reporter gene assays

The present invention provides a cell-based assay to detect anti-CD3 homodimers present in a composition comprising TDB, wherein an antigen-binding fragment of TDB binds CD3 and activates T cells.

A. T cell activation

TDB's mechanism of action is to specifically deplete target antigen-expressing cells. Simultaneous binding of TDB to the CD3e subunit of the T cell receptor (TCR) and the target antigen expressed on the surface of target cells leads to TCR clustering, resulting in T cell activation and target cell cytotoxicity reduction. Many TDBs are already in clinical use (αCD3/αCD19, αCD3/αCD20, αCD3/αHER2; de Gast GC et al., 1995, Cancer Immunol Immunother. 40(6):390-396; Buhmann R et al., 2009 Bone Marrow Transplant. 43(5):383-397; Chan JK et al., 2006, Clin Cancer Res. 12(6):1859-1867), and novel TDB-like bispecifics are being evaluated to improve clinical efficacy (Chames, P. and Baty, D. 2009, MAbs 1(6):539-547; Fournier, P. and Schirrmacher, V., 2013, BioDrugs 27(1):35-53). TDB bispecifics can activate both CD4 ⁺ and CD8 ⁺ T cell lineages, provided that the correct target expressing cells are present. Activation of CD4 ⁺ T cells leads to the induction of cytokine gene expression (IL-2, etc.), which in turn leads to the recruitment and activation of other immune cells, including the expansion and proliferation of CD8 ⁺ T cells. Activation of CD8 ⁺ CTLs by forming an immune synapse-like structure with target cells through TDB-mediated cell bridging leads to CTL activation, induction of perforin and granzyme (A, B, C; depending on the CTL subtype) transcription, degranulation, and local release of perforin and granzyme at the 'immune synapse'-like interface between target and effector cells, resulting in the killing of target cells (Pores-Fernando, Pores-Fernando AT, Zweifach A, 2009, Immunol Rev., 231(1):160-173; Pipkin, ME et al., 2010, Immunol Rev., 235(1):55-72). Effector cell-mediated cell killing is a relatively slow process that requires synaptic stabilization for several hours and requires transcription-dependent activation of the prf1 gene and granzyme genes to ensure complete cell killing. Alternatively, CTL-mediated target cell killing has also been shown to occur through Fas-mediated apoptosis (Pardo, J et al., 2003, Int Immunol., 15(12):1441-1450). The transcriptional regulation of the prf1, grB, and Fas-mediated cell killing mechanisms depends on the NFAT, NFκB, and STAT enhancer elements located within the promoters of genes required for mediating B cell depletion (Pipkin, ME et al., 2010, Immunol Rev., 235(1):55-72; Pardo, J et al., 2003, Int Immunol., 15(12):1441-1450). The strength of the interaction between the target and the effector cell (immune synapse) depends on the signaling from other co-stimulatory molecules that are also necessary to stabilize and maintain the interaction between the target and the effector cell (Krogsgaard M et al., 2003, Semin Immunol. 15 (6): 307-315; Pattu V et al., 2013, Front Immunol., 4: 411; Klieger Y et al., 2014, Eur J Immunol. 44 (1): 58-68; Schwartz JC et al., 2002, Nat Immunol. 3 (5): 427-434). Therefore, monitoring the transcriptional induction of the target gene by using a reporter gene assay is an alternative assay system for observing the activation of T cells by TDB.

T cell activation requires the spatial and dynamic reorganization of cell surface proteins and signaling molecules at the contact site of antigen presenting cells to form an immune synapse. The coordination of activation and signal transduction of T cell receptors (TCRs) and co-stimulatory receptors (CD28, CD40, ICOS, etc.) and ligands regulates the duration and signal transduction required for T cell activation. Antigen presentation by MHC on the surface of antigen presenting cells (APCs) is recognized by TCRs on the surface of T cells. MHC and TCR clustering initiates the recruitment and activation of signal transduction pathways that can cause T cell activation, which depends on the expression of co-stimulatory and immunomodulatory receptors that play a key role in regulating T cell activation. Antibodies against TCR subunits, such as CD3e (OKT3; Brown, WM, 2006, Curr Opin Investig Drugs 7:381-388; Ferran, C et al., 1993 Exp Nephrol 1:83-89) can induce T cell activation by cross-linking TCR, thereby simulating the clustering of TCR at the immune synapse, and have been used clinically and as surrogate activators for in vitro studies of TCR signaling for many years. TCR clustering by anti-CD3 antibodies rather than co-stimulation weakly activates T cells, but still causes T cell activation and limited cytokine transcription and release. Anti-CD3-mediated signaling has been shown to activate several transcription factors, including NFAT, AP1, and NFκB (MF et al., 1995, J. Leukoc. Biol. 57: 767-773; Shapiro VS et al., 1998, J. Immunol. 161(12): 6455-6458; Pardo, J et al., 2003, Int Immunol., 15(12): 1441-1450). Co-stimulation regulates the level and type of cytokine release by modulating signaling that affects transcriptional regulation of cytokine expression, which affects the nature of T cell activation responses (Shannon, MF et al., 1995, J. Leukoc. Biol. 57: 767-773). The TDB bispecific cluster TCR on the cell surface of T cells forms a bridge between the T cell and the target antigen-expressing cell. Transcriptional regulatory elements that drive expression of a reporter gene induced by T cell activation, which may be transcriptional, are tested in T cell lines to determine which events are activated by the TDB in the presence and absence of target cells.

B. Reporter molecules

Reporter gene assay is an analytical method for biological characterization of stimulation by induction of reporter gene expression in monitoring cells. Stimulation causes the induction of intracellular signal transduction pathways, which results in a cellular response generally including regulatory gene transcription. In some instances, the stimulation of cell signal transduction pathways causes regulation of gene expression by regulating and recruiting transcription factors to the upstream non-coding regions of the DNA required for the initiation of RNA transcription that causes protein production. It is necessary to control gene transcription and translation in response to stimulation to trigger most biological reactions, such as cell proliferation, differentiation, survival, and immune response. These non-coding regions of DNA (also referred to as enhancers) contain specific sequences that are recognition elements for transcription factors, and the specific sequences regulate the efficiency of gene transcription, and therefore regulate the amount and type of protein produced by cells in response to stimulation. In reporter gene assays, enhancer elements and minimal promoters in response to stimulation are engineered to drive reporter gene expression using standard molecular biology methods. DNA is then transfected into cells containing all mechanisms that specifically respond to stimulation, and the level of reporter gene transcription, translation, or activity is measured as a surrogate measure of biological response.

In some aspects, the present invention provides a method for detecting the anti-CD3 homodimer in the composition comprising TDB by contacting a T cell colony with a composition, wherein the T cell comprises a nucleic acid encoding a reporter gene, and the reporter gene is operably connected to a promoter activated in response to CD3 so that the expression of the reporter gene indicates the presence of anti-CD3 homodimers. The reporter molecule can be any molecule for which an assay method can be developed to measure the amount of the molecule produced by the cell in response to stimulation. For example, the reporter molecule can be a reporter protein encoded by a reporter gene in response to stimulation (e.g., T cell activation). Common examples of reporter molecules include, but are not limited to, luminescent proteins (such as luciferases), which emit light as a by-product of substrate catalysis, and the light can be measured experimentally. Luciferase is a class of luminescent proteins derived from many sources, including firefly luciferase (from species North American firefly (Photinus pyralis)), from the sea renilla luciferase of ocean pansy (Renilla reniformis), click bug luciferase (from Pyrearinus termitilluminans), marine copepod Gaussia luciferase (from Gaussia princeps) and deep-sea shrimp nano-luciferase (from under the fine horn thorn insect (Oplophorus gracilirostris)). Firefly luciferase catalyzes luciferin oxidation to oxyluciferin, thereby causing emission photons, and other luciferases (such as sea renilla) emit light by catalyzing coelenterazine. Different filtering systems can be used to read the wavelength of the light emitted by different luciferase forms and variants, which is conducive to multiplexing. The amount of fluorescence is proportional to the amount of the luciferase expressed in the cell, and the luciferase gene has been used as a sensitive reporter gene to assess the impact of stimulation-induced biological response. Reporter gene assays have been used for many years for a wide range of purposes, including basic research, HTS screening and efficacy (Brogan J et al., 2012, Radiat Res. 177(4):508-513; Miraglia LJ et al., 2011, Comb Chem High Throughput Screen. 14(8):648-657; Nakajima Y and Ohmiya Y. 2010, Expert Opin Drug Discovery, 5(9):835-849; Parekh BS et al., 2012, Mabs, 4(3):310-318; Svobodova K and Cajtham L T., 2010, Appl Microbiol Biotechnol., 88(4):839-847).

In some embodiments, the present invention provides a cell-based assay to detect anti-CD3 homodimers in TDB compositions, wherein T cells encode reporter gene constructs in response to T cell activation. In some embodiments, the reporter gene construct comprises luciferase. In some embodiments, the luciferase is a firefly luciferase (e.g., from species Firefly pyralis), a Renilla luciferase from marine pansy (e.g., from species Renilla reniformis), a click worm luciferase (e.g., from species Pyrearinus termitilluminans), a marine copepod Gaussia luciferase (e.g., from species Gaussia princeps), and a deep-sea shrimp nanoluciferase (e.g., from species Stenoceratidae). In some embodiments, the expression of luciferase in engineered T cells indicates the presence of anti-CD3 homodimers in the TDB compositions. In other aspects, the reporter construct encodes β-glucuronidase (GUS); a fluorescent protein, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and variants thereof; chloramphenicol acetyltransferase (CAT); β-galactosidase; β-lactamase; or secreted alkaline phosphatase (SEAP).

