US 20160280795A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0280795 A1 Wang (43) Pub. Date: Sep. 29, 2016

(54) BISPECIFIC ANTIBODY WITH TWO (52) U.S. Cl. SINGLE-DOMAIN ANTIGEN-BINDING CPC ...... C07K 16/3007 (2013.01); C07K 16/283 FRAGMENTS (2013.01); C07K 16/2809 (2013.01); C07K (71) Applicant: Zhong Wang, Foster City, CA (US) 16/40 (2013.01); C07K 23.17/35 (2013.01); C07K 23.17/31 (2013.01); C07K 2317/524 (72) Inventor: Zhong Wang, Foster City, CA (US) (2013.01); C07K 2317/526 (2013.01); C07K (21) Appl. No.: 15/178,169 2317/569 (2013.01); C07K 2317/76 (2013.01) y x- - - 9 (22) Filed: Jun. 9, 2016 (57) ABSTRACT Related U.S. Application Data (63) Continuation of application No. PCT/US2014, Provided are bivalent bispecific antibody comprising a first 070985, filed on Dec. 17, 2014. polypeptide comprising a first Fc fragment and a first (60) Provisional application No. 61/918,383, filed on Dec. single-domain antigen-binding (VHH) fragment and a sec 19, 2013. ond polypeptide comprising a second Fc fragment and a Publication Classification second single-domain antigen-binding (VHH) fragment, wherein the first VHH fragment has specificity to a tumor (51) Int. Cl. cell or a microorganism and the second VHH fragment has C T 2% 3.08: specificity to an immune cell, and wherein the first fragment C07K 6/28 (2006.01) is N-terminal to the second fragment. Patent Application Publication Sep. 29, 2016 Sheet 1 of 10 US 2016/0280795 A1

Patent Application Publication Sep. 29, 2016 Sheet 3 of 10 US 2016/0280795 A1

SEQ ID Aligned Sequences No. ER4 -- 1. 2 O 2 7

23. ss 9 WP------O2 WGOGTOVTVSS-- 23 WGPGTWTWSSGR 28 8 WGKGTOVTVSS-- 2.

FIG. 2 (Cont'd) Patent Application Publication Sep. 29, 2016 Sheet 4 of 10 US 2016/0280795 A1

FIG. 3 Patent Application Publication Sep. 29, 2016 Sheet 5 of 10 US 2016/0280795 A1

Anti-His wester Anti-Flag western Detect anti-CEA Detect Anti-CD6

FIG. 4 Patent Application Publication Sep. 29, 2016 Sheet 6 of 10 US 2016/0280795 A1

->anti-his tag

-> anti-flag tag

FIG. 5 Patent Application Publication Sep. 29, 2016 Sheet 7 of 10 US 2016/0280795 A1

s' WB v. is. Sisticose treatient M(išax MgCl2: *FB; as fiew after binding: ww2: :orax iris, Siavi Natipi 7.3: i:i-i?: 2 in eitation of {}, \t Glycine pi;3.7 with {{}ai Xi ris.

FIG. 6

w wixie kis: S:stics sysk treatises: & ( iš:x: > iFB: Fas low after binding: wi.w3; taxi ris, 50axi NaCl pi 7.5; i:i-E7: 2 in eities of 0. M Gisciae pi i2.7 witi 200a M ris.

FIG. 7 Patent Application Publication Sep. 29, 2016 Sheet 8 of 10 US 2016/0280795 A1

W: wei: ces:S sacrisk six: xii. Six vigi: it: Fast low after hiding: wi.: taxi ris, Stax NaCl pi;7.3: 7.2 eation of (). v. Glycine pii 2, with 20 a visis

FIG. 8 Patent Application Publication Sep. 29, 2016 Sheet 9 of 10 US 2016/0280795 A1

a ------120 :- 3. 88 fumor ces of ly S$ aw : Eumor ce: NK ce. s 3O Sa & fumor ce-NK cell Bispecific 60 C : x fumor ce--NK ce: Monospecific 2 a. anti-CEA s fumor ce?--NK cel-Monospecific & 20 anti-C)

O .

SKOW3 29 (CEA negative) (CEA positive)

60 ------

140 - 20 SS : : unor cells Way : s :- &Tusmor cells + ce's s 3umor cells +8specifictug/n}} 8 8; unor--ceils-Bispecific (ig/in) 68 3: Tuforces +8i specific{Cugn) 2 & Tumor-- cets--Bispecific Qug/mi) Ae al

20 O SKOW3 -29 (CEA negative) (CEA positive) FIG. 10 Patent Application Publication Sep. 29, 2016 Sheet 10 of 10 US 2016/0280795 A1

140 ;

120 : Tunior ceils orily

OO . : Trnor & NK ce:

80 & Tutor re-K ce+Bispecific SO xi or ce-NK 40 ce:+Morospecificanti-Her2 incr ce+NK O - ce3+Morospecific anti-CD16

MD-WAB231 SK-BR (Her2 negative) Her2 positive) FIG. 11

: morces &timor cells + ces 3 Eurmor ceils +Bispecific(3.ug/mi) & Rumor cells-Bispecific lug/mi) 3: Tumor cells +Bispecific Oug/n}} Šumor + cells--Bispecific {10ug/ml)

VO-MAB231 SK-BR Her2 negative) (Her2 positive)

