Modular bispecific antibodies and methods of using them to screen for bispecific antibody combinations

Abstract

The present disclosure provides modular, bispecific antibodies and methods of using them to rapidly screen antibodies and antibody fragments to identify whether a tested antibody or antibody fragment contains antigen specific sequences for incorporation into therapeutic bispecific antibodies. The modular elements of the bispecific antibodies of the present disclosure can be exchanged for other, similar elements for use according to the present disclosure.

Description

Attorney Docket Number: MS-0051-01-WOModular bispecific antibodies and methods of using them to screen for bispecific antibody combinationsCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Application No. 63 / 740,254, filed December 30, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing.xml file, identified as MS-0051-WO-01, is 37,220 bytes in size and was created on December 16, 2025.TECHNICAL FIELD

[0003] The present disclosure relates generally to compositions and methods for screening bispecific antibody combinations.BACKGROUND

[0004] T-cell redirecting therapies (TRT) are useful tools for cancer therapy that can have a profound impact on hematologic and solid tumors. One type of TRT uses T-cell-engaging bispecific antibodies (BsAbs). The binding regions of endogenous and monoclonal antibodies target the same epitope, but BsAbs are engineered antibodies that can simultaneously bind to two different epitopes of an antigen, or two different antigens. For T-cell-engaging BsAbs, the antibodies simultaneously bind to tumor cell specific antigens and T cell specific antigens. This simultaneous binding process leads to T cell recruitment into the tumor microenvironment, followed by T-cell activation, and ultimately resulting in tumor cell elimination through tumor cell apoptosis via perforin and granzyme release.

[0005] One advantage of T-cell-engaging BsAbs is that T-cell cytotoxicity mediated by them is independent of major histocompatibility complex (MHC) restrictions. As a result. T-cell-engaging BsAbs possess anti-tumor activity against tumor cells that exhibit down-regulated MHC class I expression. In addition, evidence suggests that TBsAb -mediated tumor cell killing requires a lower level of tumor associated antigens, compared to other therapeutic regimens. The approval of multiple TBsAbs in recent years has led to the recognition of theirAttorney Docket Number: MS-0051-01-WOpotential for the treatment of hematologic malignancies and certain solid tumors. Their effectiveness has led to the expectation that they will become standard frontline therapeutic options within 5 years.

[0006] Clinical therapies based on T-cell engaging BsAbs face certain hurdles, however. For example, the use of T cell-redirecting BsAbs in solid tumors has been slower than in hematological malignancies. One possible reason for this is the limited number of tumor specific antigens to prevent antigen specific off-tumor toxicity. In addition, most clinical responses to these types of therapy, are transient. Causes of relapse and resistance include insufficient T cell activation and loss of the targeted antigen (Ag) on the targeted tumor. Some data has described CD20 loss after bsAB treatment in 67-80% of relapsing patients, and outcomes for CD20" patients are dismal. Additional potential complications include the presence of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), infusion-related reactions (IRRs) and opportunistic infections in patients receiving BsAbs. Moreover, translating this technology to clinical therapies faces multiple challenges, including the complexity of antigen selection, immune escape mechanisms, and the high cost of developing new treatments.

[0007] Thus, there is a need to address these deficiencies and provide a means for rapidly screening Abs and Ab binding regions to develop new therapeutic bispecific antibodies.SUMMARY

[0008] The present disclosure addresses these issues by providing a modular system (FlexiBiTE) that enables rapid, iterative assessment of different BsAb combinations for the development of bispecific therapeutic antibodies that modulate on-target and bystander killing of cancer cells and their molecular requirements.

[0009] In an aspect, the present disclosure provides a composition comprising a bispecific antibody comprising a first antigen binding site specific for an immune cell specific epitope and at least a second antigen binding site specific for an IgG specific epitope. In an embodiment, the immune cell specific epitope is for an antigen present on a protein selected from the group consisting of: CD3; CD16; NKp46; NKG2D; NKp30; CD64; SIRPa; CD89; CD40; PD-L1; CD137; 0X40; CD16a; CD32a; MerTK; CLEC5A; CD47; CD38; CD5L; Vy9 TCR; BTN3A1; BTN2A1; NGK2D; NKp30; TNF-a; LRP1; Macl; CLEC9A; CD205; CDllc. In an embodiment, the antigen is present on CD3. In an embodiment the antigen binding site is specific for an IgG selected from the group consisting of: IgG2a, IgG2b, and IgGl. In an embodiment, the bispecific antibody is encoded by the polypeptide sequence selected from theAttorney Docket Number: MS-0051-01-WOgroup consisting of: Seq ID No. 1; Seq ID No. 2; Seq ID No. 4; Seq ID No. 5; Seq ID No. 6; Seq ID No. 7; or Seq ID No. 8. In an embodiment, the bispecific antibody is encoded by Seq ID No 1. In an embodiment, the bispecific antibody is encoded by Seq ID No 2. In an embodiment, the bispecific antibody is encoded by Seq ID No 4. In an embodiment, the bispecific antibody is encoded by Seq ID No 5. In an embodiment, the bispecific antibody is encoded by Seq ID No 6. In an embodiment, the bispecific antibody is encoded by Seq ID No 7. In an embodiment, the bispecific antibody is encoded by Seq ID No 8. In an embodiment, said first epitope or said second epitope is encoded by a sequence from a: mouse, rat, rabbit, hamster, donkey, horse, or human. In an embodiment, the disclosure provides a method of using said bispecific antibody to identify antibodies or antibody fragments that contain antigen binding sites for incorporation into a therapeutic bispecific antibody.

[0010] In an aspect, the present disclosure provides composition comprising a bispecific antibody comprising a first antigen binding site specific for an epitope on a first type of IgG and at least a second antigen binding site specific for an epitope on a different type of IgG. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on lgG2b. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgG2b. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the first antigen binding site is specific for an epitope on IgG2b and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the epitope is encoded by a sequence from a: mouse, rat, rabbit, hamster, donkey, horse, or human. In an embodiment, at least one IgG epitope is modified. In an embodiment, the bispecific antibody is encoded by the polypeptide sequence selected from the group consisting of: Seq ID No 3 or Seq ID No 9. In an embodiment, the bispecific antibody is encoded by Seq ID No 3. In an embodiment, the bispecific antibody is encoded by Seq ID No 9. In an embodiment, the disclosure provides a method of using said bispecific antibody to identify antibodies or antibody fragments that contain antigen binding sites for incorporation into a therapeutic bispecific antibody.

[0011] In an aspect, the present disclosure provides a composition comprising a polynucleotide encoding a bispecific antibody comprising a first antigen binding site specific for an immune cell specific epitope and at least a second antigen binding site specific for an IgG specific epitope. In an embodiment, the immune cell specific epitope is for an antigen present on aAttorney Docket Number: MS-0051-01-WOprotein selected from the group consisting of: CD3; CD16; NKp46; NKG2D; NKp30; CD64; SIRPa; CD89; CD40; PD-L1; CD137; 0X40; CD16a; CD32a; MerTK; CLEC5A; CD47; CD38; CD5L; Vy9 TCR; BTN3A1; BTN2A1; NGK2D; NKp30; TNF-a; LRP1; Macl; CLEC9A; CD205; CDllc. In an embodiment, the antigen is present on CD3. In an embodiment, the epitope is encoded by a sequence from a: mouse, rat, rabbit, hamster, donkey, horse, or human. In an embodiment, at least one IgG epitope is modified.

[0012] In an aspect, the present disclosure provides a composition comprising a polynucleotide encoding a bispecific antibody comprising a first antigen binding site specific for an epitope on a first type of IgG and at least a second antigen binding site specific for an epitope on a different type of IgG. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgG2b. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgG2b. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the first antigen binding site is specific for an epitope on IgG2b and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the epitope is encoded by a sequence from a: mouse, rat, rabbit, hamster, donkey, horse, or human. In an embodiment, at least one IgG epitope is modified.

[0013] In an aspect, the present disclosure provides a composition comprising a bispecific antibody comprising a first antigen binding site specific for an immune cell specific epitope and at least a second antigen binding site specific for a different immune cell specific epitope. In an embodiment, each of said first antigen binding site and said second antigen binding site is present on a protein selected from the group consisting of: CD3; CD16; NKp46; NKG2D; NKp30; CD64; SIRPa; CD89; CD40; PD-L1; CD137; 0X40; CD16a; CD32a; MerTK; CLEC5A; CD47; CD38; CD5L; Vy9 TCR; BTN3A1; BTN2A1; NGK2D; NKp30; TNF-a: LRP1; Macl; CLEC9A; CD205; CDllc.

[0014] In an aspect, the present disclosure provides a polynucleotide encoding a bispecific antibody comprising a first antigen binding site specific for an immune cell specific epitope and at least a second antigen binding site specific for a different immune cell specific epitope. In an embodiment, each of said first antigen binding site and said second antigen binding site is present on a protein selected from the group consisting of: CD3; CD 16; NKp46; NKG2D; NKp30; CD64; SIRPa; CD89; CD40; PD-L1; CD 137; 0X40; CD 16a; CD32a;Attorney Docket Number: MS-0051-01-WOMerTK; CLEC5A; CD47; CD38; CD5L; Vy9 TCR; BTN3A1; BTN2A1; NGK2D; NKp30; TNF-a: LRP1; Macl; CLEC9A: CD205; CDllc.