In some aspects of the present invention, the nucleic acid encoding reporter molecules (for example, reporter protein) is operably connected to a promoter and/or enhancer in response to T cell activation. In some embodiments, the promoter and/or enhancer in response to T cell activation is an expression control sequence. Expression and cloning vectors are generally contained by host organisms and are operably connected to the promoter of the nucleic acid of the coded polypeptide (for example, reporter polypeptide). Suitably, expression control sequences are eukaryotic promoter systems in the vector that can transform or transfect eukaryotic host cells (for example, T cells). Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences after T cell activation.

Promoter sequences are known for eukaryotic organisms. In fact all eukaryotic genes all have the district that is rich in AT, and described district is positioned at about 25 to 30 base places of the upstream of transcription start site.Another sequence that is found in the upstream 70 to 80 base places that the transcription of many genes begins is the CNCAAT district, and wherein N can be any Nucleotide.At the 3 ' end of most eukaryotic genes is AATAAA sequence, and it can be the signal that is used for poly A tail is added to the 3 ' end of encoding sequence.All these sequences are suitably inserted in the eukaryotic expression vector.

In some aspects of the invention, the invention provides a T cell comprising a nucleic acid encoding a reporter molecule under the control of a promoter responsive to T cell activation. Promoters responsive to T cell activation are known in the art.

In other embodiments, the invention provides T cells comprising nucleic acids encoding reporter molecules, the reporter molecules are under the control of a minimal promoter operably linked to an enhancer element in response to T cell activation. In some embodiments, the minimal promoter is a thymidine kinase (TK) minimal promoter, a minimal promoter from cytomegalovirus (CMV), a promoter derived from SV40, or a minimal elongation factor 1 alpha (EF1 alpha) promoter. In some embodiments, the nucleic acid encoding the reporter molecules is under the control of a minimal TK promoter regulated by a T cell activation responsive DNA recognition element. In some embodiments, the T cell activation responsive DNA recognition element is a NFAT (nuclear factor of activated T cells) enhancer, AP-1 (Fos/Jun) enhancer, NFAT/AP1 enhancer, NFκB enhancer, FOXO enhancer, STAT3 enhancer, STAT5 enhancer, and IRF enhancer. The enhancer can be spliced into the vector at the 5' or 3' position of the polypeptide coding sequence, but in some embodiments, is located at the 5' site of the promoter. In some embodiments, the present invention provides T cells wherein a luciferase gene is operably linked to a minimal TK promoter, which is in turn operably linked to an NFκB-responsive enhancer element.

In some embodiments of the present invention, expression reporter vectors for use in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for terminating transcription and stabilizing the mRNA. Such sequences are commonly available from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

In some embodiments, the present invention provides a vector for expressing a reporter molecule in a T cell. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, a multiple cloning site containing recognition sequences for many restriction endonucleases, an enhancer element, a promoter (e.g., an enhancer element and/or promoter in response to T cell activation), and a transcription termination sequence. In some embodiments, the vector is a plasmid. In other embodiments, the vector is a recombinant viral genome; for example, a recombinant lentiviral genome, a recombinant retroviral genome, a recombinant adeno-associated viral genome. A vector containing a polynucleotide sequence (e.g., a reporter gene operably linked to a T cell response promoter/enhancer) can be transferred into a host T cell by a known method. For example, calcium phosphate treatment, electroporation, lipofection, a gene gun, or a virus-based transfection can be used. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd edition, 1989). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection.

C. Cells

In some aspects, the present invention provides a cell-based assay to detect anti-CD3 homodimers in a composition comprising TDB by contacting a T cell population comprising a reporter complex in response to T cell activation. In some embodiments, the T cells of the population are CD4 ⁺ T cells. In some embodiments, the T cells are CD8 ⁺ T cells. In other embodiments, the T cells are CD4 ⁺ /CD8 ⁺ T cells. In some embodiments, CD4 ⁺ and/or CD8 ⁺ T cells exhibit an increase in the release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukins. In some embodiments, the T cell population is an immortalized T cell population (e.g., an immortalized T cell line). In some embodiments, the T cell population is an immortalized CD4 ⁺ and/or CD8 ⁺ cell population expressing TCR/CD3e. In some embodiments, the T cell is a Jurkat cell. In some embodiments, the T cell is a CTLL-2T cell.

In some embodiments, the T cells of the present invention comprise a T cell receptor. The T cell receptor exists as a complex of several proteins. The T cell receptor itself is composed of two independent peptide chains encoded by independent T cell receptor α and β (TCRα and TCRβ) genes. Other proteins in the complex include CD3 proteins: CD3ε (also known as CD3e), CD3γ, CD3δ, and CD3ζ. CD3 proteins are found as CD3εγ and CD3εδ heterodimers and CD3ζ homodimers. CD3ζ homodimers allow signaling complexes to aggregate around these proteins. In some embodiments, one arm of TDB binds to the T cell receptor complex. In some embodiments, TDB binds to CD3. In some embodiments, TDB binds to CD3ε (CD3e) protein.

In some embodiments, the present invention provides a composition comprising T cells, which is used in a cell-based assay to detect and/or quantify anti-CD3 homodimers in a TDB composition. In some embodiments, the T cells of the composition are CD4 ⁺ T cells. In some embodiments, the T cells of the composition are CD8 ⁺ T cells. In other embodiments, the T cells of the composition are CD4 ⁺ /CD8 ⁺ T cells. In some embodiments, the T cells of the composition are immortalized T cells. In some embodiments, the T cells of the composition are Jurkat cells. In some embodiments, the T cells of the composition are CTLL-2T cells. In some embodiments, the T cells of the composition include a reporter complex in response to T cell activation. In some embodiments, the reporter complex includes a polynucleotide encoding a luciferase. In some embodiments, the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase. In some embodiments, the polynucleotide encoding a reporter gene (e.g., luciferase) is operably linked to a T cell activation responsive regulatory element (e.g., a T cell activation responsive promoter and/or enhancer). In some embodiments, the promoter responsive to T cell activation is a NFAT promoter, an AP-1 promoter, a NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter.

In some embodiments, the T cells (reporter T cells) into which T cell activation responsiveness reporter gene constructs have been introduced are screened for activation of anti-CD3 homodimers. For example, stable clones can be isolated by limiting dilution, and their responses to purified anti-CD3 homodimers are screened. In some embodiments, stable reporter T cells are screened with purified anti-CD3 homodimers of any one greater than about 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL, or 10 μg/mL.

In some embodiments, the present invention provides compositions of T cells engineered with T cell activation reporter complexes. In some embodiments, the reporter gene is luciferase, fluorescent protein (such as GFP, YFP, etc.), alkaline phosphatase or beta galactosidase. In some embodiments, luciferase is firefly luciferase, Renilla luciferase or nanoluciferase. In some embodiments, the promoter in response to T cell activation is NFAT promoter, AP-1 promoter, NFκB promoter, FOXO promoter, STAT3 promoter, STAT5 promoter or IRF promoter. In some embodiments, the promoter in response to T cell activation includes any one or more T cell response elements from NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. In some embodiments, the composition of T cells includes CD4 ⁺ T cells and/or CD8 ⁺ T cells. In some embodiments, T cells are Jurkat cells or CTLL-2 cells. In some embodiments, the T cell is a Jurkat cell comprising a polynucleotide encoding luciferase operably linked to an NFκB promoter.

D. Methods for Identifying CD3 Homodimers

In some aspects, the present invention provides a method for detecting anti-CD3 homodimers in a composition comprising a TDB, wherein the TDB antibody comprises a target antigen binding fragment and a CD3 binding fragment, the method comprising contacting a population of T cells with the composition, wherein the T cells comprise a nucleic acid encoding a reporter gene operably linked to a promoter responsive to T cell activation, and wherein the T cell population does not comprise the target antigen, wherein expression of the reporter gene indicates the presence of anti-CD3 homodimers. In some embodiments, the T cell population does not comprise cells expressing the target antigen of the TDB (a non-T cell antigen).

In some embodiments, a population of T cells is contacted with a composition comprising TDB at a concentration ranging from about 0.01 ng/mL to about 50 ng/mL, about 0.05 ng/mL to about 50 ng/mL, about 0.1 ng/mL to about 50 ng/mL, about 0.5 ng/mL to about 50 ng/mL, about 1 ng/mL to about 50 ng/mL, about 5 ng/mL to about 50 ng/mL, about 10 ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 40 ng/mL, about 0.01 ng/mL to about 3 ... About 0.01 ng/mL to about 20 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01 ng/mL to about 5 ng/mL, about 0.01 ng/mL to about 1 ng/mL, about 0.01 ng/mL to about 0.5 ng/mL, about 0.01 ng/mL to about 0.1 ng/mL, about 0.01 ng/mL to about 0.05 ng/mL, about 0.1 ng/mL to about 10 ng/mL, about 0.5 ng/mL to about 10 ng/mL, about 1 ng/mL to about 10 ng/mL, or about 5 ng/mL to about 50 ng/mL.

In some embodiments, the reporter gene is detected after the cell is contacted with the composition for greater than about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, or about 24 hours. In some embodiments, the reporter gene is detected after the cell is contacted with the composition for between about 1 hour and about 24 hours, about 1 hour and about 12 hours, about 1 hour and about 8 hours, about 1 hour and about 6 hours, about 1 hour and about 4 hours, about 1 hour and about 2 hours, about 4 hours and about 24 hours, about 4 hours and about 12 hours, about 4 hours and about 8 hours, about 8 hours and about 24 hours, about 8 hours and about 12 hours, about 16 hours and about 24 hours, about 16 hours and about 20 hours, or about 20 hours and about 24 hours.