FIG. 12 US 2016/0280795 A1 Sep. 29, 2016

BSPECIFIC ANTIBODY WITH TWO polypeptide comprising a first Fc fragment and a first SINGLE-DOMAN ANTIGEN-BINDING single-domain antigen-binding (VHH) fragment and (b) a FRAGMENTS second polypeptide comprising a second Fc fragment and a second single-domain antigen-binding (VHH) fragment, CROSS REFERENCE TO RELATED wherein the first VHH fragment has specificity to a tumor APPLICATIONS cell or a microorganism and the second VHH fragment has 0001. This application is a continuation of International specificity to an immune cell. Application No. PCT/US2014/070985, filed Dec. 17, 2014, 0008. In some aspects, the first VHH fragment has speci which claims the benefit under 35 U.S.C. S 119(e) of U.S. ficity to a tumor antigen. In some aspects, the tumor antigen Provisional Application No. 61/918,383 filed on Dec. 19, is selected from the group consisting of CEA, EGFR. Her2. 2013, which is hereby incorporated by reference in its EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, entirety. gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding pro tein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, CVB3, BACKGROUND c5|B1, ERBB2, ERBB3, MET, IGFIR, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin. In some aspects, the 0002 Bispecific antibodies (BSMAb, BSAb) are artificial tumor antigen is CEA. proteins composed of fragments of two different monoclonal 0009. In some aspects, the first VHH fragment comprises antibodies and consequently binds to two different types of the amino acid sequence of SEQID NO:1, or an amino acid antigens. In cancer immunotherapy, for instance, BSMAbs having at least about 95% sequence identity thereto. are engineered that simultaneously bind to a cytotoxic cell 0010. In some aspects, the first VHH fragment has speci and a target like a tumor cell to be destroyed. ficity to a virus or a bacterium. In some aspects, the first 0003. At least three types of bispecific antibodies have VHH fragment has specificity to an endotoxin. been proposed or tested, including trifunctional antibody, 0011. In some aspects, the second VHH fragment has chemically linked Fab and bi-specific T-cell engager. In specificity to an antigen selected from the group consisting order to overcome manufacturing difficulties, a first-genera of CD3, CD16, CD19, CD28 and CD64. In some aspects, tion BSMAb, called trifunctional antibody, has been devel the antigen is CD16. oped. It consists of two heavy and two light chains, one each 0012. In some aspects, the second VHH fragment com from two different antibodies. The two Fab regions are prises the amino acid sequence of one of SEQ ID NO:2-5, directed against two antigens. The Fc region is made up from or an amino acid having at least about 95% sequence identity the two heavy chains and forms the third binding site; hence thereto. the name. 0013. In some aspects, the first VHH fragment and/or the 0004. Other types of bispecific antibodies have been second VHH fragment does not contain Val, Gly, Leu, and designed to overcome certain problems, such as short half Trp residues at Kabat positions 37, 44, 45, and 47, respec life, immunogenicity and side-effects caused by cytokine tively. liberation. They include chemically linked Fabs, consisting 0014. In some aspects, each of the Fc fragments com only of the Fab regions, and various types of bivalent and prises a CH2 domain and a CH3 domain. trivalent single-chain variable fragments (scFVs), fusion 0015. In some aspects, the two polypeptides are con proteins mimicking the variable domains of two antibodies. nected with two disulfide bonds. In some aspects, the The furthest developed of these newer formats are the disulfide bonds are connected between cysteine residues bi-specific T-cell engagers (BiTEs) and trifunctional anti located at a hinge region between each of the VHH fragment bodies. and the Fc fragment. 0005. Despite these advancements, there are still major 0016. In some aspects, the Fc fragments comprise one or challenges with bispecific antibodies, such as improving more Substitutions, as compared to a wild-type Fc fragment, manufacturing efficiency, retaining immunogenicity and that form an ionic bond between the Fc fragments. maintaining half-life. 0017. In some aspects, the Fc fragments comprises one or more Substitutions, as compared to a wild-type Fc frag SUMMARY ments, that form a knob-into-the-hole conformational pair 0006. The present disclosure provides a bispecific anti ing between the heavy chain and the Fc fragment. body that includes immunoglobulin Fc fragments connected 0018. Also provided, in one embodiment, is a polynucle to two single-domain antigen-binding fragments (or otide comprising a nucleic acid sequence encoding an anti domains), each of which targets a different antigen. Such a body of any preceding claim. In some aspects, provided is a bispecific antibody, without light chains and with its reduced host cell comprising the polynucleotide of the present dis molecule weight as compared to a conventional antibody, closure. In some aspects, the host cell is a bacterial cell or presents a significant advantage in antibody production and yeast cell. In some aspects, the host cell is E. coli. purification. Unexpectedly, Such a bispecific antibody can 0019. Yet in another embodiment, provided is a method still bind to both antigens effectively, carrying out its of treating a tumor in a patient, comprising administering to intended biological functions. Also unexpectedly, even the patient an antibody of present disclosure, wherein the though these disclosed bispecific antibodies are still het first fragment has specificity to a tumor antigen expressed on erodimmers, they can be readily expressed in bacterial cells a tumor cell in the patient. Such as E. coli, leading to production of Soluble proteins, 0020. Another embodiment provides a polypeptide com while other known bispecific antibodies produced by E. coli prising the amino acid sequence of SEQID NO:13 or having are hardly soluble. at least 95% sequence identity to SEQID NO:13 (or having 0007 Thus, one embodiment of the present disclosure one, two or three amino acid addition, deletion or Substitu provides a bivalent bispecific antibody comprising (a) a first tion as compared to SEQ ID NO:13), wherein the polypep US 2016/0280795 A1 Sep. 29, 2016 tide has binding specificity to a mammalian CD3 protein, shares less than 40% identity, though preferably less than Such as a human CD3 protein. 25% identity, with one of the sequences of the present 0021. In some embodiments, the present disclosure pro disclosure. vides a bivalent antibody comprising (a) a first polypeptide 0032. A polynucleotide or polynucleotide region (or a comprising a first Fc fragment and a first single-domain polypeptide or polypeptide region) has a certain percentage antigen-binding (VHH) fragment and (b) a second polypep (for example, 60%. 65%, 70%, 75%, 80%, 85%, 90%, 95%, tide comprising a second Fc fragment and a second VHH 98% or 99%) of “sequence identity” to another sequence fragment. The two polypeptides can be identical (bivalent, means that, when aligned, that percentage of bases (or amino monospecific antibody) or have different binding specificity acids) are the same in comparing the two sequences. This (bivalent, bispecific antibody). alignment and the percent homology or sequence identity BRIEF DESCRIPTION OF THE DRAWINGS can be determined using software programs known in the art. 0022 FIG. 1 illustrates a bispecific antibody that includes 0033. The term “an equivalent polynucleotide' refers to a first single-domain antigen-binding fragment (VHH1) and a nucleic acid sequence having a certain degree of homol a second VHH (VHH2) each connected to the N-terminus of ogy, or sequence identity, to a reference nucleotide sequence an Fc fragment, forming a light chain-less bispecific anti or the complement thereof. In one aspect, homologs of body. nucleic acids are capable of hybridizing to the nucleic acid 0023 FIG. 2 presents a multiple sequence alignment of or complement thereof. Likewise, “an equivalent polypep the sequences of Table 1, with related domains annotated. tide' refers to a polypeptide having a certain degree of 0024 FIG. 3 shows Commassiue blue staining of homology, or sequence identity, with the amino acid insoluble and soluble fractions of the antibody expressed in sequence of a reference polypeptide. In some aspects, the the bacterial cells. sequence identity is at least about 70%, 75%, 80%, 85%, 0025 FIG. 4 shows the staining of each of the two 90%. 95%, 98%, or 99%. In some aspects, the equivalent antibody chains separately, using antibodies against the His sequence retains the activity (e.g., epitope-binding) or struc tag and the Flag tag, respectively. ture (e.g., Salt-bridge) of the reference sequence. 0026 FIG. 5 shows Western blots with anti-His tag 0034) For each polypeptide or polynucleotide disclosed antibody (middle panel) or with anti-Flag tag antibody herein, its equivalents are also contemplated. In one aspect, (lower panel), as compared to Commassiue blue staining of an equivalent of a polypeptide includes an alteration (i.e., a the purified antibodies (upper panel). deletion, an addition or a Substitution) of an amino acid 0027 FIG. 6-8 present Commassiue blue staining images residue. In one aspect, an equivalent of a polypeptide for obtained bispecific antibodies at each stage of purifica includes no more than two alterations of amino acid resi tion, for the anti-CEA-Fc:anti-CD3-Fc, the anti-Her2-Fc: dues. In one aspect, an equivalent of a polypeptide includes anti-CD16-Fc and the anti-Her2-Fc:anti-CD3-Fc bispecific no more than 3, 4, or 5 alterations of amino acid residues. In antibodies, respectively. Some aspects, the amino acid alterations are at residues not 0028 FIG. 9-11 present bar charts showing the cytotox critical to the activity of the reference polypeptide. Residues icity of three bispecific antibodies, anti-CEA-Fc:anti-CD 16 critical to the activity of a polypeptide can be readily tested Fc (FIG. 9) anti-CEA-Fc:anti-CD3-Fc (FIG. 10) anti-CEA by site-specific mutation analysis, or even sequence align Fc:anti-CD16-Fc (FIG. 11) in a killing manner dependent on ments as such residues are highly reserved. bispecific antibodies. 0035. As used herein, an “antibody' or “antigen-binding 0029 FIG. 12 presents a bar chart showing the dose polypeptide' refers to a polypeptide or a polypeptide com dependent and immune cell-dependent nature of the cyto plex that specifically recognizes and binds to one or more toxicity in a killing manner dependent on bispecific anti antigens. An antibody can be a whole antibody and any body. antigen binding fragment or a single chain thereof. Thus the term “antibody includes any protein or peptide containing DETAILED DESCRIPTION molecule that comprises at least a portion of an immuno globulin molecule having biological activity of binding to Definitions the antigen. Examples of Such include, but are not limited to 0030. It is to be noted that the term “a” or “an entity a complementarity determining region (CDR) of a heavy or refers to one or more of that entity; for example, “a bispecific light chain or a ligand binding portion thereof, a heavy chain antibody,” is understood to represent one or more bispecific or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion antibodies. As such, the terms “a” (or “an”), “one or more.” thereof, or at least one portion of a binding protein. The term and "at least one' can be used interchangeably herein. antibody also encompasses polypeptides or polypeptide 0031) “Homology” or “identity” or “similarity” refers to complexes that, upon activation, possess antigen-binding sequence similarity between two peptides or between two capabilities. nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be 0036. The terms “antibody fragment” or “antigen-bind aligned for purposes of comparison. When a position in the ing fragment’, as used herein, is a portion of an antibody compared sequence is occupied by the same base or amino such as F(ab'), F(ab), Fab', Fab, Fv, scFv and the like. acid, then the molecules are homologous at that position. A Regardless of structure, an antibody fragment binds with the degree of homology between sequences is a function of the same antigen that is recognized by the intact antibody. The number of matching or homologous positions shared by the term “antibody fragment includes aptamers, Spiegelmers, sequences. An "unrelated' or “non-homologous' sequence and diabodies. The term “antibody fragment” also includes US 2016/0280795 A1 Sep. 29, 2016 any synthetic or genetically engineered protein that acts like 0041. In naturally occurring antibodies, the six “comple an antibody by binding to a specific antigen to form a mentarity determining regions” or “CDRs' present in each complex. antigen-binding domain are short, non-contiguous 0037 Antibodies, antigen-binding polypeptides, vari sequences of amino acids that are specifically positioned to ants, or derivatives thereof of the disclosure include, but are form the antigen-binding domain as the antibody assumes its not limited to, polyclonal, monoclonal, multispecific, three dimensional configuration in an aqueous environment. human, humanized, primatized, or chimeric antibodies, The remainder of the amino acids in the antigen-binding single chain antibodies, epitope-binding fragments, e.g., domains, referred to as “framework” regions, show less Fab, Fab' and F(ab'), Fd. Fvs, single-chain Fvs (sclv), inter-molecular variability. The framework regions largely single-chain antibodies, disulfide-linked FVs (sdFv), frag adopt a f-sheet conformation and the CDRs form loops ments comprising either a VK or VH domain, fragments which connect, and in some cases form part of the B-sheet produced by a Fab expression library, and anti-idiotypic structure. Thus, framework regions act to form a scaffold (anti-Id) antibodies (including, e.g., anti-Id antibodies to that provides for positioning the CDRs in correctorientation LIGHT antibodies disclosed herein). Immunoglobulin or by inter-chain, non-covalent interactions. The antigen-bind antibody molecules of the disclosure can be of any type ing domain formed by the positioned CDRs defines a surface (e.g., IgG, IgE. IgM, Ig), IgA, and IgY), class (e.g., IgGI. complementary to the epitope on the immunoreactive anti IgG2, IgG3, IgG4, IgA1 and IgA2) or Subclass of immuno gen. This complementary Surface promotes the non-covalent globulin molecule. binding of the antibody to its cognate epitope. The amino acids comprising the CDRS and the framework regions, 0038 Light chains are classified as either kappa or respectively, can be readily identified for any given heavy or lambda (K, w). Each heavy chain class may be bound with light chain variable region by one of ordinary skill in the art, either a kappa or lambda light chain. In general, the light and since they have been precisely defined (see “Sequences of heavy chains are covalently bonded to each other, and the Proteins of Immunological Interest, Kabat, E., et al., U.S. “tail portions of the two heavy chains are bonded to each Department of Health and Human Services, (1983); and other by covalent disulfide linkages or non-covalent linkages Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which when the immunoglobulins are generated either by hybrido are incorporated herein by reference in their entireties). mas, B cells or genetically engineered host cells. In the 0042. In the case where there are two or more definitions heavy chain, the amino acid sequences run from an N-ter of a term which is used and/or accepted within the art, the minus at the forked ends of the Y configuration to the definition of the term as used herein is intended to include C-terminus at the bottom of each chain. all Such meanings unless explicitly stated to the contrary. A 0039. Both the light and heavy chains are divided into specific example is the use of the term "complementarity regions of structural and functional homology. The terms determining region” (“CDR) to describe the non-contigu “constant and “variable' are used functionally. In this ous antigen combining sites found within the variable region regard, it will be appreciated that the variable domains of of both heavy and light chain polypeptides. This particular both the light (VK) and heavy (VH) chain portions deter region has been described by Kabat et al., U.S. Dept. of mine antigen recognition and specificity. Conversely, the Health and Human Services, “Sequences of Proteins of constant domains of the light chain (CK) and the heavy Immunological Interest' (1983) and by Chothia et al., J. chain (CH1, CH2 or CH3) confer important biological Mol. Biol. 196:901-917 (1987), which are incorporated properties such as secretion, transplacental mobility, Fc herein by reference in their entireties. The CDR definitions receptor binding, complement binding, and the like. By according to Kabat and Chothia include overlapping or convention the numbering of the constant region domains Subsets of amino acid residues when compared against each increases as they become more distal from the antigen other. Nevertheless, application of either definition to refer binding site or amino-terminus of the antibody. The N-ter to a CDR of an antibody or variants thereof is intended to be minal portion is a variable region and at the C-terminal within the scope of the term as defined and used herein. The portion is a constant region; the CH3 and CK domains appropriate amino acid residues which encompass the CDRS actually comprise the carboxy-terminus of the heavy and as defined by each of the above cited references are set forth light chain, respectively. in the table below as a comparison. The exact residue 0040. As indicated above, the variable region allows the numbers which encompass a particular CDR will vary antibody to selectively recognize and specifically bind depending on the sequence and size of the CDR. Those epitopes on antigens. That is, the VK domain and VH skilled in the art can routinely determine which residues domain, or Subset of the complementarity determining comprise a particular CDR given the variable region amino regions (CDRs), of an antibody combine to form the variable acid sequence of the antibody. region that defines a three dimensional antigen-binding site. 0043 Kabat et al. also defined a numbering system for This quaternary antibody structure forms the antigen-bind variable domain sequences that is applicable to any anti ing site present at the end of each arm of the Y. More body. One of ordinary skill in the art can unambiguously specifically, the antigen-binding site is defined by three assign this system of "Kabat numbering to any variable CDRs on each of the VH and VK chains (i.e. CDR-H1, domain sequence, without reliance on any experimental data CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In beyond the sequence itself. As used herein, “Kabat number Some instances, e.g., certain immunoglobulin molecules ing refers to the numbering system set forth by Kabat et al., derived from camelid species or engineered based on cam U.S. Dept. of Health and Human Services, “Sequence of elid immunoglobulins, a complete immunoglobulin mol Proteins of Immunological Interest' (1983). ecule may consist of heavy chains only, with no light chains. 0044. By “specifically binds” or “has specificity to.' it is See, e.g., Hamers-Casterman et al., Nature 363:446-448 generally meant that an antibody binds to an epitope via its (1993). antigen-binding domain, and that the binding entails some US 2016/0280795 A1 Sep. 29, 2016