[0015] In an aspect, the present disclosure provides a method of using a bispecific antibody comprising a first antigen binding site specific for an immune cell specific epitope and at least a second antigen binding site specific for an IgG specific epitope to identify antibodies or antibody fragments that contain antigen binding sites for incorporation into a therapeutic bispecific antibody. In an embodiment, the immune cell specific epitope is for an antigen present on a protein selected from the group consisting of: CD3; CD1; NKp46; NKG2D; NKp30; CD64; SIRPa; CD89; CD40; PD-L1; CD137; 0X40; CD16a; CD32a; MerTK; CLEC5A; CD47; CD38; CD5L; Vy9 TCR; BTN3A1; BTN2A1; NGK2D: NKp30; TNF-a: LRP1; Macl; CLEC9A; CD205; CDllc. In an embodiment, the antigen is present on CD3. In an embodiment the antigen binding site is specific for an IgG selected from the group consisting of: IgG2a, IgG2b, and IgGl. In an embodiment, the bispecific antibody is encoded by the polypeptide sequence selected from the group consisting of: Seq ID No. 1; Seq ID No.2; Seq ID No. 4; Seq ID No. 5; Seq ID No. 6; Seq ID No. 7: or Seq ID No. 8. In an embodiment, the bispecific antibody is encoded by Seq ID No 1. In an embodiment, the bispecific antibody is encoded by Seq ID No 2. In an embodiment, the bispecific antibody is encoded by Seq ID No 4. In an embodiment, the bispecific antibody is encoded by Seq ID No 5. In an embodiment, the bispecific antibody is encoded by Seq ID No 6. In an embodiment, the bispecific antibody is encoded by Seq ID No 7. In an embodiment, the bispecific antibody is encoded by Seq ID No 8. In an embodiment, said first epitope or said second epitope is encoded by a sequence from a: mouse, rat, rabbit, hamster, donkey, horse, or human. In an embodiment, at least one IgG epitope is modified. In an embodiment, method of using said bispecific antibody to identify antibodies or antibody fragments for use in a therapeutic bispecific antibody.

[0016] In an aspect, the present disclosure provides a method of using a bispecific antibody comprising a first antigen binding site specific for an epitope on a first type of IgG and at least a second antigen binding site specific for an epitope on a different type of IgG to identify antibodies or antibody fragments that contain antigen binding sites for incorporation into a therapeutic bispecific antibody. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgG2b. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the secondAttorney Docket Number: MS-0051-01-WOantigen binding site is specific for an epitope on IgG2b. In an embodiment, the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the first antigen binding site is specific for an epitope on IgG2b and the second antigen binding site is specific for an epitope on IgGl. In an embodiment, the epitope is encoded by a sequence from a: mouse, rat, rabbit, hamster, donkey, horse, or human. In an embodiment, at least one IgG epitope is modified. In an embodiment, the bispecific antibody is encoded by the polypeptide sequence selected from the group consisting of: Seq ID No 3 or Seq ID No 9. In an embodiment, the bispecific antibody is encoded by Seq ID No 3. In an embodiment, the bispecific antibody is encoded by Seq ID No 9.

[0017] In an aspect, the present disclosure provides a method for using a composition comprising a bispecific antibody comprising a first antigen binding site specific for an immune cell specific epitope and at least a second antigen binding site specific for a different immune cell specific epitope to identify antibodies or antibody fragments that contain antigen binding sites for incorporation into a therapeutic bispecific antibody. In an embodiment, each of said first antigen binding site and said second antigen binding site is present on a protein selected from the group consisting of: CD3; CD16; NKp46; NKG2D; NKp30; CD64; SIRPa; CD89; CD40; PD-L1; CD137; 0X40; CD16a; CD32a; MerTK; CLEC5A; CD47; CD38; CD5L; Vy9 TCR; BTN3A1; BTN2A1; NGK2D; NKp30; TNF-a; LRP1; Macl; CLEC9A; CD205; CD 11c.BRIEF DESCRIPTION OF THE FIGURES

[0018] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate various embodiments of the disclosure and, together with the description, explain the principles of the disclosure. The figures are not to be construed as limiting the invention, but rather as illustrative of certain embodiments thereof.

[0019] Figure 1 provides an illustration of an embodiment of the FlexiBiTE bispecific antibody and illustrates how the different binding sites present on it can be altered to target different antigens or epitopes (in this instance, one binding site can be designed to be specific for any IgG2a and the other can be designed to be specific for any IgG2b).

[0020] Figure 2 provides embodiments of the present disclosure showing the use of different FlexiBiTE bispecific antibody variants for efficiently screening for different antibody combinations that can then be incorporated into bispecific antibodies.Attorney Docket Number: MS-0051-01-WO

[0021] Figure 3 provides certain embodiments of the present disclosure and depicts 3 variants, designated as: FlexiA; FlexiB; and Double FlexiA / B.

[0022] Figure 4 provides an illustration of some of the different embodiments encompassed by the present disclosure. Figure 4a provides variations on the FlexiBiTe bispecific antibody structure (e.g., FlexiA, FlexiB, and Double FlexiA / B) and different antigens that present in lymphoma cells and that can be used as targets for testing different antibody combinations (e.g., those that detect CD20, PDL1, CD 16 / 32, BAFFR, etc.) for incorporation into a new, therapeutic bispecific antibody.

[0023] Figure 5 provides an illustration of an in vivo assay that tests an embodiment of the FlexiBiTe bispecific antibody (FlexiA) for mediating T-cell killing. T-cell-mediated cytotoxic activity of the Flexi A T-cell engager was evaluated using a flow cytometry-based killing assay.

[0024] Figure 6 provides a Biolayer Interferometry (BLI) Affinity Measurement of binding kinetics of different FlexiBiTE bispecific antibody embodiments. Binding kinetics and affinity of Flexi bispecific antibodies (Flexi-B; FlexiB-Fc23; and FlexiB-albumin) were determined by biolayer interferometry (BLI) using an Octet instrument (Sartorius) with Ni-NTA biosensors.

[0025] Figure 7 provides an illustration of the system used to test whether an embodiment of the FlexiBiTE bispecific antibody is effective at screening antibodies in an in vivo system.

[0026] Figure 8 provides the results of testing whether an embodiment of the FlexiBiTE bispecific antibody is effective at screening antibodies in an in vivo system.DETAILED DESCRIPTION

[0027] Compositions, systems and methods for screening and identifying antibody sequences for inclusion into therapeutic bispecific antibodies are provided.

[0028] The description of the various embodiments is for purposes of illustration only and is not intended to be exhaustive or limiting. Modifications and variations will be apparent to one of ordinary' skill in the art in light of the teachings herein and may be made without departing from scope of the present disclosure.

[0029] For example, the use of a singular term, such as, "a' is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, ’‘top,” “bottom,” “left,” “right,” “upper,” “lower,” "down." “up,” and “side,” are used in the description for clarity' in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims. Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood toAttorney Docket Number: MS-0051-01-WOinclude an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms ‘'about” or ‘'approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%. such as less than or equal to ± 1 %, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0030] The term “about"’ or “approximately,” as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary’ skill in the art. which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” can mean an acceptable error range for the value, such as 10% of the value modified by the term "about.” As used herein, the term “about,” can mean relative to the recited value, e g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0031] Notwithstanding that the disclosed numerical ranges and parameters are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive ol) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0032] The terms “comprising,"’ “including” and “having"’ are used interchangeably in this disclosure. The terms “comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described, and mean that there may be additional elements other than the listed elements. Wherever the terms '‘comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of’ the disclosed elements, in which additional elements other than the listed elements are not included. The terms “or” and “and / or,” as used herein, are toAttorney Docket Number: MS-0051-01-WObe interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A. B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0033] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0034] The nomenclatures used in connection with, and the laboratory procedures and techniques of biochemistry7, immunology7, microbiology7, molecular biology7, and virology described herein are those well-known and commonly used in the art. The phrases and terminology employed herein are for the purpose of description and should not be regarded as limiting.

[0035] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary7skill in the art. Generally, nomenclature used in connection with, and techniques of, pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology7, genetics and protein and nucleic acid chemistry, described herein, are those well- known and commonly used in the art. In case of conflict, the present specification, including definitions, will control.

[0036] Unless defined otherwise herein, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology7(Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.). Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology7(1991), all of which are incorporated by reference herein. As used herein, the following terms have the meanings ascribed to them below7, unless specified otherwise.

[0037] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology7, cell biology, biochemistry7and immunology7, w7hich are w ithin the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al.. 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology:Attorney Docket Number: MS-0051-01-WOA Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.. 1994); Sambrook and Russell. Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology7, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0038] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect.” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein.

[0039] Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be. or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0040] Where aspects or embodiments are described in terms of a Markush group or other grouping of alternatives, the present application encompasses not only the entire group listedAttorney Docket Number: MS-0051-01-WOas a whole, but each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present application also envisages the explicit exclusion of one or more of any of the group members in the Markush group or other grouping of alternatives.