In some aspects, the present invention provides a method for quantifying the amount of anti-CD3 homodimer antibodies in a composition comprising a TDB, wherein the TDB comprises a target antigen binding fragment and a CD3 binding fragment, the method comprising contacting a T cell population with the composition at one or more TDB concentrations, wherein the T cells comprise a nucleic acid encoding a reporter gene operably linked to a promoter responsive to T cell activation, and wherein the T cell population does not comprise the target antigen; and correlating the expression of the reporter gene as a function of antibody concentration with a standard curve generated by contacting the T cells with varying concentrations of purified anti-CD3 homodimers. Dilutions of an anti-CD3 homodimer assay standard (a purified anti-CD3 homodimer of known concentration), an anti-CD3 homodimer control, and a TDB test sample are prepared and added to the reporter T cells. After timed incubation, the amount of reporter gene activity induced by the homodimer assay standard, the homodimer control, and the TDB test sample is measured. The amount of bioactive anti-CD3 homodimers in the TDB test sample is determined based on the standard curve generated by the anti-CD3 homodimer assay standard. The percentage of anti-CD3 homodimer present in the test sample is determined by the ratio of the amount of anti-CD3 homodimer present relative to the total amount of TDB present in the test sample.

In some embodiments, a population of T cells is contacted with a composition comprising TDB at a concentration ranging from about 0.01 ng/mL to about 50 ng/mL, about 0.05 ng/mL to about 50 ng/mL, about 0.1 ng/mL to about 50 ng/mL, about 0.5 ng/mL to about 50 ng/mL, about 1 ng/mL to about 50 ng/mL, about 5 ng/mL to about 50 ng/mL, about 10 ng/mL to about 50 ng/mL, about 0.01 ng/mL to about 40 ng/mL, about 0.01 ng/mL to about 3 ... About 0.01 ng/mL to about 20 ng/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01 ng/mL to about 5 ng/mL, about 0.01 ng/mL to about 1 ng/mL, about 0.01 ng/mL to about 0.5 ng/mL, about 0.01 ng/mL to about 0.1 ng/mL, about 0.01 ng/mL to about 0.05 ng/mL, about 0.1 ng/mL to about 10 ng/mL, about 0.5 ng/mL to about 10 ng/mL, about 1 ng/mL to about 10 ng/mL, or about 5 ng/mL to about 50 ng/mL.

In some embodiments, a standard curve for an anti-CD3 homodimer assay standard is generated by contacting reporter T cells with anti-CD3 homodimers at concentrations ranging from about 0.01 ng/mL to about 50 ng/mL. In some embodiments, the multiple concentrations of anti-CD3 homodimer standards include any one of 100/mL ng, 150 ng/mL, 200 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, 1 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, or 500 μg/mL. In some embodiments, the multiple concentrations of anti-CD3 homodimer standards are about 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 concentrations.

The accuracy of the method is assessed by spiking known amounts of purified anti-CD3 homodimer into a TDB preparation and measuring the percent recovery of the anti-CD3 homodimer. In some embodiments, one or more mixtures of anti-CD3 homodimer and TDB are produced by adding greater than about 100 ng, 150 ng, 200 ng, 250 ng, 500 ng, 750 ng, 1 μg, 2.5 μg, 5 μg, 10 μg, 25 μg, 50 μg, 100 μg, 250 μg, or 500 μg of purified anti-CD3 homodimer to about 1 mg/mL of a stock solution of αCD20/αCD3 TDB.

In some embodiments, the reporter gene is detected after the cell is contacted with the composition for greater than about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, or about 24 hours. In some embodiments, the reporter gene is detected after the cell is contacted with the composition for between about 1 hour and about 24 hours, about 1 hour and about 12 hours, about 1 hour and about 8 hours, about 1 hour and about 6 hours, about 1 hour and about 4 hours, about 1 hour and about 2 hours, about 4 hours and about 24 hours, about 4 hours and about 12 hours, about 4 hours and about 8 hours, about 8 hours and about 24 hours, about 8 hours and about 12 hours, about 16 hours and about 24 hours, about 16 hours and about 20 hours, or about 20 hours and about 24 hours.

E. Measurement

The following is an exemplary but non-limiting method for developing a cell-based assay to detect anti-CD3 homodimers in TDB preparations.

DNA construct: A lentivirus is used to generate a stable cell line for evaluating the purity of the TDB bispecific antibody. A lentiviral vector is constructed that expresses the reporter gene firefly luciferase, Renilla luciferase, or nanoluciferase under the control of a minimal TK promoter regulated by DNA recognition elements of NFAT (nuclear factor of activated T cells), AP-1 (Fos/Jun), NFAT/AP1, NFκB, FOXO, STAT3,5, and IRF. The lentiviral expression cassette used to generate a stable reporter gene cell line can be a third-generation self-inactivating bicistronic vector that expresses various antibiotic selection markers under the control of a constitutive promoter/enhancer (EF1α or SV40) to enable the generation of a stable cell line. The reporter gene lentiviral vector used is modified from the commercially available vector pCDH.MCS.EF1a.Puro (SBI biosciences; catalog number CD510B-1). Promoter modifications included removal of the CMV minimal promoter and replacement with various enhancer elements (NFAT, NFκB, etc.), addition of the minimal core RNA polymerase promoter (TATA box) from pRK5.CMV.luciferase (Osaka, G et al., 1996 J Pharm Sci. 1996, 85: 612-618), and replacement of different selection cassettes from internal DNA (neomycin resistance gene from pRK5.tk.neo, hygromycin resistance gene from pRK5.tk.hygro; and blasticidin resistance gene from pRK5.tk.blasticidin). The effect of the constitutive promoter used for selection on the activation of the enhancer element was minimal, due to the incorporation of a non-coding stretch of DNA designed to minimize promoter/enhancer crosstalk. Firefly luciferase from pRK5.CMV.luciferase (Osaka, 1996) is cloned into the HindIII-NotI site of the modified lentiviral parent vector. Other luminescent proteins including Renilla luciferase and nanoluciferase can also be subcloned into the HindIII-NotI site. Lentiviral packaging constructs (pCMV.HIVΔ, pCMV.VSV-G and pCMV.Rev) for transient transfection of 293s (293 suspension adapted cell line) cells are available (pCMV.VSV-G) or produced (pCMV.HIVΔ, pCMV.REV). HIV strain MN (Nakamura, GR et al., 1993, J. Virol. 67(10):6179-6191) can be used to generate the pCMV.HIVΔ packaging vector and contains an internal EcoRI partial digestion deletion to inactivate the HIV viral envelope by deletion and contains modifications to the 5' and 3' LTRs for safety purposes. HIV Rev was cloned from 293s cell RNA transfected with pCMV.HIVΔ by RT-PCR and introduced into the ClaI-Xho sites of pRK5.tk.neo. VSV-G was used to pseudotype the lentiviral reporter gene (replacing HIV env with VSV-G) to infect any cell type. Lentivirus expression plasmids and packaging constructs were amplified in Stbl2 competent cells (Life Technologies, catalog number 10268-019) and DNA was purified using the Qiagen Maxi Prep kit (catalog number 12662). All DNA constructs were confirmed by DNA sequencing.

Reporter gene assay cell line development: Jurkat CD4+ T cell line (DSMZ, catalog number ACC 282) and CTLL-2CD8 ⁺ T cell line (Life Technologies, catalog number K1653) were used to evaluate the feasibility of reporter gene assay monitoring by TDB activated T cells. A lentiviral vector was constructed, which expresses the reporter gene firefly luciferase, sea renilla luciferase or nanoluciferase under the control of a minimal TK promoter regulated by DNA recognition elements of NFAT (nuclear factor of activated T cells), AP-1 (Fos/Jun), NFAT/AP1, NFκB, FOXO, STAT3,5 and IRF. Reporter gene virus stock solution was generated by transient transfection of 293s cells and pseudotyped with VSV-G, concentrated, and titrated using standard methods (Naldini, L. et al., 1996 Science, 272: 263-267). By centrifugal inoculation (spinoculation) with 10 MOI with slow virus reporter gene virus stock solution infection Jurkat CTLL-2 cells, and after 3 days, infected cells were selected for antibiotic resistance. After 2 weeks, stable pools were produced and their response to purified TDB was assessed. The qPCR method using assessment copy number and integration was used to prove that all stable pools were stably infected by reporter gene constructs. Purified anti-CD3 homodimers can activate NFAT and NFκB Jurkat reporter gene pools. Similar responses were also observed in other TDB. Based on these experiments, limiting dilution of Jurkat/NFκB-luciferase and Jurkat/NFAT-luciferase was set up to realize the generation of single cell clones and single stable reporter cell lines.

Development and evaluation of T cell activation impurity assays: Anti-CD3 homodimers are product-related impurities that can activate T cells in the absence of target cells and therefore represent separate activities from TDB. The anti-CD3 homodimer species present as impurities in TDB purified preparations can be covalently or non-covalently linked, and therefore can be used to cross-link the variants to activate the TCR on the surface of T cells. Since TDB has only one anti-CD3 arm, TDB cannot cross-link TCR and has no activity when incubated with T cells alone. In vivo, TDB may be able to cross-link TCR on T cells through FcgR-mediated cross-linking mediated by effector cells (monocytes, macrophages, NK cells).