complementarity between the antigen-binding domain and tion, about half (50%) of paired antibodies would be the the epitope. According to this definition, an antibody is said desired bispecific antibody. Given the lack of light chains to “specifically bind to an epitope when it binds to that alone, therefore, the present technology leads to greatly epitope, via its antigen-binding domain more readily than it improved production efficiency compared to conventional would bind to a random, unrelated epitope. The term “speci Fab-Fc bispecific antibodies. ficity' is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, A. Unexpected Properties of the Presently antibody “A” may be deemed to have a higher specificity for Disclosed Bispecific Antibodies a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it 0051. As a VHH fragment is smaller and much shorter has for related epitope “D’. than conventional Fab fragments, it was Suspected that a 0045. As used herein, the terms “treat' or “treatment bispecific antibody that includes two VHH fragments could refer to both therapeutic treatment and prophylactic or be too short to render functional dual specificity. This is preventative measures, wherein the object is to prevent or because a bispecific antibody, such as one that simultane slow down (lessen) an undesired physiological change or ously targets two cells, an immune cell and a tumor cell or disorder, Such as the progression of cancer. Beneficial or bacterium, needs access to specific antigens on two separate desired clinical results include, but are not limited to, large cells. Too short a connection between the two antigen alleviation of symptoms, diminishment of extent of disease, binding sites on a bispecific antibody, therefore, would stabilized (i.e., not worsening) state of disease, delay or subject the antibody to steric hindrance between the two slowing of disease progression, amelioration or palliation of cells. the disease state, and remission (whether partial or total), 0.052 Unexpectedly, however, it is discovered that the whether detectable or undetectable. "Treatment can also Suspected Steric hindrance had limited impact on the func mean prolonging Survival as compared to expected Survival tion of the presently disclosed bispecific antibodies. Further, if not receiving treatment. Those in need of treatment the overall bispecific affinity of the antibody, without the include those already with the condition or disorder as well need of light chains, was comparable to conventional bispe as those prone to have the condition or disorder or those in cific antibodies (see Example 3). which the condition or disorder is to be prevented. 0053. The advantage of the present technology also 0046 By “subject” or “individual” or “animal” or applies to cell expression efficiency and protein stability. "patient’ or “mammal,” is meant any subject, particularly a Even though it was generally thought that Small antibodies mammalian Subject, for whom diagnosis, prognosis, or are easier to produce in cells than larger antibodies, bispe therapy is desired. Mammalian Subjects include humans, cific antibodies present a special challenge for antibody domestic animals, farm animals, and Zoo, sport, or pet production. This is at least because the two (or three or four) animals such as dogs, cats, guinea pigs, rabbits, rats, mice, different protein chains can interact with one another inside horses, cattle, cows, and so on. and outside cells, leading to interference and thus decreased 0047. As used herein, phrases such as “to a patient in efficiency of a host cells expression system and increased need of treatment' or “a subject in need of treatment protein instability. Therefore, bispecific antibodies with full includes Subjects, such as mammalian Subjects, that would heavy chains and light chains are typically expressed in two benefit from administration of an antibody or composition of separate cells, or use a common light chain to reduce the present disclosure used, e.g., for detection, for a diag mis-pairing of light chains with heavy chains, which pro nostic procedure and/or for treatment. duce non-functional antibodies. 0054 Even bispecific antibodies that are smaller than Bivalent Antibodies those presently disclosed are associated with Such problems. 0048. The present disclosure, in one embodiment, pro For instance, Bi-specific T-cell engagers (BITEs) are fusion vides bispecific antibodies targeting two different antigens, proteins consisting of two typical single chain variable one of which is present on a tumor cell or a microorganism, fragments. Although much smaller (typically 55 KDa) than and another on an immune cell. Upon administration to an the bispecific antibodies disclosed herein (about 100 KDa), individual, the bispecific antibody specifically binds to a BITEs cannot be expressed soluble in bacterial cells. Pres tumor cell or microorganism and at the same time specifi ently, BITEs are expressed in mammalian systems, which cally binds immune cells, such as a cytotoxic cell. Such dual are much more expensive. binding can lead to killing of the bound tumor or microor 0055 Unexpectedly, as demonstrated in Examples 1-2, ganism by the host’s immune system. when expressed in E. coli cells, up to about 20% the 0049. The bispecific antibody of the present technology bispecific antibodies of the present disclosure were soluble. includes two single-domain antigen-binding fragments As explained above, these antibodies are much larger than (VHH fragments or VHH domains), each having specificity BITEs. Nevertheless, while bacterium-expressed BITEs are to one of the antigens. VHH fragments are known in the art entirely insoluble, the bispecific antibodies of the present and are further described below. Each of the VHH fragments disclosure can be readily produced from bacterial cells. Such is connected, optionally through a hinge region, to an Fc a result, therefore, is Surprising and unexpected. fragment of a conventional antibody. 0056 Furthermore, since the bispecific antibodies of the 0050. As a VHH fragment is independently capable of present technology have relatively small sizes compared to specifically recognizing and binding an antigen without a conventional antibodies, and they are efficient to produce in paired light chain, Such an antibody includes only two particular in a large-scale setting, Such as from yeast and polypeptide chains. Accordingly, during production of the bacterial hosts. The stability, solubility and half-life of these bispecific antibody, there are only two possible pairings for bispecific ligands are also much Superior to the bispecific each chain. In other words, even without any paring selec antibodies being developed in the field. US 2016/0280795 A1 Sep. 29, 2016