[0041] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the various aspects and embodiments. The materials, methods, and examples are illustrative only and not intended to be limiting.Definitions

[0042] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0043] As used herein, and unless otherwise specified, the term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues See, e.g., Batzer et al., Nucleic Acid Res.19:5081 (1991), the disclosure of which is incorporated in its entirety herein.

[0044] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longerAttorney Docket Number: MS-0051-01-WOchains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0045] The terms “antibody” or “antibodies” as used herein encompasses various antibody structures, including, but not limited to: monoclonal antibodies, polyclonal antibodies, antibody protein structures, multispecific antibodies (e g., bispecific antibodies), and antibody fragments, so long as these structures exhibit the necessary antigen-binding activity.

[0046] The term “BiTE” as used herein describe bispecfic antibodies, where at least one antigen binding domain specifically identifies and binds to a T-cell specific antigen, or an immune cell specific antigen, and at least one antigen binding domain specifically identifies and binds to an antigen present on a tumor cell.

[0047] An “antibody fragment” refers to a molecule, other than an intact antibody, that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, cross-Fab fragments; linear antibodies; singlechain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. scFv antibodies are described in Houston, J. S., Methods in Enzymol.203 (1991) 46-96) for example. In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full-length antibodies.

[0048] As used herein, “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CHI) of a heavy chain. In one embodiment, bispecific antibodies of the present disclosure comprise at least one Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged.

[0049] A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following ordersAttorney Docket Number: MS-0051-01-WOin N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 and d) VL-CH1-linker-VH-CL, are stabilized via the natural disulfide bond between the CL domain and the CHI domain. In addition, these single chain Fab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering). The term “N-terminus denotes the last amino acid of the N-terminus. The term “C-terminus denotes the last amino acid of the C-terminus.

[0050] By '‘fused” or ‘'connected” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0051] The term “linker’ as used herein refers to a peptide linker and is preferably a peptide with an amino acid sequence with a length of at least 5 amino acids, preferably with a length of 5 to 100, more preferably of 10 to 50 amino acids. In one embodiment said peptide linker is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, 4, 5, 6, or 7, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3), preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In one embodiment said peptide linker is (G4S)4. In one embodiment, said peptide linker is encoded by the amino acid sequence GSTSGSGKPGSEGSTKG. In one embodiment, said peptide linker is encoded by EPRGPTIKPCPPCKCP.

[0052] The term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL). also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an immunoglobulin may beAttorney Docket Number: MS-0051-01-WOassigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.

[0053] An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the “antibody that binds” to its antigen in a competition assay by 50% or more.

[0054] The term “antigen binding domain” refers to the part of an antigen binding molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0055] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See e.g. Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U. S. Pat. Nos. 5.202,238 and 5,204.244.

[0056] “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells; down regulation of cell surface receptors (e g. B cell receptor); and B cell activation.

[0057] As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes, forAttorney Docket Number: MS-0051-01-WOexample, modifications of: the amino acid sequence, the glycosylation pattern, the crosslinking, or the side chain group of individual amino acids, as well as combinations of these approaches.

[0058] The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid sequence deletions and insertions include amino- and / or carboxy -termin al deletions and insertions of amino acids. Particular amino acid mutations are amino acid substitutions.

[0059] Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. Methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful.

[0060] The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy' chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0061] “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1. FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0062] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.Attorney Docket Number: MS-0051-01-WO

[0063] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0064] The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the disclosure have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

[0065] A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest. Fifth Edition. NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

[0066] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0067] The term “hypervariable region” or “HVR,” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurallyAttorney Docket Number: MS-0051-01-WOdefined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2. H3), and three in the VL (LI, L2. L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2). 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-LL CDR-L2, CDR-L3, CDR-Hl, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of Hl, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Sendee, National Institutes of Health, Bethesda, Md. (1991).) Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al., U. S. Dept, of Health and Human Senices, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of ammo acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0068] With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-Ll, a-CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 ofL2, 89-96 ofL3, 31-35B of Hl, 50-58 ofH2, and 95-102 ofH3. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008).) Unless otherwise indicated. HVR residues and other residues in the variable domain (e g., FR residues) are numbered herein according to Kabat et al., supra.

[0069] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e g., cows, sheep, cats, dogs, and horses), primates (e.g.. humans andAttorney Docket Number: MS-0051-01-WOnon-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0070] An "isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g.. Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0071] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0072] “Isolated nucleic acid encoding a bispecific antibody that specifically binds” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0073] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which ty pically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0074] A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0075] “Native antibodies” or “natural antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of twoAttorney Docket Number: MS-0051-01-WOidentical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus. each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

[0076] '‘No substantial cross-reactivity” means that a molecule (e.g., an antibody) does not recognize or specifically bind an antigen different from the actual target antigen of the molecule (e.g. an antigen closely related to the target antigen), particularly when compared to that target antigen. For example, an antibody may bind less than about 10% to less than about 5% to an antigen different from the actual target antigen, or may bind said antigen different from the actual target antigen at an amount consisting of less than about 10%, 9%, 8% 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%, 1%, or 0.5%, and most preferably less than about 0.2% or 0.1% antigen different from the actual target antigen.

[0077] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6thed., W. H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al.. Nature 352:624-628 (1991).

[0078] The terms “antigen-binding site” or “antigen binding site of an antibody” when used herein refer to the amino acid residues of an antibody which are responsible for antigenbinding. The antigen-binding portion of an antibody comprises amino acid residues from the “complementary determining regions” or “CDRs”. “Framework” or “FR” regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody's properties. CDR and FR regions are determined according to the standardAttorney Docket Number: MS-0051-01-WOdefinition of 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”.

[0079] Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

[0080] “Bispecific antibodies”or “BsAbs” according to the disclosure are antibodies which have at least two different antigen-binding specificities. Antibodies of the present disclosure are specific for different epitopes in various combinations. BsAbs are broadly categorized into Fc based (IgG-like and IgG-appended) or fragment based on the presence or absence of the Fc region in their structure. Fc-based bsAbs commonly mirror the modular structure of endogenous IgG molecules. Fragment-based bsAbs often represent a simpler structure with a smaller size and at least two variable domains. Generally, bsAbs are synthesized by fusing two different heavy chains, and two different light chains. The two variable binding domains allow simultaneous targeting of two epitopes that are leveraged for therapeutic effects. The term “bispecific” antibody as used herein denotes an antibody that has at least two binding sites each of which bind to different epitopes of the same antigen or at least two binding sites each of which bind to different epitopes from different antigens.

[0081] The term “therapeutic bispecific antibody” as used herein denotes a bispecific antibody that modulates on-target and bystander killing of cancer cells and their molecular requirements and that is being developed for targeted treatment of a disease or disorder, such as cancer, in a subj ect.

[0082] The term “FlexiBiTE” refers to the modular bispecific antibodies of the present disclosure that can be used to rapidly screen for different antibody combinations, which can be used to develop and generate therapeutic bispecific antibodies. Embodiments include interchangeable, modular polypeptide sequences that enable the use of the FlexiBiTE bispecific antibodies in the screening process. The modular polypeptide sequences of the FlexiBiTE antibodies can encode, for example, the different elements in different combinations, such as, for example: a signal sequence, at least one antibody light chain, at least one antibody heavy chain, at least one polypeptide linker, an albumin D3 sequence, and polypeptide tags for isolation and purification (e g., His Tag). The bispecific antibodies of the present disclosure have two or more binding sites and are at least bispecific. That is, the antibodies mayAttorney Docket Number: MS-0051-01-WObe bispecific even in cases where there are more than two binding sites (i.e. that the antibody is trivalent or multivalent).

[0083] The term “valent” as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antibody molecule. The bispecific antibodies according to the disclosure are at least “bivalent” and may be “trivalent” or “multivalent” (e.g.“tetravalent” or “hexavalent”).

[0084] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0085] The term “amino acid” as used within this application denotes the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val. V).

[0086] As used herein, the expressions “cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transfectants” and “transfected cells” include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0087] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art.Attorney Docket Number: MS-0051-01-WO

[0088] As used herein, the term "binding" or “specifically binding’' refers to the binding of the antibody to an epitope of the antigen in an in-vitro assay. The affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody / antigen complex), kD (dissociation constant), and KD (kD / ka).

[0089] The term “epitope” includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.

[0090] The term “immune cell specific antigen” as used herein refers to an antigen present on a polypeptide that is expressed on the surface of an immune cell and that can be used to identify that polypeptide and that immune cell-type. Similarly, the term “immune cell specific epitope” as used herein refers to an epitope present on a polypeptide that is expressed on the surface of an immune cell and that can be used to identify that polypeptide and that immune cell-ty pe.Nucleic Acid Sequences and Vectors:

[0091] In further objects the present disclosure relates to amino acid sequences comprising, for example, a sequence encoding a heavy’ chain of a bispecific antibody of the present disclosure, an amino acid sequence comprising a sequence encoding a light chain of a bispecific antibody of the present disclosure, a sequence encoding a signal sequence, a sequence encoding a hinge section, a sequence encoding a linker, a sequence encoding a tag, and a sequence encoding an albumin polypeptide.