In order to quantify the amount of aCD3 homodimer variants present, the amount of luciferase activity observed in the TDB sample was calculated based on the best fit curve of luciferase activity observed when a purified aCD3 homodimer standard of known concentration was incubated with the Jurkat/NFκB-firefly luciferase clone 2 cell line (Figure 7). In order to assess the influence of the matrix effect and impurity concentration in each sample, serial dilutions were prepared, and dilution linearity was assessed in the endpoint assay. The total amount of homodimer present was determined by the impurity mass present in the TDB gross mass and expressed as anti-CD3 homodimer %. The method can quantitatively detect anti-CD3 homodimers as low as 0.1 microgram (0.1ppm) of purified in the purified TDB preparation. The assay format also demonstrates the ability to detect impurity activity in TDB materials that have not been purified after the initial purification step of the current process and has been used to assess purification methods for TDB to remove impurities. In the case of incorporation of purified anti-CD3 homodimers, the assay format showed accurate recovery rates of as low as 0.5% homodimer spiked material in the TDB test material (Figure 7). The method has been used in combination with other orthogonal tests to demonstrate that the current purification process of TDB removes homodimers and other T cell activating substances to below the quantitative limit of the assay. However, during the process, various samples showed T cell activation activity in the assay, which was not correlated with the anti-CD3 homodimer mass spectrometry assay. Correlation with other orthogonal analytical methods suggests that these other species may be some form of aggregates or HMWS (Figure 9). Aggregates of TDB will induce TCR clustering and activity. It has been observed that HMWS as low as 1.5% can induce significant activity in the assay, as observed during the evaluation of various formulation studies. The purification and evaluation of these other species enables the use of various impurity reference standards to carry out impurity assays to assess the purity and safety of TDB. The use of different reporter gene cell lines allows the classification of the different species present. Therefore, as a result of these efforts, the currently reported value for % anti-CD3 homodimer may be revised to another value.The sensitivity of reporter gene assays to detect biologically active impurities is a useful general method for classifying product variants and assessing acceptable levels that may be present in therapeutics.

III. Kit

In some aspects of the present invention, a kit or article is provided comprising a container for accommodating a composition comprising an engineered T cell containing a reporter complex in response to T cell activation as described herein, and optionally providing instructions for use. In some embodiments, the kit provides an anti-CD3 homodimer assay standard (a purified anti-CD3 homodimer of known concentration) and/or an anti-CD3 homodimer control. The container holds the formulation, and a label on or associated with the container can indicate instructions for use. The article may also include other materials desired from a commercial and user perspective, including other buffers, diluents, culture dishes, reagents for detecting reporter molecules, and a package insert with instructions for use.

IV. Peptides

Recombinant technology is generally used to produce polypeptides to be analyzed using the methods described herein. The method for producing recombinant proteins is described in, for example, U.S. Patent Nos. 5,534,615 and 4,816,567, which are specifically incorporated herein by reference. In some embodiments, the protein of interest is produced in CHO cells (see, for example, WO 94/11026). In some embodiments, the polypeptide of interest is produced in Escherichia coli cells. See, for example, U.S. Patent No. 5,648,237; U.S. Patent No. 5,789,199 and U.S. Patent No. 5,840,523, which describe translation initiation regions (TIR) and signal sequences for optimizing expression and secretion. Also referring to Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C.Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describe the expression of antibody fragments in E. coli. When using recombinant techniques, the polypeptide can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium.

The polypeptide can be recovered from the culture medium or from the host cell lysate. The cells used in expressing the polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents. If the polypeptide is produced intracellularly, then as a first step, particulate debris (host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating polypeptides secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) over approximately 30 minutes. Cell debris can be removed by centrifugation. When the polypeptide is secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using commercially available polypeptide concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration units). Protease inhibitors (e.g., PMSF) may be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

In some embodiments, the polypeptide in a composition comprising a polypeptide and one or more contaminants has been purified or partially purified prior to analysis by the methods of the present invention. For example, the polypeptide of the methods is in the eluate from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography. In some embodiments, the polypeptide is in the eluate from Protein A chromatography.

Examples of polypeptides that can be analyzed by the methods of the present invention include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, enzymes, hormones, fusion proteins, Fc-containing proteins, immunoconjugates, cytokines, and interleukins.

(A) Antibodies

In some embodiments of any of the methods described herein, the polypeptide used in any of the methods of analyzing polypeptides and preparations comprising the polypeptides by the methods described herein is an antibody. In some embodiments, the polypeptide is a T cell-dependent bispecific (TDB) antibody.

Molecular targets of the antibodies include CD proteins and their ligands, such as, but not limited to: (i) CD3, CD4, CD8, CD19, CD11a, CD20, CD22, CD34, CD40, CD79α (CD79a) and CD79β (CD79b); (ii) members of the ErbB receptor family, such as EGF receptor, HER2, HER3 or HER4 receptor; (iii) cell adhesion molecules, such as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and αv/β3 integrin. Proteins, including their α or β subunits (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); (iv) growth factors, such as VEGF; IgE; blood group antigens; flk2/flt3 receptors; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, beta7, etc.; (v) cell surface and transmembrane tumor-associated antigens (TAAs), such as those described in U.S. Patent No. 7,521,541, and (vi) other targets, such as FcRH5, LyPD1, TenB2. In some embodiments, the antibody is an anti-CD20/anti-CD3 antibody. Exemplary bispecific antibodies are provided in Table 1.

Table 1. Exemplary Antibodies

Other exemplary antibodies include those selected from, but not limited to, anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2/neu antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9/p24 antibodies, anti-CD10 antibodies, anti-CD11a antibodies, anti-CD11c antibodies, anti-CD13 antibodies, anti-CD14 antibodies, anti-CD16 antibodies, anti-CD17 antibodies, anti-CD18 antibodies, anti-CD19 ... 5 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA/CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-κ light chain antibody, anti-λ light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrillin antibody, anti-keratin antibody and anti-Tn-antigen antibody.

(i) Monoclonal antibodies

In some embodiments, the antibody is a monoclonal antibody. A monoclonal antibody is an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants that arise during the production of the monoclonal antibody (such variants generally exist in small amounts). Thus, the modifier "monoclonal" indicates the characteristic of the antibody as not being a mixture of discrete or polyclonal antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (U.S. Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal (such as a hamster) is immunized as described herein to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The prepared hybridoma cells are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of cells lacking HGPRT.

In some embodiments, myeloma cells are those that fuse efficiently, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to a culture medium such as HAT medium. In some embodiments, among these cell lines, the myeloma cell line is a murine myeloma cell line, such as cell lines derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The production of monoclonal antibodies directed against the antigen is assayed in the culture medium in which the hybridoma cells are grown. In some embodiments, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, polypeptide A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The DNA encoding the monoclonal antibody is easily separated and sequenced using conventional procedures (e.g., using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of murine antibodies). In some embodiments, hybridoma cells serve as the source of this DNA. Once separated, the DNA can be placed in an expression vector and then transfected into a host cell (such as an E. coli cell, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) that does not otherwise produce immunoglobulin polypeptides, to obtain the synthesis of the monoclonal antibody in a recombinant host cell. Reviews on recombinant expression of the DNA encoding the antibody in bacteria include Skerra et al., Curr. Opinion in Immunol. 5: 256-262 (1993) and Plückthun, Immunol. Revs., 130: 151-188 (1992).

In another embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries produced using the technology described in McCafferty et al., Nature 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe the use of phage libraries to separate mouse and human antibodies, respectively. Subsequent publications describe the generation of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for building very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques represent suitable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA can also be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody comprising one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody.

(ii) Humanized antibodies

In some embodiments, the antibody is a humanized antibody. The method of humanizing non-human antibodies has been described in the art. In some embodiments, the humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "input" residues, which are generally taken from the "input" variable domain. Humanization can be basically carried out according to the method of Winter and collaborators (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by replacing the corresponding sequence of human antibodies with hypervariable region sequences. Therefore, this type of "humanized" antibody is a chimeric antibody (U.S. Patent number 4,816,567), in which significantly less than a complete human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The selection of human variable domain light and heavy chains for preparing humanized antibodies is very important for reducing antigenicity. According to the so-called "best fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human framework region (FR) of the humanized antibody (Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol. 196: 901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chain variable regions. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)).

It is also important that the antibody is humanized to retain high affinity and other favorable biological properties to the antigen. To achieve this goal, in some embodiments of the method, the three-dimensional model of the parental sequence and the humanized sequence is used to prepare humanized antibodies by the analytical method of the parental sequence and various conceptually humanized products. The three-dimensional immunoglobulin model is normally available and is familiar to those skilled in the art. Available computer programs can be used to illustrate and demonstrate the possible three-dimensional conformational structure of selected candidate immunoglobulin sequences. The inspection of these demonstrations allows analysis of the possible effects of residues in the function of candidate immunoglobulin sequences, i.e., analysis of the residues that affect the ability of candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and incorporated to from recipient and input sequence, thereby realizing required antibody characteristics, such as increased affinity to the target antigen. Usually, hypervariable region residues directly and most substantially relate to influencing antigen binding.

(iii) Human antibodies

In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that can produce a full spectrum of human antibodies after immunization in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain joining region ( _JH ) gene in chimeric and germline mutant mice has been described to completely suppress endogenous antibody production. The transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies after antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggermann et al., Year in Immuno. 7: 33 (1993); and U.S. Patent Nos. 5,591,669; 5,589,369; and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from the immunoglobulin variable (V) domain gene repertoire of unimmunized donors. According to this technology, antibody V domain genes are cloned in frame into either a major or minor coat polypeptide gene of a filamentous bacteriophage (such as M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of genes encoding antibodies exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats; for their review, see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) A range of different anti-oxazolone antibodies were isolated from a small random combinatorial library of V genes derived from the spleens of immunized mice. V gene libraries from unimmunized human donors can be constructed and antibodies against a range of different antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

Human antibodies can also be generated from in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

(iv) Antibody fragments

In some embodiments, the antibody is an antibody fragment. Various techniques have been developed for producing antibody fragments. Traditionally, these fragments are produced by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be directly produced by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically linked to form F (ab) ₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another method, F (ab') ₂ fragments can be directly separated from recombinant host cell cultures. Other techniques for producing antibody fragments will be obvious to skilled practitioners. In other embodiments, the selected antibody is a single-chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. For example, an antibody fragment may also be a "linear antibody," as described, for example, in U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of: a Fab fragment, a Fab' fragment, a F(ab') ₂ fragment, a scFv, a Fv, and a diabody.