B. Bivalent, Monospecific or Bispecific Antibodies (2007)). Single-domain antibodies do not include CH1 domains which, in a conventional antibody, interact with the 0057. From the foregoing, it is apparent that the antibod light chains. ies of the structures as illustrated in FIG. 1, in general, 0060 VHHs contain four framework regions (FR1-FR4) exhibit high bacterial production, stability and binding affin that form the core structure of the immunoglobulin domain ity. In some embodiments, therefore, the present disclosure and three complementarity-determining regions (CDR1 provides bivalent antibody comprising (a) a first polypeptide CDR3) that are involved in antigen binding (see, e.g., FIG. comprising a first Fc fragment and a first single-domain 2). As compared to human VH domains. The VHH frame antigen-binding (VHH) fragment and (b) a second polypep work regions show a high sequence homology (>80%) to tide comprising a second Fc fragment and a second VHH human VH domains. See Harmsen and Haard, 2007, which fragment. Such a bivalent antibody can be monospecific further describes that the “most characteristic feature of (when the two polypeptides are identical or have the same VHHs is the presence of amino acid substitutions at four binding specificity) or bispecific (when the two polypeptides FR2 positions (positions 37, 44, 45, and 47; Kabat number ing) that are conserved in conventional VH domains and that have different binding specificity). are involved in forming the hydrophobic interface with VL 0058 Such a bispecific antibody can be configured to domains.” VHHs typically have different amino acid resi target different antigen pair. For instance, one VHH frag dents at these and other positions that are highly reserved in ment can have specificity to a first immune cell while the the conventional VHs (e.g., Leu11 Ser, Val 37Phe or Tyr, other target a second immune cell; one VHH fragment can Gly44Glu, Leu45Arg or Cys, Trp47Gly). have specificity to a first tumor cell while the other has 0061 Also as described in Harmsen and Haard, 2007, specificity to a second tumor cell; one VHH fragment can CDRs of VHHs have certain known characteristic features. have specificity to an immune cell and the other to a The N-terminal part of CDR1, for instance, is more variable microorganism, an infected cell, a tumor cell, a inflamed and a conventional antibody. Further, some VHHs have an cell, an apoptotic cell, or a foreign cell, without limitation. extended CDR3 that is often stabilized by an additional disulfide bond with a cysteine in CDR1 or FR2, resulting in C. Single-Domain Antigen-Binding Fragments the folding of the CDR3 loop across the former VL interface. (VHH) A particular subfamily of llama VHHs (VHH3) also contains an extended CDR3 that is stabilized by an additional disul 0059 A “single-domain antigen-binding fragment,” or fide bond with a cysteine at position 50 in FR2. “single-domain antibody fragment” or “VHH, is an anti 0062. Many scAbs are known in the art, and can be gen-binding fragment that is able to bind to an antigen readily prepared from animals such as camelids. From these without pairing with a light chain. VHH was originally sdAb, their VHHs can be readily identified and prepared. isolated from single-domain antibodies (sdAb) as the sole Table 1 lists a number of non-limiting examples for VHHs antigen-binding fragment. The first known single-domain and SdAbs. Accordingly, in Some embodiments, the present antibodies were isolated from camelids (Hamers-Casterman disclosure provides polypeptides comprising each of Such et al., Nature 363:446-8 (1993) and later from cartilaginous disclosed sequences, the equivalents thereof, and polynucle fish. Camelids produce functional antibodies without light otides encoding each. In one aspect, the polypeptide com chains and their single N-terminal domain (VHH) binds prises an amino acid sequence of SEQ ID NO:13, or an antigen without requiring domain pairing (reviewed in amino acid sequence having one, two or three amino acid Harmsen and Haard, App Microbiol Biotechnol., 77:13-22 addition/deletion/substitution. TABLE 1. Example Single-Domain Antigen-Binding Fragments (VHHs) and Single Domain Antibodies (sdAbs) 1. Anti- CEA. WH (SEQ ID NO: 1) EVOLVESGGG FWOAGESLTL SCTSSTLTFT PYRMAWYROA PGKORDLVAD ISSGDGRTTN YADFAKGRFT ISR DNIKNTV FLRMTNLKPE DTAVYYCNTF WSFWGIARSW GOGTOWTVSS 2. Anti-CD16. W H (SEQ ID NO: 2) EVOLVESGGG LVO SCSFPGSIFS LTMGWYROAP GKERELWTSA TPGGDTNYAD FVKGRFTISR DNARSIIYLO MNSLKIPEDTA WYYCYARTRN WG 3. Anti-CD16. W H (SEQ ID NO : 3) EVOLVESGGE LVOAGGSLRL SCAASGLTFS SYNMGWFRRA PGKEREFWAS ITWSGRDTFY ADSWKGRFTI SRDNAKNTWY LOMSSLKPED TAWYYCAANP WP

4. Anti-CD16. W H (SEQ ID NO: 4) EVOLVESGGG LVO PGESLTL SCW WAGSIFS FAMSWYROAP GKERELWARI GSDDRWTYAD SWKGRFTISR DNI KRTAGLQ MNSLKIPEDTA WYYCNAOTDL RD

5. Anti-CD16 W H (SEO ID NO; 5) EVOLVESGGG LVO PGGSLTL SCWAAGSIFT FAMSWYROAP RKERELWARI GTDDETMYKD SWKGRFTISR DNW KRTAGLQ MNNLKIPEDTA WYYCNARTDY RD

6. Anti-Her2 scAb (SEQ ID NO : 6) QVOLVOSGGG LVOAGGSLRL SCAASGRTFS SYAMAWFROA PGKEREFWAA ISWSGANIYW ADSVKGRFTI SRDNAKDTVY LOMNSLKPED TAWYYCAWKL GFAPVEEROY DYWGOGTOVT WSS US 2016/0280795 A1 Sep. 29, 2016

TABLE 1 - continued Example Single-Domain Antigen-Binding Fragments (WHHS) and Single Domain Antibodies (scAbs)

7. Anti-EGFR1 VHH (SEO ID NO: 7) MAEWOOASGG GLVOAGGSLR LSCAASGRTE TTSAIAWFRO APGKEREFWA QISASGLGIN YSGTVKGRFT ISRDADKTTV YLOMNSLTPE DTAVYYCAAG FHYIAAIRRT TDFHFWGPGT LWTWSSGR

8. Anti-F4 + ETEC bacteria WHH (SEQ ID NO: 8) QWOLOESGGG LVOAGGSLRL SCEASGNVDR IDAMGWFROA PGKOREFWGY ISEGGILNYG DFWKGRFTIS RDNAKNTVYL OMSNLKSEDT GWYFCAASHW GTLLIKGIEH WGKGTOVTVS S

9. Anti-PS2- 8 WHH (SEQ ID NO: 9) EVOLVESGGG LVOAGGSLRL SCAASGRSFS RDAMGWFROA PGKERDWWAA INLNGGRTYS ADSVKGRFTI SRDNDKNTVY LOMSNLKPED TAVYYCAARE GDVGLVSYKR SSNYPYWGOG TOVTVSS 10. Anti-Huntavirus WHH (SEQ ID NO : 10) MAEVOLOASG GGLVOAGGSL RLSCAASGRT SSMYSMWWFR QAPGKEREFW AGIIWTSSLT YYADSLKGRF TISRDNAKNT WYLOMNSLKP EDTAIYYCAA DTKTGGGGYE YWGOVTVTVS S 11. Anti-Huntavirus WHH (SEQ ID NO: 11) MAEVOLOASG GGLVOPGGSL RLSCAASGSI FSSDWMGWFR QAPGKERELV AFITDDGGTN YADSVKGRFT ISRDNAENTV SLOMNSLKPE DTAVYYCNAR YYSGGYRNYW GOVTVTVSS 12. Anti-CD16 WHH (SEQ ID NO: 12) EVOLVESGGG LVOPGGSLRL SCSFPGSIFS LTMGWYROAP GKERELVTSA TPGGDTNYAD FWKGRFTISR DNARSIIYLO MNSLKPEDTA WYYCYARTRN WGTVWGOGTOWTVSS 13. Anti-CD3 WHH (SEQ ID NO: 13) QWOLOESGGG LVOAGGSLRL SCAASGRTFS NYHMGWFROA PGKERELVAA ISGSGGSTYY TDSVKGRFTI SRNNAKNTMS LOMSNLKPED TOWYYCTTPT EKGSSIDYWG OGTOVTVSSG RYPYDWPDY