[0092] The disclosure also relates to expression vectors comprising a nucleic acid sequence of the present disclosure and to a prokaryotic or eukaryotic host cell comprising a vector of the present disclosure. In addition, a method of producing an antibody comprising culturing the host cell so that the antibody’ is produced is provided.

[0093] The disclosure further provides isolated polynucleotides or isolated polypeptides encoding a bispecific antibody as described herein or a fragment thereof. The polynucleotides encoding bispecific antibodies of the disclosure may be expressed as a single polynucleotide that encodes the entire bispecific antigen binding molecule or as multiple (e g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are coexpressed may associate through, e g., disulfide bonds or other means to form a functional bispecific antibody. For example, the light chain portion of a Fab fragment may be encoded by a separate polynucleotide from the portion of the bispecific antibody comprisingAttorney Docket Number: MS-0051-01-WOthe heavy chain portion of the Fab fragment, an Fc domain subunit and optionally (part of) another Fab fragment. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the Fab fragment. In another example, the portion of the bispecific antibody provided therein comprising one of the two Fc domain subunits and optionally (part of) one or more Fab fragments could be encoded by a separate polynucleotide from the portion of the bispecific antibody provided therein comprising the other of the two Fc domain subunits and optionally (part of) a Fab fragment. When co-expressed. the Fc domain subunits will associate to form the Fc domain.Polypeptide Sequences and Bispecific Antibody Variants:

[0094] The present disclosure relates to polypeptide sequences that encode a bispecific antibody that facilitates the screening of antibodies and antibody sequences for incorporation into a therapeutic bispecific antibody.

[0095] Embodiments of the present disclosure contain interchangeable, modular elements that are identifiable by a person of skill and that can be altered or swapped for other elements that can perform the same or a similar purpose. In certain embodiments, the modular elements can include polypeptides that encode, for example, the following: a signal sequence; at least one antigen specific light chain; at least one antigen specific heavy chain; a polypeptide linker; an albumin D3 sequence; a polypeptide for isolation and purification (such as a His tag); and other sequences.

[0096] In a specific embodiment, designated as Flexi A, the polypeptide comprises a sequence that is shown in SEQ ID No 1, which encodes the following modular elements: a signal peptide (MGWSCIILFLVATATGVHS); an anti-CD3 heavy chain (EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV TVSS); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-CD3 light chain (DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK); a 218s linker (GSTSGSGKPGSEGSTKG); an anti-rat IgG2a light chain (DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDG VPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQDDEFPRTFGGGTKLEIK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-rat IgG2a heavy chain (EVQLLQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHEKSLEWIGYINPYSGAttorney Docket Number: MS-0051-01-WODTIYNHKFKDKATLTVDKSSNIAYMELRSLTSEDTAVYYCARGGYDYGDHWGQGTT LTVSS); and a His tag (HHHHHH).

[0097] In a specific embodiment, designated as Flexi B, the polypeptide comprises a sequence that is shown in SEQ ID No 2, which encodes the following modular elements: a signal peptide (MGWSCIILFLVATATGVHS); an anti-CD3 light chain (DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-CD3 heavy chain (EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV TVSS); a 218s linker (GSTSGSGKPGSEGSTKG); an anti-rat IgG2b light chain (QIVLSQSPVVLSGSPGEKVTMTCRATSSVSYMHWYQQKPGSSPKPWIYATSNLASGV PARFSGSGSGTSYSLTISRVEAADAATYYCQQWSSNPPTFGAGTTLELK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-rat IgG2b heavy chain (QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGTGLEWLGVIWSGGST EYNAAFISRLTITKDNSKSQVLFTMDSLQPDDTAIYYCARIYGNTYTTTWYFDVWGT GTTVTVSS); and a His tag (HHHHHH).

[0098] In a specific embodiment, designated as Flexi 2a-2b, the polypeptide comprises a sequence that is shown in SEQ ID No 3, which encodes the following modular elements: a signal peptide (MGWSCIILFLVATATGVHS); an anti-rat IgG2a light chain (DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDG VPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQDDEFPRTFGGGTKLEIK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-rat IgG2a heavy chain (EVQLLQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHEKSLEWIGYINPYSG DTIYNHKFKDKATLTVDKSSNIAYMELRSLTSEDTAVYYCARGGYDYGDHWGQGTT LTVSS); a 218s linker (GSTSGSGKPGSEGSTKG); an anti-rat IgG2b light chain (QIVLSQSPVVLSGSPGEKVTMTCRATSSVSYMHWYQQKPGSSPKPWIYATSNLASGV PARFSGSGSGTSYSLTISRVEAADAATYYCQQWSSNPPTFGAGTTLELK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-rat IgG2b heavy chain (QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGTGLEWLGVIWSGGST EYNAAFISRLTITKDNSKSQVLFTMDSLQPDDTAIYYCARIYGNTYTTTWYFDVWGT GTTVTVSS); and a His tag (HHHHHH).

[0099] In a specific embodiment, designated as Flexi 1, the polypeptide comprises a sequence that is shown in SEQ ID No 4, which encodes the following modular elements: a signal peptideAttorney Docket Number: MS-0051-01-WO(MGWSCIILFLVATATGVHS); an anti-CD3 heavy chain (EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV TVSS); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-CD3 light chain (DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK); a 218s linker (GSTSGSGKPGSEGSTKG); an anti-rat IgGl light chain (DIQMTQTTSSLSASLGDRVTINCSASQGIRNYLNWYQQKPDGTVKLLIYYTSSLHSG VPSRFSGGGSGTDYSLTISNLEPEDIATYYCQQYSRLPWTFGGGTKLEIK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-rat IgGl heavy chain (QIQLVQSGPELKKPGETVKVSCKASGYTFTTYGMSWVQQAPGKGFKWIGWIDTYS GVPTYADDFKGRFAFSLETSAGTAYMQINNLKNEDTATYLCARWGYYGSNSYWTFD VWGTGTTVTVSS); and a His tag (HHHHHH).

[0100] In a specific embodiment, designated as FlexiB Fc23, the polypeptide comprises a sequence that is shown in SEQ ID No 5, which encodes the following modular elements: a signal peptide (MGWSCIILFLVATATGVHS); an anti-CD3 light chain (DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-CD3 heavy chain (EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV TVSS); a 218s linker (GSTSGSGKPGSEGSTKG); an anti-rat IgG2b light chain (QIVLSQSPVVLSGSPGEKVTMTCRATSSVSYMHWYQQKPGSSPKPWIYATSNLASGV PARFSGSGSGTSYSLTISRVEAADAATYYCQQWSSNPPTFGAGTTLELK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-rat IgG2b heavy chain (QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGTGLEWLGVIWSGGST EYNAAFISRLTITKDNSKSQVLFTMDSLQPDDTAIYYCARIYGNTYTTTWYFDVWGT GTTVTVSS); a GGGGS linker (GGGGS GGGGSGGGGS); a mouse IgG2b hinge (EPSGPISTINPCPPCKECHKCP); a mouse IgG2b Fc23 fusion (APNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRA PQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGS YFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK); and a GSA-His tag (GSAHHHHHH).Attorney Docket Number: MS-0051-01-WO

[0101] In a specific embodiment, designated as Flexi Albumin, the polypeptide comprises a sequence that is shown in SEQ ID No 6. which encodes the following modular elements: a signal peptide (MGWSCIILFLVATATGVHS); an anti-CD3 light chain (DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-CD3 heavy chain (EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV TVSS); a 218s linker (GSTSGSGKPGSEGSTKG); an anti-rat IgG2b light chain (QIVLSQSPVVLSGSPGEKVTMTCRATSSVSYMHWYQQKPGSSPKPWIYATSNLASGV PARFSGSGSGTSYSLTISRVEAADAATYYCQQWSSNPPTFGAGTTLELK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-rat IgG2b heavy chain (QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGTGLEWLGVIWSGGST EYNAAFISRLTITKDNSKSQVLFTMDSLQPDDTAIYYCARIYGNTYTTTWYFDVWGT GTTVTVSS); a GGGGS linker (GGGGSGGGGSGGGGS); an Albumin D3 sequence (LVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTK CCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVD ETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFA QFLDTCCKAADKDTCFSTEGPNLVT); and a GSA-His tag (GSAHHHHHH).

[0102] In a specific embodiment, designated as FlexiB Fc23 LALA mut. the polypeptide comprises a sequence that is shown in SEQ ID No 7, which encodes the following modular elements: a signal peptide (MGWSCIILFLVATATGVHS); an anti-CD3 light chain (DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-CD3 heavy chain (EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV TVSS); a 218s linker (GSTSGSGKPGSEGSTKG); an anti-rat IgG2b light chain (QIVLSQSPVVLSGSPGEKVTMTCRATSSVSYMHWYQQKPGSSPKPWIYATSNLASGV PARFSGSGSGTSYSLTISRVEAADAATYYCQQWSSNPPTFGAGTTLELK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-rat IgG2b heavy chain (QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGTGLEWLGVIWSGGST EYNAAFISRLTITKDNSKSQVLFTMDSLQPDDTAIYYCARIYGNTYTTTWYFDVWGT GTTVTVSS); a GGGGS linker (GGGGSGGGGSGGGGS); a mouse IgG2a hingeAttorney Docket Number: MS-0051-01-WO(EPRGPTIKPCPPCKCP); a mouse IgG2a-Fc23 LALA mutated (APNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ TQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRA PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSD GSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK); and aGSA-His tag (GSAHHHHHH).