(v) Bispecific antibodies

In some embodiments, the antibody is a bispecific antibody. A bispecific antibody is an antibody with binding specificity for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes. Alternatively, the bispecific antibody binding arm can be combined with an arm that binds to a triggering molecule on a leukocyte (such as a T cell receptor molecule (e.g., CD2 or CD3)) or an Fc receptor (FcγR) of IgG (such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)), thereby concentrating the cellular defense mechanism on the cell. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab') ₂ bispecific antibodies). In some embodiments, the antibody is a T cell-dependent bispecific (TDB) antibody. In some embodiments, the TDB comprises a target antigen binding fragment and a T cell receptor binding fragment. In some embodiments, the TDB comprises a target antigen binding fragment and a CD3 binding fragment. In some embodiments, the TDB comprises a target antigen binding fragment and a CD3 e binding fragment.

The method for preparing bispecific antibodies is known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Millstein et al., Nature 305:537-539 (1983)). Due to the random distribution of immunoglobulin heavy chains and light chains, these hybridomas (quadrivalent tumors) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecules usually completed by affinity chromatography steps is quite cumbersome, and the product yield is low. WO 93/08829 and Traunecker et al., EMBO J., 10: Similar procedures are disclosed in 3655-3659 (1991).

According to different methods, the antibody variable domains with required binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is with the immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, CH2 region and CH3 region. In some embodiments, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. The DNA encoding immunoglobulin heavy chain fusion and (if necessary) immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. When the unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides in embodiments the large flexibility of regulating the mutual ratios of the three polypeptide fragments. However, when the expression of at least two polypeptide chains of equal ratios results in high yields or when the ratio does not have a specific meaning, the coding sequences of two or all three polypeptide chains can be inserted into an expression vector.

In some embodiments of this method, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in another arm. It has been found that this asymmetric structure is conducive to separating the required bispecific compound from unwanted immunoglobulin chain combinations because the presence of immunoglobulin light chains in only half of the bispecific molecule provides an easy separation mode. This method is disclosed in WO 94/04690. For more details about producing bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the proportion of heterodimers that can be recovered from recombinant cell culture. In some embodiments, the interface comprises at least a portion of a _CH3 domain of an antibody constant domain. In this approach, one or more small amino acid side chains at the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" having the same or similar size as the large side chains are created at the interface of a second antibody molecule by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be connected to avidin and the other antibody can be connected to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and for treating HIV infection (WO91/00360, WO 92/200373 and EP 0308936). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with many cross-linking techniques.

The technology for producing bispecific antibodies from antibody fragments has also been described in the literature. For example, chemical linkage can be used to prepare bispecific antibodies. Brennan et al., Science 229: 81 (1985) described a method in which intact antibodies are proteolytically cleaved to produce F(ab') ₂ fragments. In the presence of the dithiol complexing agent sodium arsenite, these fragments are reduced to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted into thionitrobenzoic acid (TNB) derivatives. One of the Fab'-TNB derivatives is then converted back into Fab'-thiol by reduction with mercaptoethylamine, and mixed with an equimolar amount of other Fab'-TNB derivatives to form bispecific antibodies. The resulting bispecific antibodies can be used as reagents for selective enzyme immobilization.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zippers have been used to produce bispecific antibodies. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portion of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. These fragments include a heavy chain variable domain ( _VH ) linked to a light chain variable domain ( _VL ) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the _VH and _VL domains of one fragment are forced to pair with the complementary _VL and _VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

(v) Multivalent Antibodies

In some embodiments, the antibody is a multivalent antibody. Compared with bivalent antibodies, multivalent antibodies can be internalized (and/or alienated) faster by cells expressing the antigen to which the antibody is bound. The antibodies provided herein can be multivalent antibodies (e.g., tetravalent antibodies) (different from the IgM class) with three or more antigen binding sites, which can be easily produced by recombinantly expressing nucleic acids encoding the polypeptide chains of the antibodies. Multivalent antibodies can include a dimerization domain and three or more antigen binding sites. Preferred dimerization domains include an Fc region or a hinge region (or are composed of the regions). In this case, the antibody will include an Fc region and three or more antigen binding sites at the amino terminus of the Fc region. Preferred multivalent antibodies herein include three to about eight (but preferably four) antigen binding sites (or are composed of the antigen binding sites). The multivalent antibody includes at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain includes two or more variable domains. For example, a polypeptide chain may include VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, a polypeptide chain may include: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for example, comprise about 2 to about 8 light chain variable domain polypeptides. The light chain variable domain polypeptides encompassed herein comprise a light chain variable domain and, optionally, further comprise a CL domain. In some embodiments, the multivalent antibody comprises a T cell binding fragment. In some embodiments, the multivalent antibody comprises a T cell receptor binding fragment. In some embodiments, the multivalent antibody comprises a CD3 binding fragment. In some embodiments, the multivalent antibody comprises a CD3 epsilon binding fragment.

In some embodiments, the antibody is a multispecific antibody. Examples of multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain ( _VH ) and _a light chain variable domain ( _VL ) (wherein the _VHVL unit has polyepitopic specificity), antibodies having two or more _VL and _VH domains (wherein each _VHVL unit binds to a different _epitope ), antibodies having two or more single variable domains (wherein each single variable domain binds to a different epitope), full-length antibodies, antibody fragments (such as Fab, Fv, dsFv, scFv), diabodies, bispecific diabodies, tribodies, trifunctional antibodies, and covalently or non-covalently linked antibody fragments. In some embodiments, the antibody has polyepitopic specificity; for example, the ability to specifically bind to two or more different epitopes on the same or different targets. In some embodiments, the antibody is monospecific; for example, an antibody that binds only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

(vi) Other Antibody Modifications

In terms of effector function, it may be necessary to modify the antibodies provided herein, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or in addition thereto, cysteine residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The resulting homodimeric antibodies can have improved internalization ability and/or enhanced complement-mediated cell killing and antibody-dependent cellular toxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B.J., Immunol. 148: 2918-2922 (1992). As described in Wolff et al., Cancer Research 53: 2560-2565 (1993), heterodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking agents. Alternatively, antibodies with dual Fc regions can be engineered and thus have enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half-life of an antibody, amino acid changes may be made in the antibody as described in US 2006/0067930, which is herein incorporated by reference in its entirety.

(B) Polypeptide Variants and Modifications

Amino acid sequence modifications of the polypeptides (including antibodies) described herein can be used in methods of purifying the polypeptides (eg, antibodies) described herein.

(i) Variant polypeptides

"Polypeptide variant" means a kind of polypeptide, preferably as defined herein with the full-length native sequence of a polypeptide, a polypeptide sequence lacking a signal peptide, an extracellular domain of a polypeptide with or without a signal peptide having at least about 80% amino acid sequence identity. Such polypeptide variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of a full-length native amino acid sequence. Typically, TAT polypeptide variants will have at least about 80% amino acid sequence identity, or at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the extracellular domain of a polypeptide with or without a signal peptide. Optionally, the variant polypeptide has no more than one conservative amino acid substitution compared to the native polypeptide sequence, or has no more than about 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to the native polypeptide sequence.

For example, when compared to a full-length native polypeptide, a variant polypeptide may be truncated at the N-terminus or C-terminus, or may lack internal residues. Certain variant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These variant polypeptides with truncation, deletions, and insertions can be prepared by any of many conventional techniques. The desired variant polypeptide can be chemically synthesized. Another suitable technique involves separating and amplifying nucleic acid fragments encoding the desired variant polypeptides by polymerase chain reaction (PCR). Oligonucleotides defining the desired ends of the nucleic acid fragments are employed at the 5' and 3' primers in PCR. Preferably, the variant polypeptide shares at least one biological and/or immunological activity with the native polypeptides disclosed herein.

Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions with single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include fusion of the N-terminus or C-terminus of the antibody with an enzyme or polypeptide that increases the serum half-life of the antibody.

For example, it may be necessary to improve the binding affinity and/or other biological properties of the polypeptide. Amino acid sequence variants of the polypeptide may be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the polypeptide. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes may also alter the post-translational processing of the polypeptide (e.g., antibody), such as changing the number or position of glycosylation sites.

Guidance in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the polypeptide sequence to the sequences of homologous known polypeptide molecules and minimizing the number of amino acid sequence changes that occur in regions of high homology.

The method that can be used to identify certain residues or regions as preferred positions for mutagenesis in polypeptides (e.g., antibodies) is called "alanine scanning mutagenesis", as described by Cunningham and Wells, Science 244:1081-1085 (1989). Here, a residue or a group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys and Glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of amino acids with antigens. Those amino acid positions that exhibit functional sensitivity are then refined by introducing additional or other variants at or for the substitution site. Therefore, although the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the performance of the mutation at a given site, ala scanning or random mutagenesis is carried out at the target codon or region, and the antibody variants expressed for the desired activity screening are screened.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue replaced by a different residue in the antibody molecule. The most interesting site of substitution mutagenesis includes the hypervariable region, but also encompasses FR changes. Conservative substitutions are shown under the heading "Exemplary Substitutions" in Table 2 below. If this type of substitution causes a change in biological activity, then a more substantial change named "substitution" in Table 2 or as further described below about the amino acid class can be introduced, and the product screened.

Table 2.

Substantial changes in the biological properties of a polypeptide can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, e.g., in a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (in A. L. Lehninger, Biochemistry 2nd ed., pp. 73-75, Worth Publishers, New York (1975)):

(1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) Uncharged polarity: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q)

(3) Acidic: Asp (D), Glu (E)

(4) Basic: Lys(K), Arg(R), His(H)

Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect 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.

Any cysteine residues not involved in maintaining the correct conformation of the antibody may also be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to a polypeptide to improve its stability (particularly when the antibody is an antibody fragment (such as an Fv fragment)).