0063 As shown in FIG. 2, certain regions of these pairing between them. Knob-into-hole designs are known in sequences are highly conserved, such as FR1-3, while the the art. See, e.g., Ridgway et al. “Knob-into-holes engi CDRs are more variable. neering of antibody C3 domains for heavy chain heterodi merization.” Protein Engineering 9(7):617-21 (1996). D. Fc Modifications to Enhance Heterodimer 0067. In one aspect, K366 on one of the Fc fragment is Paring Substituted with a relatively large amino acid residue. Such 0064. The VHH fragments each is connected, optionally as tyrosine (Y) or tryptophan (W). Then Y407 on the other through a hinge region or linker, to the N-terminus of an Fc Fc fragment can be substituted with a relatively small amino fragment, which is preferably a human Fc fragment or acid residue. Such as threonine (T), alanine (A) or valine (V). humanized Fc fragment. Modifications to the Fc fragments E. Binding Targets can be introduced to improve paring between two different antibody chains to form the desired bispecific antibody, or to 0068. In some embodiments, the first VHH (e.g., VHH1 further stabilize or improve activity of the antibodies. For of FIG. 1) of the bispecific antibody has binding specificity instance, either or both of the Fc fragments can include one to a tumor antigen. or more substitutions, as compared to a wild-type antibody 0069. A “tumor antigen' is an antigenic substance pro Fc fragment, that form an ionic bond between them. duced in tumor cells, i.e., it triggers an immune response in 0065. In one aspect, one of the Fc fragments contains one the host. Tumor antigens are useful in identifying tumor cells or more Substitutions with amino acid residues having a and are potential candidates for use in cancer therapy. positive charge under physiological conditions and the other Normal proteins in the body are not antigenic. Certain Fc fragment contains one or more Substitutions with one or proteins, however, are produced or overexpressed during more amino acid residues having a negative charge under tumorigenesis and thus appear “foreign' to the body. This physiological conditions. In one aspect, the positively may include normal proteins that are well sequestered from charged amino acid residue can be arginine (R), histidine (H) the immune system, proteins that are normally produced in or lysine (K). In another aspect, the negatively charged extremely small quantities, proteins that are normally pro amino acid residue can be aspartic acid (D) or glutamic acid duced only in certain stages of development, or proteins (E). Amino acid residues that can be substituted include, whose structure is modified due to mutation. without limitation, D356, E357, L368, K370, K392, D399 0070 An abundance of tumor antigens are known in the and K409. art and new tumor antigens can be readily identified by 0066. In some aspects, the Fc fragments can include one screening. Non-limiting examples of tumor antigens include or more substitutions, as compared to a wild-type antibody EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, Fc fragment, that form a knob-into-the-hole conformational CD133, CEA, gp A33, Mucins, TAG-72, CIX, PSMA, US 2016/0280795 A1 Sep. 29, 2016 folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, signal peptide to direct secretion of the encoded polypeptide, Integrin, CVB3, a5 B1, ERBB2, ERBB3, MET, IGF1R, antibody constant regions as described herein, or other EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin. heterologous polypeptides as described herein. 0071. In some aspects, the first VHH has specificity to a (0079. It will also be understood by one of ordinary skill protein that is overexpressed on a tumor cell as compared to in the art that antibodies as disclosed herein may be modified a corresponding non-tumor cell. A "corresponding non Such that they vary in amino acid sequence from the natu tumor cell as used here, refers to a non-tumor cell that is of rally occurring binding polypeptide from which they were the same cell type as the origin of the tumor cell. It is noted derived. For example, a polypeptide or amino acid sequence that such proteins are not necessarily different from tumor derived from a designated protein may be similar, e.g., have antigens. Non-limiting examples include carcinoembryonic a certain percent identity to the starting sequence, e.g., it antigen (CEA), which is overexpressed in most colon, may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or rectum, breast, lung, pancreas and gastrointestinal tract 99% identical to the starting sequence. carcinomas; heregulin receptors (HER-2, neu or c-erbB-2), 0080 Furthermore, nucleotide or amino acid substitu which is frequently overexpressed in breast, ovarian, colon, tions, deletions, or insertions leading to conservative Sub lung, prostate and cervical cancers; epidermal growth factor stitutions or changes at “non-essential amino acid regions receptor (EGFR), which is highly expressed in a range of may be made. For example, a polypeptide or amino acid Solid tumors including those of the breast, head and neck, sequence derived from a designated protein may be identical non-Small cell lung and prostate; asialoglycoprotein recep to the starting sequence except for one or more individual tor; transferrin receptor, serpin enzyme complex receptor, amino acid Substitutions, insertions, or deletions, e.g., one, which is expressed on hepatocytes; fibroblast growth factor two, three, four, five, six, seven, eight, nine, ten, fifteen, receptor (FGFR), which is overexpressed on pancreatic twenty or more individual amino acid substitutions, inser ductal adenocarcinoma cells; vascular endothelial growth tions, or deletions. In certain embodiments, a polypeptide or factor receptor (VEGFR), for anti-angiogenesis gene amino acid sequence derived from a designated protein has therapy; folate receptor, which is selectively overexpressed one to five, one to ten, one to fifteen, or one to twenty in 90% of nonmucinous ovarian carcinomas; cell Surface individual amino acid Substitutions, insertions, or deletions glycocalyx; carbohydrate receptors; and polymeric immu relative to the starting sequence. noglobulin receptor, which is useful for gene delivery to I0081. In certain embodiments, an antigen-binding poly respiratory epithelial cells and attractive for treatment of peptide comprises an amino acid sequence or one or more lung diseases such as Cystic Fibrosis. moieties not normally associated with an antibody. Exem 0072. In one aspect, the first VHH has specificity to CEA plary modifications are described in more detail below. For or Her2. A representative sequence for this VHH is provided example, a fragment of the disclosure may comprise a as SEQ ID NO:1 or 6 (Table 1). In some aspects, the first flexible linker sequence, or may be modified to add a VHH includes an amino acid sequence of SEQ ID NO:1 or functional moiety (e.g., PEG, a drug, a toxin, or a label). 6 with one or two or three addition, deletion or substitution. 0082 Antibodies, variants, or derivatives thereof of the In one aspect, the first VHH includes an amino acid disclosure include derivatives that are modified, i.e., by the sequence of SEQ ID NO:7 (anti-EGFR1) optionally with covalent attachment of any type of molecule to the antibody one or two or three addition, deletion or substitution. Such that covalent attachment does not prevent the antibody 0073. In some aspects, the first VHH has specificity to an from binding to the epitope. For example, but not by way of microorganism (e.g., virus or bacterium). Non-limiting limitation, the antibodies can be modified, e.g., by glycosy examples of microorganism include microorganism Surface lation, acetylation, pegylation, phosphorylation, phosphory receptors and endotoxins. Examples of endotoxins include, lation, amidation, derivatization by known protecting/block without limitation, lipopolysaccharide (LPS) and lipooli ing groups, proteolytic cleavage, linkage to a cellular ligand gosaccharide (LOS). or other protein, etc. Any of numerous chemical modifica 0074. In some aspects, the first VHH includes an amino tions may be carried out by known techniques, including, acid sequence selected from SEQID NO: 8-11 (Table 1), or but not limited to specific chemical cleavage, acetylation, optionally with one or two or three addition, deletion or formylation, metabolic synthesis of tunicamycin, etc. Addi substitution. tionally, the antibodies may contain one or more non 0075. In some aspects, the second VHH (e.g., VHH2 of classical amino acids. FIG. 1) has specificity to an immune cell. In one aspect, the I0083. In other embodiments, the antigen-binding poly immune cell is selected from the group consisting of a T cell, peptides of the present disclosure may contain conservative a B cell, a monocyte, a macrophage, a neutrophil, a dendritic amino acid Substitutions. cell, a phagocyte, a natural killer cell, an eosinophil, a 0084. A “conservative amino acid substitution' is one in basophil, and a mast cell. which the amino acid residue is replaced with an amino acid 0076. In one aspect, the second VHH specifically recog residue having a similar side chain. Families of amino acid nizes an antigen selected from the group consisting of CD3. residues having similar side chains have been defined in the CD16, CD19, CD28 and CD64. In one aspect, the second art, including basic side chains (e.g., lysine, arginine, histi VHH specifically recognizes CD3 or CD16. dine), acidic side chains (e.g., aspartic acid, glutamic acid), 0077. In one aspect, the second VHH has specificity to uncharged polar side chains (e.g., glycine, asparagine, glu CD16 or CD3. Representative sequences for this VHH are tamine, serine, threonine, tyrosine, cysteine), nonpolar side provided as SEQID NO: 2, 3, 4, 5, 12 and 13 (Table 1), or chains (e.g., alanine, Valine, leucine, isoleucine, proline, optionally with one or two or three addition, deletion or phenylalanine, methionine, tryptophan), beta-branched side substitution. chains (e.g., threonine, Valine, isoleucine) and aromatic side 0078. Any of the antibodies or polypeptides described chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). above may further include additional polypeptides, e.g., a Thus, a nonessential amino acid residue in an immunoglobu US 2016/0280795 A1 Sep. 29, 2016 lin polypeptide is preferably replaced with another amino disclosure may encode portions of the antibody or VHH, acid residue from the same side chain family. In another variants or derivatives thereof on the same polynucleotide embodiment, a string of amino acids can be replaced with a molecule or on separate polynucleotide molecules. structurally similar string that differs in order and/or com position of side chain family members. 0091. In certain embodiments, the prepared antibodies 0085. Non-limiting examples of conservative amino acid will not elicit a deleterious immune response in the animal substitutions are provided in the table below, where a to be treated, e.g., in a human. In one embodiment, antigen similarity score of 0 or higher indicates conservative sub binding polypeptides, variants, or derivatives thereof of the stitution between the two amino acids. disclosure are modified to reduce their immunogenicity