[0103] In a specific embodiment, designated as FlexiB CRossMab LALAmut sequence 1. the polypeptide comprises a sequence that is shown in SEQ ID No 8, which encodes the following modular elements: a signal peptide (MGWSCIILFLVATATGVHS); an anti-CD3 light chain (DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK); a GGGGS linker (GGGGSGGGGSGGGGSGGGGS); an anti-CD3 heavy chain (EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV TVSS); a GGGGS linker (GGGGSGGGGSGGGGS); a mouse IgG2a hinge (EPRGPTIKPCPPCKCP); a mouse IgG2a-Fc23 knob in hole LAEA mutated sequence (APNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ TQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRA PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSD GSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK); and aGSA-His tag (GSAHHHHHH).

[0104] In a specific embodiment, designated as FlexiB CrossMab LALAmut sequence 2, the polypeptide comprises a sequence that is shown in SEQ ID No. 9, which encodes the following modular elements: a signal peptide (MGWSCIILFLVATATGVHS); an anti-rat IgG2b light chain (QIVLSQSPVVLSGSPGEKVTMTCRATSSVSYMHWYQQKPGSSPKPWIYATSNLASGV PARFSGSGSGTSYSLTISRVEAADAATYYCQQWSSNPPTFGAGTTLELK); a GGGGS linker (GGGGSGGGGSGGGGSGGGS); an anti-rat IgG2b heavy chain (QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGTGLEWLGVIWSGGST EYNAAFISRLTITKDNSKSQVLFTMDSLQPDDTAIYYCARIYGNTYTTTWYFDVWGT GTTVTVSS); a GGGGS linker (GGGGSGGGGSGGGGS); a mouse IgG2a hinge (EPRGPTIKPCPPCKCP); a mouse IgG2a-Fc23 knob in hole LALA mutated sequence (APNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ TQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAAttorney Docket Number: MS-0051-01-WOPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSD GSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK); and aGSA-His tag (GSAHHHHH).

[0105] In some embodiments, the bispecific antibody of the present disclosure can include any of: a signal sequence; an anti-IgGl light chain; an anti-IgGl heavy chain; an anti-IgG2a light chain; an anti-IgG2a heavy chain; an anti-IgG2b light chain; an anti-IgG2b heavy chain; a GGGGS polypeptide linker; a polypeptide linker sequence; an IgG hinge fragment; an anti-CD3 light chain; an anti-CD3 heavy chain; an anti-immune cell specific antigen light chain; an anti-immune cell specific antigen heavy chain; an albumin sequence; a His tag.

[0106] In some embodiments, antibody chains that can be used according to the present disclosure can be chosen from any of: Seq ID No. 11; Seq ID No. 12; Seq ID No. 15; Seq ID No. 16; Seq ID No. 18; Seq ID No. 19; Seq ID No. 20; Seq ID No. 21; Seq ID No. 23; and Seq ID No. 7.

[0107] In some embodiments of the present disclosure, the bispecific antibody includes at least one antibody sequence that is specific for an antigen on an immune cell surface. These immune cell specific antigens can be specifically bound or identified by any of the following polypeptide sequence combinations: an anti-CD3 light chain and an anti-CD3 heavy chain; an anti-CD16 light chain and an anti-CD16 heavy chain; an anti-NKp46 light chain and an anti-NKp46 heavy chain; an anti-NKG2D light chain and an anti-NKG2D heavy chain; an anti NKp30 light chain and an anti NKp30 heavy chain; an anti-CD64 light chain and an anti-CD64 heavy chain; an anti-SIRPa light chain and an anti-SIRPa heavy chain; an anti-CD89 light chain and an anti-CD89 heavy chain; an anti-CD40 light chain and an anti-CD40 heavy chain; an anti-PDLl light chain and an anti-PDLl heavy chain; an anti-CD137 light chain and an antiCD 137 heavy chain; an anti-OX40 light chain and an anti-OX40 heavy chain; an anti-CD16a light chain and an anti-CD 16a heavy chain; an anti -CD32a light chain and an anti-CD32a heavy chain; an anti-MerTK light chain and an anti -MerTK heavy chain; an anti-CLEC5Alight chain and an anti-CLEC5Aheavy chain; an anti-CD47 light chain and an anti-CD47 heavy chain; an anti-CD38 light chain and an anti-CD38 heavy chain; an anti-CD5L light chain and an anti-CD5L heavy chain; an anti-Vy9 TCR light chain and an anti- anti-V’ / 9 TCR heavy chain: an anti- BTN3A1 light chain and an anti-BTN3Al heavy chain; an anti- BTN2A1 light chain and an anti-BTN2Al heavy chain; an anti-NGK2D light chain and an anti- NGK2D heavy chain; an anti-NKp30 light chain and an anti-NKp30 heavy chain; an anti-TNF-a light chain and an anti- TNF-a heavy chain; an anti-LRPl light chain and an anti-LRPl heavy chain; an anti-Macl light chain and an anti-Macl heavy chain; an anti-CLEC9A light chain and an antiAttomey Docket Number: MS-0051-01-WOCLEC9A heavy chain; an anti-CD205 light chain and an anti-CD205 heavy chain; an anti-CDllc light chain and an anti-CDllc heavy chain; an anti-CDllc light chain and an anti-CDllc heavy chain; an anti-Fc receptor type light chain and an anti-Fc receptor type heavy chain; an anti-CD20 light chain and an anti-CD20 heavy chain; anti-CDllb heavy chain and an anti-CDllb heavy chain; anti -BAFFR heavy chain and an anti-BFFR light chain; anti-FolR2 heavy chain and an anti-FolR2 light chain; an anti-EpCAM heavy chain and an anti-EpCAM light chain; an anti-CD138 heavy chain and an anti-CD138 light chain.

[0108] In certain embodiments, amino acid sequence variants of the bispecific antibodies provided herein are contemplated in addition to those described above. For example, it may be desirable to improve the binding affinity and / or other biological properties of the bispecific antibody. Amino acid sequence variants of a bispecific antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the bispecific antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arnve at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

[0109] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0110] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variants) selected for further study will have modifications (e.g, improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display - based affinity' maturation techniques such as those known in the art

[0111] For example, alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity'. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a-Attorney Docket Number: MS-0051-01-WOCDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology / 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N. J., (2001).) In some embodiments of affinity maturation, diversity' is introduced into the variable genes chosen for maturation by any of a variety' of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted.

[0112] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs Such alterations may be outside of HVR ‘“hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three ammo acid substitutions.

[0113] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.Recombinant methods

[0114] Bispecific antibodies of the disclosure may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the bispecific antibodies (or fragments), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment, a vector comprising one or more of the polynucleotides of the disclosure is provided. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence ofAttorney Docket Number: MS-0051-01-WOa bispecific antibody (fragment) along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N. Y ( 1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the bispecific antibody (fragment) (i.e. the coding region) is cloned in operable association with a promoter and / or other transcription or translation control elements.

[0115] As used herein, a “coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5′ and 3′ untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present disclosure may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the disclosure may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the bispecific antibody (fragment) of the disclosure, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0116] An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, aAttorney Docket Number: MS-0051-01-WOpromoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.

[0117] The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus).

[0118] Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit a-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and / or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

[0119] Polynucleotide and nucleic acid coding regions of the present disclosure may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present disclosure. For example, if secretion of the bispecific antibody is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding a bispecific antibody of the disclosure or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which isAttorney Docket Number: MS-0051-01-WOcleaved from the translated polypeptide to produce a secreted or ‘‘mature” form of the polypeptide.

[0120] In certain embodiments, the native signal peptide, e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse β-glucuronidase DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the bispecific antibody may be included within or at the ends of the bispecific antibody (fragment) encoding polynucleotide. In a further embodiment, a host cell comprising one or more polynucleotides of the disclosure is provided. In certain embodiments a host cell comprising one or more vectors of the disclosure is provided The polynucleoti des and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such embodiment a host cell comprises (e. g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) a bispecific antibody of the disclosure

[0121] As used herein, the term “host cell” refers to any kind of cellular system which can be engineered to generate the bispecific antibodies of the disclosure or fragments thereof. Host cells suitable for replicating and for supporting expression of bispecific antibodies are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the bispecific antibody for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates).Attorney Docket Number: MS-0051-01-WOExamples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. U. S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3 A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N. Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr⁻ CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N. J.). pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain such as an antibody, may be engineered so as to also express the other of the antibody chains such that the expressed product is an antibody that has both a heavy and a light chain.

[0122] In one embodiment, a method of producing a bispecific antibody according to the disclosure is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the bispecific antibody, as provided herein, under conditions suitable for expression of the bispecific antibody, and recovering the bispecific antibody from the host cell (or host cell culture medium).Attorney Docket Number: MS-0051-01-WO

[0123] In one embodiment, the components of the bispecific antibody are genetically fused to each other. Bispecific antibodies can be designed such that its components are fused directly to each other or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of bispecific antibodies are found in the sequences provided herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence.