Particularly preferred types of substitution variants involve replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). Generally speaking, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were produced. A convenient way to produce such substitution variants involves affinity maturation using phage display. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus produced are displayed from filamentous phage particles in a monovalent manner as a fusion of the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. In order to identify candidate hypervariable region sites to be modified, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, analyzing the crystal structure of the antigen-antibody complex may be beneficial to identify the contact points between the antibody and the target. Such contact residues and adjacent residues are candidates for substitution according to the technology set forth herein. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Another type of amino acid variant of a polypeptide alters the original glycosylation pattern of the antibody. The polypeptide may contain non-amino acid moieties. For example, the polypeptide may be glycosylated. Such glycosylation may occur naturally during expression of the polypeptide in a host cell or host organism, or may be an intentional modification resulting from human intervention. By alteration is meant deleting one or more carbohydrate moieties found in the polypeptide and/or adding one or more glycosylation sites not present in the polypeptide.

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are the discrimination sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition of glycosylation sites to a polypeptide is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding one or more serine or threonine residues to the sequence of the original antibody, or substituting one or more serine or threonine residues with one or more serine or threonine residues (for O-linked glycosylation sites).

Removal of carbohydrate moieties present on a polypeptide can be achieved chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that are targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on a polypeptide can be achieved using a variety of endoglycosidases and exoglycosidases.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of the hydroxyl group of seryl or threonyl residues; methylation of the α-amino groups of lysine, arginine, and histidine side chains; acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

(ii) Chimeric polypeptides

The polypeptides described herein can be modified in the form of chimeric molecules comprising a polypeptide fused to another heterologous polypeptide or amino acid sequence. In some embodiments, the chimeric molecule comprises a fusion of a polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is typically located at the amino or carboxyl terminus of the polypeptide. The presence of such epitope-tagged polypeptides can be detected using antibodies against the tag polypeptide. In addition, providing an epitope tag enables the polypeptide to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

In alternative embodiments, the chimeric molecule may comprise a fusion of a polypeptide with an immunoglobulin or a specific region of an immunoglobulin. The bivalent form of the chimeric molecule is referred to as an "immunoadhesin."

As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide with the effector functions of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises an amino acid sequence with a desired binding specificity that is different from the antigen recognition and binding site (i.e., "heterologous") of an antibody and a fusion of an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a continuous amino acid sequence that at least comprises the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin such as IgG-1, IgG-2, IgG-3, or IgG-4 subtype, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

Ig fusions preferably involve substituting a soluble (transmembrane domain deleted or inactivated) form of the polypeptide in place of at least one variable region within an Ig molecule. In particularly preferred embodiments, the immunoglobulin fusion comprises the hinge, _CH2 , and _CH3 or hinge, _CH1 , _CH2 , and _CH3 regions of an IgG1 molecule.

(iii) Polypeptide Conjugates

The polypeptides used in the polypeptide formulations can be conjugated to a cytotoxic agent (such as a chemotherapeutic agent), a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents suitable for producing such conjugates can be used. In addition, enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curculin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecene. A variety of radionuclides can be used to produce radioconjugated polypeptides. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re. Conjugates of polypeptides and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl diimidoadipate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to polypeptides.

Also contemplated herein are conjugates of the polypeptides with one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothenes, and CC1065, as well as toxinically active derivatives of these toxins.

Maytansine is a mitotic inhibitor that works by suppressing tubulin polymerization. Maytansine was originally separated from the East African shrub Maytansine (Maytenus serrata). Subsequently, it was found that some microorganisms also produced maytansine, such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and derivatives thereof and analogs are also contemplated. Many linking groups for preparing polypeptide-maytansinoid conjugates are known in the art, including those disclosed in, for example, U.S. Patent number 5,208,020. Linking group includes a disulfide group, a thioether group, an acid-labile group, a light-labile group, a peptidase-labile group or an esterase-labile group disclosed in the patent as described above, preferably a disulfide group and a thioether group.

Joint can be connected to the different positions of maytansinol molecule, and this depends on the type connected.For example, ester bond can be formed by using conventional coupling technology and hydroxyl reaction.Reaction can occur in the C-3 position with hydroxyl, the C-14 position modified through hydroxyl, the C-15 position modified through hydroxyl and the C-20 position with hydroxyl.In a preferred embodiment, key is formed at the C-3 position of maytansinol or maytansinol analog.

Another conjugate of interest comprises a polypeptide conjugated to one or more calicheamicin molecules. The antibiotics of the calicheamicin family are capable of producing double-stranded DNA breaks at concentrations below picomolar. For the preparation of conjugates of the calicheamicin family, see, for example, U.S. Patent No. 5,712,374. Structural analogs of calicheamicin that can be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG, and θ ₁ ^I. Another anti-tumor drug that can be conjugated to an antibody is QFA, an antifolate. Both calicheamicin and QFA have intracellular sites of action and are not easy to pass through the plasma membrane. Therefore, cellular uptake of these agents by polypeptide (e.g., antibody)-mediated internalization greatly improves their cytotoxic effects.

Other anti-tumor agents that can be conjugated to the polypeptides described herein include BCNU, streptozoicin, vincristine and 5-fluorouracil, a family of agents collectively known as the LL-E33288 complex, and esperamicins.

In some embodiments, the polypeptide can be a conjugate between the polypeptide and a compound having nucleolytic activity (eg, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

In another embodiment, polypeptides (e.g., antibodies) can be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, wherein the polypeptide receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., an antibiotic) conjugated to a cytotoxic agent (e.g., a radionucleotide).

In some embodiments, the polypeptide can be conjugated to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent) into an active anticancer drug. The enzyme component of the immunoconjugate includes any enzyme that can act on a prodrug in such a way as to convert it into a more active, cytotoxic form.

Useful enzymes include, but are not limited to, alkaline phosphatase, which can be used to convert phosphate-containing prodrugs into free drug; arylsulfatases, which can be used to convert sulfate-containing prodrugs into free drug; cytosine deaminases, which can be used to convert the non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), which can be used to convert peptide-containing prodrugs into free drug; D-alanyl carboxypeptidases, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate cleavage enzymes, such as β-galactosidase and neuraminidase, which can be used to convert glycosylated prodrugs into free drug; β-lactamases, which can be used to convert β-lactam-derivatized drugs into free drug; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which can be used to convert drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, at the amine nitrogen into free drug. Alternatively, antibodies with enzymatic activity (also known in the art as "abzymes") can be used to convert the prodrug into the free active drug.

(iv) Others

Another type of covalent modification of polypeptides includes linking the polypeptide to one of a variety of non-protein polymers (e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol). Polypeptides can also be trapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A.R., ed. (1990).

V. Obtaining Polypeptides for Use in Formulations and Methods

The polypeptides used in the analytical methods described herein can be obtained using methods well known in the art, including recombinant methods. The following section provides guidance on these methods.

(A) Polynucleotide

"Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA.

Polynucleotides encoding polypeptides can be obtained from any source, including but not limited to cDNA libraries prepared from tissues believed to possess polypeptide mRNA and express it at detectable levels. Thus, polynucleotides encoding polypeptides can be conveniently obtained from cDNA libraries prepared from human tissues. Genes encoding polypeptides can also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

For example, a polynucleotide can encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain comprises not only a heavy chain variable region ( _VH ), but also a heavy chain constant region ( _CH ), which typically will comprise three constant domains: _CH1 , _CH2 , and _CH3 ; and a "hinge" region. In some cases, the presence of a constant region is desirable. In some embodiments, a polynucleotide encodes one or more immunoglobulin molecule chains of a TDB.

Other polypeptides that can be encoded by a polynucleotide include antigen-binding antibody fragments, such as single-domain antibodies ("dAbs"), Fvs, scFvs, Fab' and F(ab') ₂ , as well as "minibodies". Minibodies are (usually) bivalent antibody fragments from which the _CH1 and _CK or _CL domains have been removed. Because minibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical/diagnostic applications, but they are bivalent and should therefore retain higher binding affinity than monovalent antibody fragments (such as dAbs). Therefore, unless the context indicates otherwise, the term "antibody" as used herein encompasses not only whole antibody molecules, but also antigen-binding antibody fragments of the types discussed above. Preferably, each framework region present in the encoded polypeptide will contain at least one amino acid substitution relative to the corresponding human acceptor framework. Thus, for example, the framework regions may contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid substitutions relative to the acceptor framework regions.

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by a feature serving the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features.

Further details of the present invention are illustrated by the following non-limiting examples.The disclosures of all references in this specification are expressly incorporated herein by reference.

Example

The following examples are intended only to illustrate the present invention and therefore should not be considered to limit the present invention in any way.The following examples and detailed description are provided by way of illustration and not by way of limitation.

Example 1. Anti-CD3 homodimer activation of T cells

The T cell-dependent bispecific (TDB) antibody (αCD20/αCD3 TDB, anti-CD20 (Mab2; VH SEQ ID NO:31/VL SEQ ID NO:32)/anti-CD3 (Mab1; VH SEQ ID NO:19/VL SEQ ID NO:20)) requires cells expressing the CD20 antigen to induce T cell activation and antigenic cell killing. As shown in Figure 1A, CD8 ⁺ T cells were isolated from human peripheral blood, incubated with an antigen-expressing target cell line at a 1:1 ratio, and stimulated with increasing concentrations of purified αCD20/αCD3 TDB antibodies. After 24 hours of timed incubation following the addition of TDB to the cells, T cells were assessed by flow cytometry for the amount of CD69 (C-type lectin protein) and CD25 (IL-2 receptor) induced on the T cell surface. CD69 and CD25 are markers of T cell activation (Shipkova M, 2012, Clin. Chim. Acta. 413: 1338-49 and Ziegler SF et al., 1994, Stem Cells 12(5): 465-465). Following stimulation with αCD20/αCD3 TDB, CD69 and CD25 cell surface expression increased in a dose-dependent manner. As evidenced by the lack of increase in CD69 and CD25 cell surface expression, in the absence of target cells (blue rectangles), there was no T cell activation. As shown in Figure 1B, T cell-mediated αCD20/αCD3 TDB is required for target cell killing. PBMCs or PBMCs depleted of CD3+ (T cell receptor/CD3e subunit) obtained by negative selection (Milteny Biotec) were incubated with a target cell line expressing CD20 at a ratio of 1:1 and then stimulated with increasing concentrations of αCD20/αCD3 TDB. After 24 hours, PBMCs showed a dose-dependent decrease in the number of target cells by flow cytometry (red circles). However, no loss of target cells was detected when CD3+ T cells were depleted from the PBMC pool (blue rectangles). Therefore, depletion of CD20-expressing target cells by αCD20/αCD3 TDB requires activation of CD3+ T cells, and αCD20/αCD3 TDB alone cannot induce target cell killing.