C G P S A T D E N Q. H K R V M Y W W -8 - 7 -6 -2 - 6 -5 - 7 -7 -4 -5 -3 -3 2 - 6 - -5 -2 O O 17 Y O -5 -5 -3 -3 -3 -4 -4 -2 -4 O -4 -5 -2 - -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 1 2 9 2 6

-5 -2 - 1 O -1 O O O 1 1 O 5 -3 -2 O -1 -1 -1 1 1 2 3 6 -5 -1 O -1 O -1 2 2 1 4 4 O -1 O O 2 1 2 -5 -1 O O O 3 4 -5 -1 O O O 4 -2 O 1 1 3 1 2 1

I0086. In some embodiments, the antibodies may be con using art-recognized techniques. For example, antibodies jugated to therapeutic agents, prodrugs, peptides, proteins, can be humanized, primatized, deimmunized, or chimeric enzymes, viruses, lipids, biological response modifiers, antibodies can be made. pharmaceutical agents, or PEG. 0092. The binding specificity of bispecific antibodies of 0087. The antibodies may be conjugated or fused to a the present disclosure can be determined by in vitro assays therapeutic agent, which may include detectable labels such Such as immunoprecipitation, radioimmunoassay (RIA) or as radioactive labels, an immunomodulator, a hormone, an enzyme-linked immunoabsorbent assay (ELISA). enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or Production Systems and Methods a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in 0093. The present disclosure also provides systems and the art. methods for producing the bispecific antibody of the present disclosure. Cells suitable for producing antibodies are well 0088. The antibodies can be detectably labeled by cou known in the art, including human cells (e.g., CHO cells), pling it to a chemiluminescent compound. The presence of mammalian cells and bacterial cells. The use of bacterial the chemiluminescent-tagged antigen-binding polypeptide cells to produce bispecific antibodies presents significant is then determined by detecting the presence of lumines challenges. Nevertheless, as shown in the examples, when cence that arises during the course of a chemical reaction. expressed in bacterial cells, the resulting antibody is largely Examples of particularly useful chemiluminescent labeling soluble, even when both peptide chains are expressed in the compounds are luminol, isoluminol, theromatic acridinium same cell. ester, imidazole, acridinium salt and oxalate ester. 0094. Therefore, in one embodiment, provided is a host cell comprising one or more polynucleotides encoding both Polynucleotides Encoding the Antibodies and Methods of chains of the disclosed bispecific antibody. In one aspect, a Preparing the Antibodies single polynucleotide construct (e.g., plasmid) includes both coding sequences. In another aspects, two separate poly 0089. The present disclosure also provides isolated poly nucleotide constructs each encodes one of the polypeptide nucleotides or nucleic acid molecules encoding the bispe chains. Also provided, in one embodiment, is a host cell cific antibodies, variants or derivatives thereof of the dis comprising both polypeptide chains of the bispecific anti closure. bodies of the present disclosure. 0090 The polynucleotides of the present disclosure may 0095. In some aspects, the host cells are human cells. In encode the entire VHH, variants or derivatives thereof on the Some aspects, the host cells are mammalian cells. In some same polynucleotide molecule or on separate polynucleotide aspects, the host cells are yeast cells. In some aspects, the molecules. Additionally, the polynucleotides of the present host cells are bacterial cells, including Gram-positive and US 2016/0280795 A1 Sep. 29, 2016

Gram-negative bacterial cells. Representative bacterial cells non-Hodgkin’s disease), multiple myeloma, Waldenstrom's include, without limitation, E. coli and S. typhylmurium. macroglobulinemia, heavy chain disease, and Solid tumors 0096. Also provided, in some aspects, is a method for including, but not limited to, sarcomas and carcinomas Such preparing a bispecific antibody of the present disclosure. In as fibrosarcoma, myxosarcoma, liposarcoma, chondrosar one aspect, the method entails expressing both peptide coma, osteogenic sarcoma, chordoma, angiosarcoma, chains of the antibody in a host cell and extracting the endotheliosarcoma, lymphangiosarcoma, lymphangioen antibody from cell lysis. Further, provided are bispecific dotheliosarcoma, synovioma, mesothelioma, Ewings antibodies obtained from such methods. tumor, leiomyosarcoma, rhabdomyo sarcoma, colon carci noma, pancreatic cancer, breast cancer, ovarian cancer, Treatment and Diagnostic Methods prostate cancer, squamous cell carcinoma, basal cell carci noma, adenocarcinoma, Sweat gland carcinoma, sebaceous 0097. As described herein, the bispecific antibodies, vari gland carcinoma, papillary carcinoma, papillary adenocar ants or derivatives of the present disclosure may be used in cinomas, cystadenocarcinoma, medullary carcinoma, bron certain treatments and diagnostic methods associated with chogenic carcinoma, renal cell carcinoma, hepatoma, bile cancer or an infectious disease. duct carcinoma, choriocarcinoma, seminoma, embryonal 0098. The present disclosure is further directed to anti carcinoma, Wilm's tumor, cervical cancer, testicular tumor, body-based therapies which involve administering the lung carcinoma, Small cell lung carcinoma, bladder carci bispecific antibodies of the disclosure to a patient such as an noma, epithelial carcinoma, glioma, astrocytoma, medullo animal, a mammal, and a human for treating one or more of blastoma, craniopharyngioma, ependymoma, pinealoma, the disorders or conditions described herein. Therapeutic compositions of the disclosure include, but are not limited hemangioblastoma, acoustic neuroma, oligodendroglioma, to, antibodies of the disclosure (including variants and menangioma, melanoma, neuroblastoma and retinoblas derivatives thereofas described herein) and nucleic acids or tOma. polynucleotides encoding antibodies of the disclosure (in 0101 The antibodies of the present disclosure can also be cluding variants and derivatives thereofas described herein). used to treat an infectious disease caused by a microorgan 0099. The antibodies of the disclosure can also be used to ism, or kill a microorganism, by targeting the microorganism treat, inhibit or prevent diseases, disorders or conditions and an immune cell to effect elimination of the microorgan including malignant diseases, disorders, or conditions asso ism. In one aspect, the microorganism is a virus including ciated with Such diseases or disorder Such as diseases RNA and DNA viruses, a Gram positive bacterium, a Gram associated with increased cell survival, or the inhibition of negative bacterium, a protozoa or a fungus. apoptosis, for example cancers (such as follicular lympho 0102. A specific dosage and treatment regimen for any mas, carcinomas with p53 mutations, and hormone-depen particular patient will depend upon a variety of factors, dent tumors, including, but not limited to colon cancer, including the particular antigen-binding polypeptide, variant cardiac tumors, pancreatic cancer, melanoma, retinoblas or derivative thereof used, the patient's age, body weight, toma, glioblastoma, lung cancer, intestinal cancer, testicular general health, sex, and diet, and the time of administration, cancer, stomach cancer, neuroblastoma, myxoma, myoma, rate of excretion, drug combination, and the severity of the lymphoma, endothelioma, osteoblastoma, osteoclastoma, particular disease being treated. Judgment of such factors by osteosarcoma, chondrosarcoma, adenoma, breast cancer, medical caregivers is within the ordinary skill in the art. The prostate cancer, Kaposi's sarcoma and ovarian cancer); amount will also depend on the individual patient to be autoimmune disorders (such as, multiple Sclerosis, Sjogren's treated, the route of administration, the type of formulation, syndrome, Grave's disease, Hashimoto's thyroiditis, auto the characteristics of the compositions used, the severity of immune diabetes, biliary cirrhosis, Behcet’s disease, the disease, and the desired effect. The amount used can be Crohn's disease, polymyositis, Systemic lupus erythemato determined by pharmacological and pharmacokinetic prin SuS and immune-related glomerulonephritis, autoimmune ciples well known in the art. gastritis, autoimmune thrombocytopenic purpura, and rheu 0103 Methods of administration of the bispecific anti matoid arthritis) and viral infections (such as herpes viruses, bodies, variants or include but are not limited to intradermal, pox viruses and adenoviruses), inflammation, graft vs. host intramuscular, intraperitoneal, intravenous, Subcutaneous, disease (acute and/or chronic), acute graft rejection, and intranasal, epidural, and oral routes. The antigen-binding chronic graft rejection. Antigen binding polypeptides, vari polypeptides or compositions may be administered by any ants or derivatives thereof of the present disclosure are used convenient route, for example by infusion or bolus injection, to inhibit growth, progression, and/or metastasis of cancers, by absorption through epithelial or mucocutaneous linings in particular those listed above or in the paragraph that (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and follows. may be administered together with other biologically active 0100 Additional diseases or conditions associated with agents. Thus, pharmaceutical compositions containing the increased cell Survival, that may be treated, prevented, antigen-binding polypeptides of the disclosure may be diagnosed and/or prognosed with the antibodies or variants, administered orally, rectally, parenterally, intracistemally, or derivatives thereof of the disclosure include, but are not intravaginally, intraperitoneally, topically (as by powders, limited to, progression, and/or metastases of malignancies ointments, drops or transdermal patch), bucally, or as an oral and related disorders such as leukemia (including acute or nasal spray. leukemias (e.g., acute lymphocytic leukemia, acute myelo 0104. The term “parenteral as used herein refers to cytic leukemia (including myeloblastic, promyelocytic, modes of administration which include intravenous, intra myelomonocytic, monocytic, and erythroleukemia)) and muscular, intraperitoneal, intrasternal, Subcutaneous and chronic leukemias (e.g., chronic myelocytic (granulocytic) intra-articular injection and infusion. leukemia and chronic lymphocytic leukemia)), poly 0105 Administration can be systemic or local. In addi cythemia Vera, lymphomas (e.g., Hodgkin’s disease and tion, it may be desirable to introduce the antibodies of the US 2016/0280795 A1 Sep. 29, 2016