[0124] In certain embodiments, the Fab fragments forming part of the bispecific antibody comprise at least an antibody variable region capable of binding an antigenic determinant. Variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods to produce polyclonal antibodies and monoclonal antibodies are well known in the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U. S. Pat. No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U. S. Pat. No. 5,969,108 to McCafferty).

[0125] Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used in the bispecific antibodies of the disclosure. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the present disclosure can be of murine, primate, or human origin. A humanized or fully human form of the antibody can also be prepared in accordance with methods w ell known in the art (see e. g. U. S. Pat. No. 5,565,332 to Winter).

[0126] Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e g., donor antibody) CDRs onto human (e.g. recipient, antibody) framework and constant regions w ith or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs: the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but “cloaking” them with a humanlike section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e g, in Almagro and Fransson. Front Biosci 13, 1619-1633 (2008), and are further described, e.g., in Riechmann et al,, Nature 332, 323-329 (1988); Queen et al.. ProcAttorney Docket Number: MS-0051-01-WONatl Acad Sci USA 86, 10029-10033 (1989); U S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321. 522-525 (1986); Morrison et al.. Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing ’‘resurfacing’'); Dall'Acqua et al.. Methods 36, 43-60 (2005) (describing ”FR shuffling”); and Osbourn et al., Methods 36. 61-68 (2005) and Klimka et al., Br J Cancer 83. 252-260 (2000) (describing the "‘guided selection” approach to FR shuffling).

[0127] Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008) Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see e.g. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions may also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variable regions may also be generated by isolating Fv clone variable region sequences selected from human- derived phage display libraries (see e.g.. Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed.. Human Press, Totowa, N. J., 2001); and McCafferty et al.. Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.

[0128] In certain embodiments, the Fab fragments useful in the present disclosure are engineered to have enhanced binding affinity. The ability of the bispecific antibody of the disclosure to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance technique (analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays may be used to identify an antibody, antibody fragment, antigen binding domain or variable domain that competes with a reference antibody for binding to a particular antigen. In certain embodiments, such a competing antibody binds to the same epitope (e.g. a linear or a conformational epitope) that is bound by the reference antibody. Detailed exemplary methods for mapping an epitope toAttorney Docket Number: MS-0051-01-WOwhich an antibody binds are provided in Morris (1996) ‘‘Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa. N. J.).

[0129] In an exemplary competition assay, immobilized antigen is incubated in a solution comprising a first labeled antibody that binds to the antigen and a second unlabeled antibody that is being tested for its ability' to compete with the first antibody for binding to the antigen. The second antibody may be present in a hybridoma supernatant. As a control, immobilized antigen is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with immobilized antigen is measured. If the amount of label associated with immobilized antigen is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory', Cold Spring Harbor, N. Y.)

[0130] Bispecific antibodies prepared as described herein may be purified by art-known techniques such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography; size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the bispecific antibody binds. For example, for affinity chromatography purification of bispecific antibodies of the disclosure, a matrix with protein A or protein G may be used. Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate a bispecific antibody essentially as described in the Examples. The purity of the bispecific antibody can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.

[0131] For the avoidance of doubt, it is intended herein that particular features (for example integers, characteristics, values, uses, diseases, formulae, compounds or groups) described in conjunction with a particular aspect, embodiment or example of the disclosure are to be understood as applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Thus, such features may be used where appropriate in conjunction with any of the definition, claims or embodiments defined herein. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, exceptAttorney Docket Number: MS-0051-01-WOcombinations where at least some of the features and / or steps are mutually exclusive. The disclosure is not restricted to any details of any disclosed embodiments. The disclosure extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.EXAMPLESExample 1: Modular bispecific antibody compositions.

[0132] Modular FlexiBiTE bispecific antibodies are disclosed herein that are useful for screening combinations of different monoclonal antibodies and combinations of monoclonal antibody and T-cell antigens for identifying different forms of therapeutic bispecific antibodies and the CDR regions that they contain. This is depicted in Figure 1, which shows binding of the FlexiBiTE bispecific antibody to the Fc regions of 2 different IgGs (an IgG2a and an IgG2b). The inset shows that even different types of light chain variants (K or ) can be utilized to achieve the desired antibody specificity depicted in this image.

[0133] Figure 2 provides embodiments of the present disclosure showing the use of different FlexiBiTE bispecific antibody variants for efficiently screening for different antibody combinations that can then be incorporated into therapeutic, bispecific antibodies. Figure 2a depicts a FlexiBiTE bispecific antibody with a first Flexi binding site that is specific for the Fc region of a rat IgG2b while a second Flexi binding site is specific for the Fc region of a rat IgG2a. This FlexiBiTE bispecific antibody can be used as depicted in this image to bind a rat IgG2b (which rat IgG specifically binds to Target 1) and a rat IgG2a (which rat IgG specifically binds to target 2). This combination can then be used to screen for the binding of those bound IgG antibodies to their respective specific targets in a therapeutic construct. Figure 2b depicts how the same FlexiBiTE bispecific antibody from Figure 2a can also be used to test a different combination of rat IgG2b and IgG2a antibodies, which are each specific for different targets. In this instance, the FlexiBiTE bispecific antibody construct from 2a is used to bind the Fc region of a rat IgG2b (which in this instance specifically binds to Target 3) and a the Fc region of a rat IgG2a (which in this instance specifically binds to target 2). In this manner, the FlexiBiTE bispecific antibody construct can be used to quickly and efficiently screen different antibody combinations to identify which combinations are potentially useful for incorporation and development into a therapeutic bispecific antibody.Expression in host cells:Attorney Docket Number: MS-0051-01-WO

[0134] In a specific embodiment, FlexiBiTE bispecific antibody expression constructs were cloned into the mammalian expression vector pTwist CMV-BG-WPRE-Neo (Twist Bioscience), incorporating a CMV promoter for high-level expression, the BG polyadenylation signal, WPRE to enhance transcript stability, and a neomycin resistance marker were included for selection. Each construct further comprised an N-terminal signal peptide to direct secretion of the recombinant protein into the extracellular space. Each construct encoded modular antibody-binding domains connected by defined peptide linkers. Within each targeting arm, heavy-chain and light-chain variable domains were genetically fused using flexible (GGGGS)n linkers to promote appropriate intramolecular folding and binding domain assembly. To generate the final bispecific Flexi structure, two distinct antigen-recognition arms were linked together using a structured 218 peptide linker, providing spatial separation and flexibility required for simultaneous target engagement. For constructs utilizing the Fc23 format, the assembly interface comprised mouse IgG2b Fc (hinge-CH2-CH3) domains, genetically fused via a (GGGGS)n linker to form a single-chain Fc heterodimerization module enabling stable intracellular pairing. All constructs were engineered to include a C-terminal 6xHis tag allowing affinity purification of secreted recombinant proteins via Ni-NTA-based immobilized metal affinity chromatography (IMAC). Recombinant Flexi antibodies were produced using mammalian expression systems selected from suspension-adapted human or hamster-derived host cell lines.

[0135] Transient expression of the constructs was achieved using Expi293F™ cells (human embryonic kidney-derived suspension line) and Turbo CHO™ cells (Chinese hamster ovary-derived production platform) following plasmid transfection. All experimental material was generated using episomal plasmid-based transient expression, including lipid-mediated delivery methods such as ExpiFectamine™ transfection reagents or functional equivalents thereof. Flexi expression using Turbo CHO™ cells was conducted by a commercial manufacturing service. Any cellular modifications implemented as part of the commercial production process, including gene amplification or selection systems routinely used in CHO manufacturing, were performed under vendor-controlled manufacturing methods and did not involve alterations to the antibody constructs described herein.

[0136] Expi293F cells were transiently transfected with plasmid vectors encoding the Flexi antibody constructs using lipid-mediated transfection reagents following standard suspension transfection protocols. Transfection of the Turbo CHO cells was conducted by the external service provider using established CHO production workflows.Attorney Docket Number: MS-0051-01-WO

[0137] Cells were cultured for several days post-transfection / expression prior to harvest. Culture supernatants were collected by centrifugation and filtration prior to downstream purification. Cell cultures were maintained under serum-free, suspension conditions. Expi293F™ cells were maintained in Expi293™ Expression Medium (ThermoFisher Scientific). Turbo CHO™ cells were maintained in proprietary CHO production media. For Expi293F cultures, cells were maintained in suspension growth format at 37°C under 8% CO2 with agitation at 110-125 rpm. At the time of transient transfection, cells were seeded at a density of approximately 2-3 × 106cells / mL. For protein produced using Turbo CHO cells, culture conditions, feeding strategies, and bioreactor parameters were controlled by the commercial manufacturing provider according to platform-standard processes.Post-Production Modification and Purification:

[0138] In a specific embodiment, proteins expressed using mammalian cell hosts underwent native post-translational modifications, including: signal peptide cleavage; disulfide bond formation; Fc region glycosylation. Intracellular folding and multimer assembly were driven by genetically encoded pairing interfaces, and no post-purification assembly reactions were employed.