Purified anti-CD3 homodimers activate human donor T cells. Human donor PBMCs from two different donors were treated with increasing concentrations of purified anti-CD3 homodimers or αCD20/αCD3 TDB bispecific antibodies, and T cell activation levels were tested by FACS 24 hours later as described above. Donor 1 (Figure 2, left panel) and donor 2 (Figure 2, right panel) were stained with anti-CD8, anti-CD69, and anti-CD25 antibodies. The percentage of CD8 ⁺ T cells positive for T cell activation markers CD69 and CD25 was plotted relative to the amount of anti-CD3 homodimers or αCD20/αCD3 TDB treatment. In the presence of target cells, anti-CD3 and αCD20/αCD3 TDB activated T cells in a dose-dependent manner, but αCD20/αCD3 TDB (EC50: 4-6 ng/mL) was a stronger T cell activator compared to anti-CD3 homodimers (EC50: 169-526 ng/mL). Despite donor variability, anti-CD3 homodimers can activate human T cells.

Anti-CD3 homodimers can reduce the efficacy of bispecific antibodies. αCD20/αCD3TDB was spiked with purified anti-CD3 homodimers at different concentrations, and the response was measured. At anti-CD3 homodimer levels above 20%, anti-CD3 homodimers significantly reduced αCD20/αCD3TDB efficacy in a dose-dependent manner at the level of T cell activation and at the level of target cell response (Figure 3A and Table 3). Using PBMCs, low levels of anti-CD3 homodimers (HD) spiked into αCD20/αCD3 TDB did not significantly reduce T cell activation (CD8 ⁺ , Figure 3B left; CD4 ⁺ , Figure 3B right). PBMCs were stimulated with increasing levels of TDB that had been fixed with a constant amount (2.5% or 5%) of purified anti-CD3 homodimers, and analyzed by flow cytometry (FACS) to assess T cell activation (stained for T cell activation markers CD69 and CD25). Anti-CD3 homodimer levels below 5% did not affect the αCD20/αCD3 TDB T cell activation potential of either CD8 ⁺ or CD4 ⁺ T cells.

Table 3

Anti-CD3 homodimers can weakly activate human CD8 ⁺ T cells from various human donors in the absence of target cells. In the presence of target cells, αCD20/αCD3 TDB is able to strongly activate most of the CD8 ⁺ T cells from PBMCs isolated from 6 human donors, and low levels of anti-CD3 homodimers (2.5% or 5%) cannot significantly activate the average T cell activation potential of TDB (Figure 4A; B+ conditions). In the absence of target cells (Figure 4A; B- conditions), anti-CD3 homodimers can weakly activate CD8 ⁺ T cells (average activation potential slightly increased). Anti-CD3 homodimer activation of human T cells showed a dose-dependent trend of increased activation of some representative cytokines. In the presence (B+ condition) or absence (B- condition) of target cells, with or without 2.5% or 5% purified anti-CD3 homodimer spike-in, PBMC isolated from 6 human donors were stimulated with 1 mg/mL αCD20/αCD3 TDB, and T cell activation potential was assessed by testing the cytokines secreted as T cell activation indicators. After 24 hours, conditioned medium was collected and the presence of cytokines was tested using a Luminex cytokine detection kit. In the absence of target cells, anti-CD3 homodimer treatment showed that certain cytokine levels (IL-10 and MCP-1) of some donor PBMCs increased significantly in a dose-dependent manner (Fig. 4B-4E; B- condition). Average cytokine level response mean was plotted.

Example 2. Anti-CD3 homodimer impurity determination

A biological impurity assay has been developed to detect the presence of T cell activation impurities in the presence of T cell dependent bispecific (TDB) antibodies. Since the anti-CD3 homodimer is bivalent, each arm of the impurity can potentially crosslink the TCR, thereby causing T cell activation. TCR-mediated cross-linking by anti-CD3 bivalent antibodies (such as OKT3) activates T cell signaling cascades, causing phosphorylation and nuclear localization of transcription factors (including NFAT and NFκB), leading to transcriptional induction of target genes such as cytokines or cell-killing agents (such as Fas, granzyme B and perforin) (Brown, WM, 2006, Curr Opin Investig Drugs 7:381-388; Ferran, C et al., 1993 Exp Nephrol 1:83-89; Shannon, MF et al., 1995, J. Leukoc. Biol. 57:767-773; Shapiro, 1998; Pardo, J et al., 2003, Int Immunol., 15(12):1441-1450). Under the transcriptional control of AP1, NFAT or NFκB, reporter genes such as firefly luciferase have been used to monitor TCR activation and T cell activation of signal transduction pathways (Shannon, MF et al., 1995, J. Leukoc. Biol. 57: 767-773; Shapiro, 1998). Since TDB does not activate T cells in the absence of target cells (Figures 1A and 1B), the reporter gene assay method is evaluated as a potential assay strategy for detecting anti-CD3 homodimers in the presence of TDB. In order to preliminarily evaluate whether anti-CD3 homodimers can activate T cells in vitro, Jurkat T cells (DSMZ, ACC 282) were infected with recombinant TCR responsive reporter gene lentiviral stock (AP1-luciferase, NFAT-luciferase or NFκB-luciferase), and the stable pool was treated with 10 μg/mL purified anti-CD3 homodimers for 4 hours. Jurkat/AP1 luciferase, Jurkat/NFAT luciferase, and Jurkat/NFκB luciferase stable pools showed dose-dependent induction of luciferase after stimulation with purified anti-CD3 homodimers. The fluorescence response (luciferase reporter gene activity) was plotted, and the highest response was observed from the Jurkat/NFκB luciferase stable pool. (Fig. 5A). Jurkat/NFκB luciferase stable clones isolated by limiting dilution were screened for their response to 10 μg/mL purified anti-CD3 homodimers. Compared to other TCR response elements, the Jurkat T cell NFκB luciferase pool showed the highest response to anti-CD3 homodimers, but other response elements may also potentially be used to detect anti-CD3 homodimers. (Fig. 5B).

To determine the relative response of this clone to αCD20/αCD3 TDB or anti-CD3 homodimers, the Jurkat/NFκB luciferase clone 2 cell line was treated with increasing concentrations of αCD20/αCD3 TDB or anti-CD3 homodimers in the presence of a CD20-expressing target cell line, and luciferase activity was plotted ( FIG6A ). Cells were stimulated with αCD20/αCD3 TDB or CD3 homodimers for 4 hours in RPMI1640 medium supplemented with 10% fetal bovine serum. Purified anti-CD3 homodimers were 1000-fold more active than purified anti-CD3 homodimers in the presence of co-stimulatory target cells. The level of T cell activation caused by anti-CD3 homodimers was lower than that caused by αCD20/αCD3 TDB, but was detectable in the presence of target cells. As measured by NFκB-dependent activation of luciferase transcription, in the absence of target cells, even at high levels of TDB, αCD20/αCD3 TDB did not lead to T cell activation in this cell line, but even at low levels of product-related impurities, anti-CD3 homodimers were able to induce luciferase induction (Figure 6B). Using other T cell activation measures, these T cell activation responses observed for the engineered Jurkat/NFκB luciferase clone 2 reporter cell line were comparable to those observed for human T cells isolated from donor peripheral blood mononuclear cells (PBMCs), indicating that monitoring T cell activation responses using reporter genes is comparable (Table 4). A cell-based assay for detecting anti-CD3 homodimer impurities in αCD20/αCD3 TDB was developed and optimized using the Jurkat/NFκB luciferase clone 2 cell line (Jurkat-NFκBLuc). In summary, these data indicate that engineered T cell reporter cell lines can be used to detect bioactive anti-CD3 homodimer product-related impurities in TDB.

Table 4

Example 3. Quantitative method for detecting anti-CD3 homodimers

A sensitive and quantitative analytical method has been developed to detect biologically active impurities in the presence of αCD20/αCD3 TDB. The αCD20/αCD3 TDB T cell activation assay detects anti-CD3 homodimers present in TDB test samples by measuring CD3e/TCR crosslinking-induced activation of the Rel/NFκB signaling pathway using the engineered T cell reporter cell line, Jurkat-NFκBLuc. Since target cells are not present in the assay, only anti-CD3 homodimers activate the T cell reporter cell line. Activated NFκB translocates to the nucleus and binds to eight NFκB response elements in a synthetic promoter that drives luciferase transcription. In this assay, dilutions of the anti-CD3 homodimer assay standard, anti-CD3 homodimer control, and αCD20/αCD3 TDB test sample are prepared and added to cultured Jurkat-NFκBLuc reporter cells in a 96-well assay plate. The aCD3 homodimer standard is a purified batch of aCD3 homodimers isolated from the αCD20/αCD3 TDB purification method. The aCD3 homodimer control is an αCD20/αCD3 TDB spiked with purified aCD3 homodimer and used as a system suitability standard in the assay. The use of the aCD3 homodimer control in the assay is specific for the method used for the impurity assay run. After a 4-hour timed incubation, the amount of luciferase activity induced by the homodimer assay standard, homodimer control, and αCD20/αCD3 TDB test samples was measured using a fluorescence plate reader. The amount of bioactive anti-CD3 homodimer in the αCD20/αCD3 TDB test samples was determined based on a fluorescence standard curve generated with the anti-CD3 homodimer assay standard from a separate set of wells (Figure 7). The percentage of anti-CD3 homodimer present in the test sample was determined by the ratio of the amount of anti-CD3 homodimer present relative to the total amount of αCD20/αCD3 TDB present in the test sample. The precision of the method was assessed by spiking known amounts of purified anti-CD3 homodimer into αCD20/αCD3 TDB formulations and measuring the percent recovery of anti-CD3 homodimer. The method showed good overall linearity (Figure 8) and had an overall precision of 6.8% (Table 5). In a 1 mg/mL αCD20/αCD3 TDB stock solution, the method was able to reproducibly detect spike levels of anti-CD3 homodimer as low as 150 ng or 0.02%. Based on the recovery studies performed, the optimized method was able to reliably quantify anti-CD3 homodimer levels from 0.25% to 35% anti-CD3 homodimer present in various TDB formulations with an overall precision of 6.8%. Precision was determined as the average CV% of recovery at each spike level.