disclosure into the central nervous system by any Suitable indicated, include in vitro cell culture assays in which a route, including intraventricular and intrathecal injection; patient tissue sample is grown in culture, and exposed to or intraventricular injection may be facilitated by an intraven otherwise administered an antibody, and the effect of such an tricular catheter, for example, attached to a reservoir, such as antibody upon the tissue sample is observed. an Ommaya reservoir. Pulmonary administration can also be 0110. In a further embodiment, the compositions of the employed, e.g., by use of an inhaler or nebulizer, and disclosure are administered in combination with an antine formulation with an aerosolizing agent. oplastic agent, an antiviral agent, antibacterial or antibiotic 0106. It may be desirable to administer the bispecific agent or antifungal agents. Any of these agents known in the antibodies or compositions of the disclosure locally to the art may be administered in the compositions of the current area in need of treatment; this may be achieved by, for disclosure. example, and not by way of limitation, local infusion during 0111. In another embodiment, compositions of the dis Surgery, topical application, e.g., in conjunction, with a closure are administered in combination with a chemothera wound dressing after Surgery, by injection, by means of a peutic agent. Chemotherapeutic agents that may be admin catheter, by means of a Suppository, or by means of an istered with the compositions of the disclosure include, but implant, said implant being of a porous, non-porous, or are not limited to, antibiotic derivatives (e.g., doxorubicin, gelatinous material, including membranes, such as Sialastic bleomycin, daunorubicin, and dactinomycin); antiestrogens membranes, or fibers. Preferably, when administering a (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, protein, including an antibody, of the disclosure, care must methotrexate, floXuridine, interferon alpha-2b, glutamic be taken to use materials to which the protein does not acid, plicamycin, mercaptopurine, and 6-thioguanine); cyto absorb. toxic agents (e.g., carmustine, BCNU, lomustine, CCNU, 0107 The amount of the antibodies of the disclosure cytosine arabinoside, cyclophosphamide, estramustine, which will be effective in the treatment, inhibition and hydroxyurea, procarbazine, mitomycin, buSulfan, cis-platin, prevention of an inflammatory, immune or malignant dis and Vincristine Sulfate); hormones (e.g., medroxyprogester ease, disorder or condition can be determined by standard one, estramustine phosphate Sodium, ethinyl estradiol, estra clinical techniques. In addition, in vitro assays may option diol, megestrol acetate, methyltestosterone, diethylstilbe ally be employed to help identify optimal dosage ranges. strol diphosphate, chlorotrianisene, and testolactone); The precise dose to be employed in the formulation will also nitrogen mustard derivatives (e.g., mephalen, chorambucil, depend on the route of administration, and the seriousness of mechlorethamine (nitrogen mustard) and thiotepa); steroids the disease, disorder or condition, and should be decided and combinations (e.g., bethamethasone sodium phosphate); according to the judgment of the practitioner and each and others (e.g., dicarbazine, asparaginase, mitotane, Vin patient’s circumstances. Effective doses may be extrapolated cristine Sulfate, vinblastine Sulfate, and etoposide). from dose-response curves derived from in vitro or animal 0112. In an additional embodiment, the compositions of model test systems. the disclosure are administered in combination with cytok 0108. As a general proposition, the dosage administered ines. Cytokines that may be administered with the compo to a patient of the antigen-binding polypeptides of the sitions of the disclosure include, but are not limited to, IL-2, present disclosure is typically 0.1 mg/kg to 100 mg/kg of the IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, patient’s body weight, between 0.1 mg/kg and 20 mg/kg of anti-CD40, CD40L, and TNF-C. the patient’s body weight, or 1 mg/kg to 10 mg/kg of the 0113. In additional embodiments, the compositions of the patient’s body weight. Generally, human antibodies have a disclosure are administered in combination with other thera longer half-life within the human body than antibodies from peutic or prophylactic regimens, such as, for example, other species due to the immune response to the foreign radiation therapy. polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the Compositions dosage and frequency of administration of antibodies of the 0114. The present disclosure also provides pharmaceuti disclosure may be reduced by enhancing uptake and tissue cal compositions. Such compositions comprise an effective penetration (e.g., into the brain) of the antibodies by modi amount of an antibody, and an acceptable carrier. In a fications such as, for example, lipidation. specific embodiment, the term "pharmaceutically accept 0109 The methods for treating an infectious or malignant able' means approved by a regulatory agency of the Federal disease, condition or disorder comprising administration of or a state government or listed in the U.S. Pharmacopeia or an antibody, variant, or derivative thereof of the disclosure other generally recognized pharmacopeia for use in animals, are typically tested in vitro, and then in Vivo in an acceptable and more particularly in humans. Further, a “pharmaceuti animal model, for the desired therapeutic or prophylactic cally acceptable carrier will generally be a non-toxic solid, activity, prior to use in humans. Suitable animal models, semisolid or liquid filler, diluent, encapsulating material or including transgenic animals, are well known to those of formulation auxiliary of any type. ordinary skill in the art. For example, in vitro assays to 0115 The term “carrier refers to a diluent, adjuvant, demonstrate the therapeutic utility of antigen-binding poly excipient, or vehicle with which the therapeutic is admin peptide described herein include the effect of an antigen istered. Such pharmaceutical carriers can be sterile liquids, binding polypeptide on a cell line or a patient tissue sample. Such as water and oils, including those of petroleum, animal, The effect of the antigen-binding polypeptide on the cell line vegetable or synthetic origin, such as peanut oil, Soybean oil, and/or tissue sample can be determined utilizing techniques mineral oil, sesame oil and the like. Water is a preferred known to those of skill in the art, such as the assays carrier when the pharmaceutical composition is adminis disclosed elsewhere herein. In accordance with the disclo tered intravenously. Saline solutions and aqueous dextrose sure, in vitro assays which can be used to determine whether and glycerol Solutions can also be employed as liquid administration of a specific antigen-binding polypeptide is carriers, particularly for injectable solutions. Suitable phar US 2016/0280795 A1 Sep. 29, 2016

maceutical excipients include starch, glucose, lactose, 0117 The compositions of the disclosure can be formu Sucrose, gelatin, malt, rice, flour, chalk, silica gel, Sodium lated as neutral or salt forms. Pharmaceutically acceptable Stearate, glycerol monostearate, talc, sodium chloride, dried salts include those formed with anions such as those derived skim milk, glycerol, propylene, glycol, water, ethanol and from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, the like. The composition, if desired, can also contain minor etc., and those formed with cations such as those derived amounts of wetting or emulsifying agents, or pH buffering from Sodium, potassium, ammonium, calcium, ferric agents such as acetates, citrates or phosphates. Antibacterial hydroxides, isopropylamine, triethylamine, 2-ethylamino agents such as benzyl alcohol or methyl parabens; antioxi ethanol, histidine, procaine, etc. dants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents Experimental Examples for the adjustment of tonicity Such as sodium chloride or dextrose are also envisioned. These compositions can take Example 1 the form of Solutions, Suspensions, emulsion, tablets, pills, capsules, powders, Sustained-release formulations and the Expression, Purification and Characterization of an like. The composition can be formulated as a Suppository, Anti-CEA-Fc:Anti-CD16-Fc Antibody with traditional binders and carriers such as triglycerides. 0118. This example tests the expression and purification Oral formulation can include standard carriers such as of an anti-CEA-Fc:anti-CD16-Fc antibody. The antibody pharmaceutical grades of mannitol, lactose, starch, magne has two chains, one with an anti-CEA VHH fragment (SEQ sium Stearate, sodium saccharine, cellulose, magnesium ID NO:1) connected to a Fc fragment (SEQID NO:14) and carbonate, etc. Examples of Suitable pharmaceutical carriers a His6 tag, and another with an anti-CD16 VHH fragment are described in Remington’s Pharmaceutical Sciences by E. (SEQ ID NO: 12) connected to a Fc fragment (SEQ ID W. Martin, incorporated herein by reference. Such compo NO:15) and a Flag tag. These two Fc fragments form a sitions will contain a therapeutically effective amount of the knob-in-the-hole pairing. TABLE 2 Protein Sequences of the Fc Fragments 1. First Fo fragment (Hedge-CH2-CH3) - with T366W modification to form a knob (SEQ ID NO: 14) DKTHTCPPCP APELLGGPSV FLEPPKPKDT LMISRTPEVT CVWWDWSHED PEWKFNWYWD GWEVHNAKTK PREEOYNSTY RVVSVLTVLH ODWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPOVYT LPPSRDELTK NOVSLWCLVK GFYPSDIAVE WESNGOPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWOOG NWFSCSVMHE ALHNHYTOKS LSLSPGK 2. Second Fo fragment (Hedge -CH2-CH3) - with T366S/L368A/Y407W modifications to form a hole (SEQ ID NO: 15) DKTHTCPPCP APELLGGPSW FLFPPKPKDT LMISRTPEWT CWWWDWSHED PEWKFNWYWD GWEVHNAKTK PREEOYNSTY RVVSVLTVLH ODWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPOVYT LPPSRDELTK NOVSLSCAVK GFYPSDIAVE WESNGOPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQOG NVFSCSVMHE ALHNHYTQKS LSLSPGK antigen-binding polypeptide, preferably in purified form, 0119) As shown in this example, about 20% of the together with a suitable amount of carrier so as to provide produced antibody was soluble, which is unexpected as the form for proper administration to the patient. The bispecific antibodies produced from a single cell are typi formulation should suit the mode of administration. The cally not soluble. parental preparation can be enclosed in ampoules, dispos able syringes or multiple dose vials made of glass or plastic. Materials and Methods 0116. In an embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical I0120 Culture A medium: TB medium supplemented with composition adapted for intravenous administration to 2% Glucose, 0.4% Glycerol. human beings. Typically, compositions for intravenous I0121 Culture B medium: TB medium supplemented with administration are solutions in sterile isotonic aqueous buf fer. Where necessary, the composition may also include a 0.4% Glycerol, 0.5 mM IPTG. solubilizing agent and a local anesthetic Such as lignocaine I0122) Lysis buffer 25 mM Tris-HCl, pH 7:250 mM. NaCl; to ease pain at the site of the injection. Generally, the 0.1% Triton-X-100. ingredients are Supplied either separately or mixed together (0123 Terrific Broth: Deionized HO, to 900 mL. Tryp in unit dosage form, for example, as a dry lyophilized tone, 12 g; Yeast extract, 24 g; Glycerol, 4 mL. Shake until powder or water free concentrate in a hermetically sealed the solutes have dissolved and then sterilize by autoclaving container Such as an ampoule or Sachette indicating the for 20 min at 15 psi (1.05 kg/cm) on liquid cycle. Allow the quantity of active agent. Where the composition is to be solution to cool to 60° C. or less, and then add 100 mL of administered by infusion, it can be dispensed with an a sterile solution of 0.17 M KHPO, 0.72 M KHPO. (This infusion bottle containing sterile pharmaceutical grade water solution was made by dissolving 2.31 g of KHPO and or saline. Where the composition is administered by injec 12.54 g of KHPO in 90 mL of HO. After the salts had tion, an ampoule of sterile water for injection or saline can dissolved, adjusted the volume of the solution to 100 mL be provided so that the ingredients may be mixed prior to with HO and sterilize by autoclaving for 20 min at 15 psi administration. 1.05 kg/cm on liquid cycle.) US 2016/0280795 A1 Sep. 29, 2016