[0139] Cell culture supernatants were harvested by centrifugation and filtered through a 0.22 μm membrane to remove cellular debris prior to purification. Clarified supernatants were incubated directly with Ni-NTA agarose beads in Expi293 culture medium under gentle agitation to allow binding of the His-tagged Flexi constructs. Following batch incubation, the bead-supernatant mixture was applied to a chromatography column pre-equilibrated with binding buffer consisting of 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole, 5% (v / v) glycerol, 1 mM β-mercaptoethanol (β-ME). The column was washed with the same binding buffer to remove unbound and nonspecifically bound proteins. Bound proteins were eluted using elution buffer composed of 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5% (v / v) glycerol, 1 mM β-mercaptoethanol supplemented with a step elution of 50-250 mM imidazole. Eluted protein fractions were collected, concentrated, and subjected to centrifugal ultrafiltration to remove residual imidazole, followed by dialysis against HBSS for buffer exchange prior to downstream analyses.Testing Specific Embodiments for Antibody Binding And Screening for Therapeutic Candidates:Attorney Docket Number: MS-0051-01-WO

[0140] The bispecific antibody constructs of the present disclosure contain modular elements that can be interchanged to rapidly test and screen different combinations of epitope binding sites to identify different combinations that can be developed into therapeutic bispecific antibodies. As noted above, the CDR regions of antibodies mediate specific binding by antibodies. The below embodiments below provide an illustration of some embodiments of how the system can be arranged for effective screening. A person of skill would understand that different components are interchangeable and can be utilized in different configurations to achieve the methods of the present disclosure.

[0141] In some embodiments, the bispecific antibody of the present disclosure is encoded by any of the following amino acid sequences: Seq ID No. 1; Seq ID No. 2; Seq ID No. 3; Seq ID No. 4; Seq ID No. 5; Seq ID No. 6; Seq ID No. 7; Seq ID No. 8; or Seq ID No. 9.FlexiBiTE bispecific antibody constructs show specific binding by off-the-shelf antibodies to their respective targets:

[0142] Three different embodiments of the bispecific antibody of the present disclosure were tested for their ability to screen for their ability to enable specific binding of off-the-shelf antibodies to their respective targets when each off-the-shelf antibody is bound to the FlexiBiTE bispecific antibody construct. This is depicted in Figure 3. The different FlexiBiTE bispecific antibody embodiments are designated as: FlexiA; FlexiB; and Double FlexiA / B. The different variations on the FlexiBiTe bispecific antibody contain the following respective elements: (a) FlexiA includes: a migG signal peptide; an anti-CD3 heavy chain; a GGGGS linker sequence; and anti-CD3 light chain (K type); a 218s linker sequence; an anti-Rat IgG2a light chain (K type); a GGGGS linker sequence; an anti-Rat IgG2a heavy chain; and a His tag; (b) FlexiB includes: a migG signal peptide; anti-CD3 light chain (K type); a GGGGS linker sequence; an anti-CD3 heavy chain; a 218s linker sequence; an anti-Rat IgG2b light chain (K type); a GGGGS linker sequence; an anti-Rat IgG2b heavy chain; and a His tag; and (c) Double Flexi A / B includes: a migG signal peptide; anti-Rat IgG2b light chain (K type); a GGGGS linker sequence; an anti-Rat IgG2b heavy chain; a 218s linker sequence; an anti-Rat IgG2a light chain (K type); a GGGGS linker sequence; an anti-Rat IgG2a heavy chain; and a His tag.

[0143] Each of the respective constructs was tested for effective binding of: (A) the FlexiA construct to a Rat IgG2a on one binding site and to a CD3 antigen on a second binding site; (B) the FlexiB construct to a Rat IgG2b on one binding site and to a CD3 antigen on a second binding site; and (C) the FlexiB construct to a Rat IgG2a on one binding site and to a Rat IgG2bAttorney Docket Number: MS-0051-01-WOon a second binding site. Effective binding of the Flexi bsAbs to their respective targets was assessed by flow cytometry using A20-GFP cells and CD3-expressing EL4 cells as target cells corresponding to the respective binding arms of the constructs. For evaluation of Flexi A and Flexi B constructs, A20-GFP cells were incubated with either rat anti-mouse B220 antibody (RA3-6B2, IgG2a isotype) or rat anti-mouse I-A / I-E antibody (M5 / 114.15.2, IgG2b isotype) in staining buffer at 4 °C for 20 minutes to load the cell surface with rat IgG isotype targets. Unbound antibodies were removed by washing. In parallel, CD3-expressing EL4 cells were used directly as CD3 target cells without antibody coating. For evaluation of Double Flexi constructs, A20-GFP cells were incubated with rat anti-mouse B220 antibody (RA3-6B2, IgG2a isotype) to load the rat IgG2a target, while EL4 cells were incubated with rat anti -mouse CD3 antibody (17A2, IgG2b isotype) to load the rat IgG2b target. Unbound antibodies were removed by washing prior to downstream binding assays. Anti body -loaded A20-GFP cells and EL4 cells were then mixed at an appropriate ratio, and Flexi bsAb was added to the mixed cell suspension. The cell mixture was incubated for 30 minutes at 4 °C to allow binding. Following incubation, cells were washed to remove unbound Flexi bsAb. To detect bound Flexi bsAb, cells were stained with a fluorescently labeled anti-His-tag detection antibody recognizing the C-terminal His tag on the Flexi constructs, washed, and resuspended in flow cytometry buffer. Samples were acquired on a flow cytometer, and Flexi binding was evaluated based on His-tag staining on GFP+A20 target cells and / or CD3+EL4 target cells.

[0144] The images at the bottom show the testing of each of the respective constructs for (A) binding of the Flexi A construct to a Rat IgG2a on one binding site and to a CD3 antigen on a second binding site; (B) binding of the FlexiB construct to a Rat IgG2b on one binding site and to a CD3 antigen on a second binding site; and (C) binding of the FlexiB construct to a Rat IgG2a on one binding site and to a Rat IgG2b on a second binding site. In representative dot plots, black dots indicate “on-target” samples (target-loaded A20-GFP and EL4 cells incubated with Flexi bsAb), whereas gray dots represent “no-target” or negative control samples lacking appropriate target presentation and Flexi bsAb, confirming that binding was dependent on the presence of the cognate target antigens.In vivo assays reveal picomolar level binding by FlexiBiTe bispecific antibody constructs to their targets and the killing of lymphomas.

[0145] FlexiBiTe bispecific antibody killing of T-cells was assessed using an in vivo assay that tests an embodiment of the FlexiBiTe bispecific antibody (FlexiA) for mediating T-cell killing. This is depicted in Figure 5. T-cell-mediated cytotoxic activity of the Flexi A T-cell engager was evaluated using a flow cytometry-based killing assay. Bell GFP-expressing cells were usedAttorney Docket Number: MS-0051-01-WOas target cells. To generate a functional anti-mouse CD20 T-cell engager complex, rat antimouse CD20 antibody (IgG2a isotype) was pre-incubated with Flexi A for 15 minutes at room temperature, followed by an additional 15 minutes at 4 °C, allowing formation of the bispecific T-cell-engaging complex through the rat IgG-binding arm of the Flexi platform.

[0146] Following complex formation, the pre-assembled engager was added to co-cultures of target cells and effector T cells. Mouse naive T cells were used as effector cells, and target cells and T cells were co-cultured at a target-to-effector (T: E) ratio of 1:20 in the presence of the pre-formed T-cell engager. Cultures were incubated for 3 days under standard cell culture conditions to allow sustained T-cell engagement and cytotoxic activity. Test reagent concentrations of 10 nM, 3.3 nM, and 1.1 nM were evaluated. Control conditions included antimouse CD20 antibody alone and Flexi A alone to assess baseline activity. At the end of the coculture period, cells were harvested and analyzed by flow cytometry. Cytotoxic activity was determined by quantify ing the number of remaining live GFP-positive target cells, while T-cell expansion was assessed by measuring total T-cell numbers in each condition. Dose-dependent target cell killing and corresponding T-cell expansion were observed across the tested concentration range, confirming functional activity of the Flexi-based T-cell engager format. This is illustrated in the bottom three panels of the image, which show T-cell killing at 1nM and 10 nM concentrations.FlexiBiTE bispecific antibodies demonstrate specific antigen binding and thermostability:

[0147] Binding kinetics and affinity of select embodiments of the present disclosure were tested were tested using biolayer interferometry. This is depicted in figure 6. Binding kinetics and affinity of Flexi bispecific antibodies (Flexi-B; FlexiB-Fc23; and FlexiB-albumin) were determined by biolayer interferometry (BLI) using an Octet instrument (Sartorius) with Ni-NTA biosensors. Biosensor tips were hydrated in Hank’s Balanced Salt Solution (HBSS) for a minimum of 10 minutes prior to use. Flexi bsAbs containing a C-terminal 6>< His tag were loaded onto the Ni-NTA biosensors at a concentration of 10 pg / mL in HBSS until a stable loading response was achieved. Following antibody immobilization, sensors were transferred to wells containing HBSS alone to establish a baseline. Association measurements were performed by dipping the antibody-loaded biosensors into wells containing serially diluted solutions of the target molecule, prepared in HBSS, covering a concentration range of 2.6 pM to 0.03 pM. The association phase was recorded for 300 seconds for each analyte concentration. Subsequently, dissociation was monitored for 900 seconds by transferring the sensors back into wells containing HBSS only. Reference subtraction was performed using control biosensorsAttorney Docket Number: MS-0051-01-WOto correct for nonspecific binding and baseline drift. Processed sensorgrams were globally analyzed using the Octet data analysis software. Binding kinetics were fit to an appropriate interaction model, including a 1:1 Langmuir binding model or heterogeneous ligand binding models where appropriate, to determine the association rate constant (k_on), dissociation rate constant (k_off), and equilibrium dissociation constant (K_D).