Table 5

The αCD20/αCD3 TDB T cell activation assay is also sensitive to the presence of another product-related impurity, anti-CD3 aggregates and αCD20/αCD3 TDB high molecular weight species. Samples containing more than 2% HMWS, as detected by SEC, can lead to T cell activation in the Jurkat/NFκB-Luc cell line. This activation by HMWS is quantified as the % homodimer from the T cell activation assay (Figure 9).

Sequence Listing

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<213>Artificial sequence

<220>

<223>Synthetic constructs

<400> 40

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

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

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

Claims (65)

1. A method for detecting anti-CD3 homodimer in a composition comprising a T-cell-dependent bispecific antibody (TDB), wherein the bispecific antibody comprises a target antigen-binding fragment and a CD3-binding fragment, the method comprising... A population of T cells is contacted with the composition, wherein the T cells contain nucleic acids encoding a reporter gene operatively linked to a response element in response to T cell activation, and wherein the population of T cells does not contain the target antigen. The expression of the reporter gene indicates the presence of anti-CD3 homodimer. 2. The method of claim 1, wherein the reporter gene is a luciferase, a fluorescent protein, an alkaline phosphatase, a β-lactamase, or a β-galactosidase. 3. The method of claim 2, wherein the luciferase is firefly luciferase, kidney luciferase, or nanoluciferase. 4. The method of claim 1, wherein the response element in response to T cell activation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 5. The method of claim 2, wherein the response element in response to T cell activation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 6. The method of claim 3, wherein the response element in response to T cell activation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 7. The method of claim 4, wherein the response element responsive to T cell activation comprises a T cell activation response element from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. 8. The method of claim 5, wherein the response element responsive to T cell activation comprises a T cell activation response element from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. 9. The method of claim 6, wherein the response element responsive to T cell activation comprises a T cell activation response element from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. 10. The method of any one of claims 1-9, wherein the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. 11. The method of any one of claims 1-9, wherein the T cell population is a population of Jurkat T cells or CTLL-2 T cells. 12. The method of any one of claims 1-9, wherein the T cell population is contacted with a composition comprising the bispecific antibody at a concentration ranging from 0.01 ng/mL to 50 ng/mL. 13. The method of claim 10, wherein the T cell population is contacted with a composition comprising the bispecific antibody at a concentration ranging from 0.01 ng/mL to 50 ng/mL. 14. The method of claim 11, wherein the T cell population is contacted with a composition comprising the bispecific antibody at a concentration ranging from 0.01 ng/mL to 50 ng/mL. 15. The method of any one of claims 1-9, wherein the reporter gene is detected after 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 16. The method of claim 10, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 17. The method of claim 11, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 18. The method of claim 12, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 19. The method of claim 13, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 20. The method of claim 14, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 21. A method for quantifying the amount of anti-CD3 homodimeric antibody in a composition comprising TDB, wherein the TDB comprises a target antigen-binding fragment and a CD3-binding fragment, the method comprising... A population of T cells is contacted with the composition at one or more of the TDB concentrations, wherein the T cells contain nucleic acids encoding a reporter gene operatively linked to a promoter in response to T cell activation, and wherein the T cell population does not contain the target antigen. The expression of the reporter gene as a function of antibody concentration was correlated with a standard curve generated by exposing the T cells to different concentrations of purified anti-CD3 homodimer. 22. The method of claim 21, wherein the reporter gene is a luciferase, a fluorescent protein, an alkaline phosphatase, a β-lactamase, or a β-galactosidase. 23. The method of claim 22, wherein the luciferase is firefly luciferase, kidney luciferase, or nanoluciferase. 24. The method of claim 21, wherein the response element in response to T cell activation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 25. The method of claim 22, wherein the response element in response to T cell activation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 26. The method of claim 23, wherein the response element in response to T cell activation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 27. The method of claim 24, wherein the response element responsive to T cell activation comprises a T cell activation response element from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. 28. The method of claim 25, wherein the response element responsive to T cell activation comprises a T cell activation response element from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. 29. The method of claim 26, wherein the response element responsive to T cell activation comprises a T cell activation response element from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. 30. The method of any one of claims 21-29, wherein the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. 31. The method of any one of claims 21-29, wherein the T cell population is a population of Jurkat T cells or CTLL-2 T cells. 32. The method of any one of claims 21-29, wherein the T cell population is contacted with a composition comprising the bispecific antibody at a concentration ranging from 0.01 ng/mL to 50 ng/mL. 33. The method of claim 30, wherein the T cell population is contacted with a composition comprising the bispecific antibody at a concentration ranging from 0.01 ng/mL to 50 ng/mL. 34. The method of claim 31, wherein the T cell population is contacted with a composition comprising the bispecific antibody at a concentration ranging from 0.01 ng/mL to 50 ng/mL. 35. The method of any one of claims 21-29, wherein the standard curve is generated by contacting the T cells with different concentrations of purified anti-CD3 antibody ranging from 0.01 ng/mL to 50 ng/mL. 36. The method of claim 30, wherein the standard curve is generated by contacting the T cells with different concentrations of purified anti-CD3 antibody ranging from 0.01 ng/mL to 50 ng/mL. 37. The method of claim 31, wherein the standard curve is generated by contacting the T cells with different concentrations of purified anti-CD3 antibody ranging from 0.01 ng/mL to 50 ng/mL. 38. The method of claim 32, wherein the standard curve is generated by contacting the T cells with different concentrations of purified anti-CD3 antibody ranging from 0.01 ng/mL to 50 ng/mL. 39. The method of claim 33, wherein the standard curve is generated by contacting the T cells with different concentrations of purified anti-CD3 antibody ranging from 0.01 ng/mL to 50 ng/mL. 40. The method of claim 34, wherein the standard curve is generated by contacting the T cells with different concentrations of purified anti-CD3 antibody ranging from 0.01 ng/mL to 50 ng/mL. 41. The method of any one of claims 21-29, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20 or 24 hours following contact of the cells with the composition. 42. The method of claim 30, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 43. The method of claim 31, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 44. The method of claim 32, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 45. The method of claim 35, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 46. The method of any one of claims 33, 34, and 36-40, wherein the reporter gene is detected after one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, or 24 hours following contact of the cells with the composition. 47. An engineered T cell for detecting anti-CD3 homodimer in a composition comprising a bispecific antibody, wherein the bispecific antibody comprises a target antigen-binding fragment and a CD3-binding fragment, wherein the T cell comprises a reporter gene operatively linked to a response element responsive to T cell activation, wherein the response element responsive to T cell activation comprises a T cell activation response element from any one or more of AP-1, NFκB, FOXO, STAT3, STAT5, and IRF. 48. The engineered T cell of claim 47, wherein the reporter gene is luciferase, fluorescent protein, alkaline phosphatase, β-lactamase, or β-galactosidase. 49. The engineered T cell of claim 48, wherein the luciferase is firefly luciferase, kidney luciferase, or nanoluciferase. 50. The engineered T cell of claim 47, wherein the response element to T cell activation is an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 51. The engineered T cell of claim 48, wherein the response element in response to T cell activation is an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 52. The engineered T cell of claim 49, wherein the response element to T cell activation is an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 53. The engineered T cells according to any one of claims 47-52, wherein the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. 54. The engineered T cells according to any one of claims 47-52, wherein the T cell population is a population of Jurkat T cells or CTLL-2 T cells. 55. A kit for detecting anti-CD3 homodimer in a composition comprising a bispecific antibody, wherein the bispecific antibody comprises a target antigen-binding fragment and a CD3-binding fragment, wherein the kit comprises engineered T cells comprising a reporter gene operatively linked to a response element responsive to T cell activation, wherein the response element responsive to T cell activation comprises a T cell activation response element from any one or more of AP-1, NFκB, FOXO, STAT3, STAT5, and IRF. 56. The kit of claim 55, further comprising an anti-CD3 homodimer assay standard and/or an anti-CD3 homodimer control. 57. The kit of claim 55, wherein the reporter gene is luciferase, fluorescent protein, alkaline phosphatase, β-lactamase, or β-galactosidase. 58. The kit of claim 56, wherein the reporter gene is luciferase, fluorescent protein, alkaline phosphatase, β-lactamase or β-galactosidase. 59. The kit of claim 57, wherein the luciferase is firefly luciferase, kidney luciferase or nanoluciferase. 60. The kit of claim 58, wherein the luciferase is firefly luciferase, kidney luciferase or nanoluciferase. 61. The kit according to any one of claims 55-60, wherein the response element responsive to T cell activation is an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. 62. The kit according to any one of claims 55-60, wherein the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. 63. The kit of claim 61, wherein the T cell population is a population of CD4 ⁺ T cells or CD8 ⁺ T cells. 64. The kit according to any one of claims 55-60, wherein the T cell population is a population of Jurkat T cells or CTLL-2 T cells. 65. The kit of claim 61, wherein the T cell population is a population of Jurkat T cells or CTLL-2 T cells.