[0148] Thermostability of the different constructs was also measured. Tycho NT.6 instrument (NanoTemper Technologies) was used to measure the melting temperature and thus thermal stability of the Flexi bispecific antibodies (Flexi bsAbs). In short, the capillaries were filled with 10ul of 5uM purified bsAbs in PBS and the intrinsic fluorescence ratio (350 nm / 330 nm) was obtained over a temperature gradient ranging from 25°C to 90°C. The first derivative curves were normalized, and the inflection curves (Ti) were calculated using the equipment software.FlexiBiTE bispecific antibodies cure lymphoma in vivo'.

[0149] Specific embodiments of the FlexiBiTE bispecific antibody of the present disclosure were tested for their ability to cure cancer in an animal model of lymphoma and thereby demonstrate the effectiveness of these bispecific antibodies in testing different antibodies or antigens for therapeutic efficacy. Experimental design is depicted in Figure 7, and the results are provided in Figure 8. Inoculation: EZB GFP+ Luciferase+ lymphoma cells (8e5 cells / mouse) were administered intravenously on Day 0 into female C57BL / 6J mice. Tumor engraftment and expansion were allowed to progress from Day 1-Day 4 post tumor inoculation. Baseline tumor burden was assessed on Day 4 post tumor inoculation, using whole body bioluminescent imaging on the IVIS Spectrum. Treatment and Controls: Treatment began Day 5 post tumor inoculation (day 0 of treatment). Mice were randomized into experimental groups, receiving the following treatments: (a) untreated — negative control; (b) anti-CD20 antibodies (in the absence of the FlexiBiTE bispecific antibody) — negative control; (c) anti-CD20 + anti-HemAgglutinin (anti-HA) antibodies (in the absence of the FlexiBiTE bispecific antibody) — negative control; (d) anti-CD20 bound to the FlexiB construct (SEQ ID No. 2); and (e) anti-CD20 bound to the FlexiB Albumin construct (SEQ ID No. 6).

[0150] FlexiBiTE constructs and antibody treatments were administered at 10ug / mouse. The below table illustrates the testing regimen.Total (n=32) Untreated lOug DoseAttorney Docket Number: MS-0051-01-WO(a) Untreated 5 mice\ (b) a-CD20 6 mice: (c) a-CD20+anti-HA 6 mice| (d) a-CD2O FlexiB 7 mice\ (e) a-CD20+FB_ALB 8 mice

[0151] IVIS Monitoring: Longitudinal tumor monitoring via bioluminescent imaging and maintained at regular intervals as indicated in accompanying IVIS imaging. Mice were assessed for tumor burden (IVIS signal) and survival (death) at these same intervals. The below table provides the survival of the different groups over time: this information is also provided in Figure 8.Treatment Day 3-6 Day 9 Day 13 Day 16 Day 22 Day 29 Day 38 Groupuntreated all alive all alive 1 / 5 4 / 5 all dead all dead all dead dead deada-CD20 all alive all alive 3 / 6 4 / 6 5 / 6 5 / 6 5 / 6dead dead dead dead dead a-CD20+ all alive 2 / 6 3 / 6 4 / 6 4 / 6 4 / 6 4 / 6 anti-HA dead dead dead dead dead dead a-CD20+ all alive all alive all alive 1 / 7 1 / 7 1 / 7 1 / 7 FlexiB dead dead dead dead a-CD20+ all alive all alive all alive all alive all alive all alive all alive FB_ALB

Claims

Attorney Docket Number: MS-0051-Ol-WOCLAIMSWhat is claimed is:

1. A composition comprising a bispecific antibody comprising a first antigen binding site specific for an immune cell specific epitope and at least a second antigen binding site specific for an IgG specific epitope.

2. The composition of claim 1, wherein the immune cell specific epitope is for an antigen present on a protein selected from the group consisting of: CD3; CD 16: NKp46; NKG2D; NKp30: CD64; SIRPa; CD89; CD40; PD-L1; CD 137: 0X40: CD16a: CD32a; MerTK: CLEC5A; CD47; CD38; CD5L; Vy9 TCR; BTN3A1; BTN2A1; NGK2D; NKp30; TNF- a; LRP1; Macl; CLEC9A; CD205; CDllc.

3. The composition of claim 2, wherein the antigen is present on CD3.

4. A composition comprising a bispecific antibody comprising a first antigen binding site specific for an epitope on a first type of IgG and at least a second antigen binding site specific for an epitope on a different type of IgG.

5. The composition of any of claims 1-3, wherein the antigen binding site is specific for an IgG selected from the group consisting of: lgG2a, IgG2b, and IgGl.

6. The composition of claim 4, wherein the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgG2b.

7. The composition of claim 4, wherein the first antigen binding site is specific for an epitope on IgG2a and the second antigen binding site is specific for an epitope on IgGl.

8. The composition of claim 4, wherein the first antigen binding site is specific for an epitope on IgG2b and the second antigen binding site is specific for an epitope on IgGl.

9. The composition of claim 4, wherein the epitope is encoded by a sequence from a: mouse, rat, rabbit, hamster, donkey, horse, or human.

10. The composition of any of claims 1-3, wherein at least one IgG epitope is modified.

11. The composition of claim 3, wherein the bispecific antibody is encoded by the polypeptide sequence selected from the group consisting of: Seq ID No. 1; Seq ID No. 2; Seq ID No. 4; Seq ID No. 5; Seq ID No. 6; Seq ID No. 7; or Seq ID No. 8.

12. The composition of claim 3 wherein the bispecific antibody is encoded by Seq ID No 1.

13. The composition of claim 3, wherein the bispecific antibody is encoded by Seq ID No 2.

14. The composition of claim 3, wherein the bispecific antibody is encoded by Seq ID No 4.

15. The composition of claim 3, wherein the bispecific antibody is encoded by Seq ID No 5.

16. The composition of claim 3, wherein the bispecific antibody is encoded by Seq ID No 6.

17. The composition of claim 3, wherein the bispecific antibody is encoded by Seq ID No 7.Attorney Docket Number: MS-0051-Ol-WO18. The composition of claim 3, wherein the bispecific antibody is encoded by Seq ID No 8.

19. The composition of claim 4, wherein the bispecific antibody is encoded by the polypeptide sequence selected from the group consisting of: Seq ID No 3 or Seq ID No 9.

20. The composition of claim 4, wherein the bispecific antibody is encoded by Seq ID No 3.

21. The composition of claim 4, wherein the bispecific antibody is encoded by Seq ID No 9.

22. A composition comprising the polynucleotide sequence encoding the bispecific antibody in any of claims 1-21.

23. A method of using the bispecific antibody of any of claims 1 -22 to screen antibodies or antibody fragments to identify antibodies or antibody fragments that contain antigen binding sites for incorporation into a therapeutic bispecific antibody.

24. The method of claim 23, using the bispecific antibody encoded by the polypeptide sequence selected from the group consisting of: Seq ID No. 1; Seq ID No. 2; Seq ID No.4; Seq ID No. 5; Seq ID No. 6; Seq ID No. 7; or Seq ID No. 8.

25. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 1.

26. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 2.

27. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 4.

28. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 5.

29. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 6.

30. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 7.

31. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 8.

32. The method of claim 23, using the bispecific antibody encoded by the polypeptide sequence selected from the group consisting of: Seq ID No 3 or Seq ID No 9.

33. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 3.

34. The method of claim 23, using the bispecific antibody encoded by Seq ID No. 9.

35. A composition comprising a bispecific antibody comprising a first antigen binding site specific for an immune cell specific epitope and at least a second antigen binding site specific for a different immune cell specific epitope.

36. The composition of claim 35, wherein each of said first antigen binding site and said second antigen binding site is present on a protein selected from the group consisting of: CD3; CD16; NKp46; NKG2D; NKp30; CD64; SIRPa; CD89; CD40; PD-L1: CD137; 0X40; CD16a; CD32a; MerTK; CLEC5A; CD47; CD38; CD5L; Vy9 TCR; BTN3A1; BTN2A1; NGK2D; NKp30; TNF-a; LRP1; Macl; CLEC9A; CD205; CDllc.Attorney Docket Number: MS-0051-Ol-WO37. A method of using the bispecific antibody of claim 35 to screen antibodies or antibody fragments to identify antibodies or antibody fragments that contain antigen binding sites for incorporation into a therapeutic bispecific antibody.

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