(0.124 LB medium: add the following to 800 ml HO: 10 against 3 at least 100 times the sample Volume. Concentra g Bacto-tryptone, 5 g yeast extract, 10 g NaCl, adjust pH to tions of the antibody were determined, and then stored as 7.5 with NaOH, adjust volume to 1 L with dHO, sterilize by aliquots at -20° C. autoclaving. I0138 Western Blot to Detect Bispecific Antibodies 0.125 Reagent and Materials for protein purification: 0.139. Twenty ul of the sample was transferred to a 1 mL binding buffer: 10 mM Tris-HCl: 150 mM NaCl, pH 7.5; tube; 5x loading buffer (5 uL) was added. The sample was elution buffer: 0.1M Glycine, pH 2.7, neutral buffer: 1M Tris then boiled for 5 min. The protein was loaded to 8% pH 9.0: 20% Ethanol. SDS-PAGE, and transferred to PVDF membrane. The 0126 Transformation sample was incubated with 5% fat-free milk powder in 0127. Competent BL21 (DE3) E. coli cells were taken out TBST for 1 hour. of -80° C. and thaw on ice. About 50 ul competent cells were pipetted to a 1.5 mL pre-chilled tube. One LL DNA (O140 Anti-His-HRP and anti-M2(Flag)-HRP antibodies construct encoding both antibody chains (concentration of were used to detect each of the bispecific antibody chains. Washing was carried out with TBST for 3x8 min. Detection DNA: is about 100 ng/uL) was added to the competent cells, solutions was added to the samples before pictures were swirled gently and was incubated on ice for 30 min. taken. 0128. Each tube then received a heat shock each, being placed into a 42°C. water bath for 45 s. The tubes were 0141 Gel Filtration to Determine Whether the Bispecific placed back on ice for 2-3 min. 450 uL media was added to Antibodies were Aggregates each tube, which was then incubated at 37° C., 100 rpm for 0142. Materials used: GF column: Superdex 2003.2/300: 1 h. molecular weight (Mr) of column: 10 000 to 600,000; 0129. Fifty uL of the resulting cells were spread on LB Eluent: 25 mM Tris HCl, pH7.5: 300 mM. NaCl; Flow rate: plates (Ampencilin, 100 ug/ml, Kanamycin, 50 lug/ml. 37 0.05 ml/min, RT. C., and grown for 12-16 h. 0.143 Concentration of the bispecific antibody was 5.4 0130 Expression mg/ml, in 20 ul. For equilibration, each sample was equili 0131) A single colony from plate was picked to 2 mL brated with at least 2 CV of room-temperatured water and TB+antibiotic (Amp+; Kan--; or both Amp+ and Kan--). Two then equilibrated with at least 2CV running buffer. Ten ul of mL cell medium was transferred to 100 mL. Culture A protein standard markers (five protein markers of molecular medium+antibiotic, 300, 220 rpm to OD-1. Cells were weights: 12.4 to 200 kd were used as standards. Each collected by centrifugation and Supernatant was discarded. bispecific antibody sample was loaded and run under the The cells were resuspended in 2 of 100 ml Culture B detection of 280 nm. medium+antibiotic and grown separately at 16O and 25O 0144. Cleaning-in-place (CIP) was conducted by wash for 16 hrs. ing the column with 1 CV of 0.1 M sodium hydroxide or 0.132. One ml cell culture of each time-point was trans alternatively 0.5 M acetic acid at a flow rate of 0.02 ml/min. ferred to eppendorf tubes and was centrifuged at 13,000 The column was immediately rinsed with 4 CV water rpm, 2 min, supernatant discarded. Each tube was added 500 ul PBS (pH7.4) and the resulting pellet was resuspended, followed by at least 4 CV eluent at a flow rate of 0.02 and centrifuged: 4O. 12000 rpm, 30 min. The supernatant ml/min. was transferred to another cold fresh tube, with 100 ul lysis buffer to resuspend the pellet. Results 0.133 Coomassie brilliant blue staining was conducted on the antibodies after run on 10 ul SDS-PAGE (15% separat 0145 Abispecific antibody with two camel VHHs (anti ing gel). Destainning was done with H2O. Another 10 ul was CEA and anti-CD16) was expressed in bacteria. FIG. 3 used for western blot using either anti-His6 or anti-Flag shows Commassiue blue staining of insoluble and soluble antibodies. For the rest of culture, the cells were collected by fractions of the antibody expressed in the bacterial cells. As centrifugation at 4000 rpm, 4° C. 20 mins. The cells were shown in the figure, about 20% of the total antibody was resuspended in 10 ml lysis buffer. The samples were frozen soluble. on dry ice and stored at -80° C. 0146 FIG. 4 shows the staining of each of the two 0134) Purification Procedure antibody chains separately, using antibodies against the His 0135 Collection tubes were prepared by adding 0.1 ml of tag and the Flag tag, respectively. As shown in FIG. 4, the 1M Tris pH 9.0 per ml of each fraction to be collected. The two chains were expressed at similar levels. FIG. 5 further tubes were centrifuged at 12000 rpm, 4° C. for 30 min or shows the staining of each antibody chain alone or when filtered. The samples were dialyzed in 10 mM Tris-HCl, 150 forming the bispecific antibody. mM NaCl, pH 7.5 for 2 hours at 4° C. 0147 The staining and gel filtration showed that the 0.136 Columns were washed with 5 bed volumes of 10 bispecific antibody has a molecular weight of about 80 KDa, mM Tris-HCl, 150 mM. NaCl, pH 7.5. The samples were with each chain of about 40 KDa. applied onto the column, and incubated at 4°C. for 2 hours. 0.148. The yield of the bispecific antibody was about 1-2 The columns were washed with 10 bed volumes of 10 mM mg per Liter cell culture. Such a yield is greater than other Tris-HCl, 150 mM. NaCl, pH 7.5, for four times to remove bispecific antibodies produced from two different batches of contaminant proteins. bacterial cells or mammalian cells to form bispecific anti 0137 The antibody was eluted with 5 bed volumes of 0.1 bodies. The purification of the present disclosure is also MGlycine. Fractions containing the antibody were collected much easier and needs less time and efforts to control the into tubes containing 1M Tris. The samples were dialyzed antibody pairing. US 2016/0280795 A1 Sep. 29, 2016 13

Example 2 0154) At day 1, the cells were thawed in plated in 10 cm dishes. At day 2, 0.25% trypsin was used to digest every cell Preparation of Other Bispecific Antibodies lines. Cells were collected and cell numbers counted. The 0149. This example demonstrates the expression and cells were then diluted to 5x10"/mL. One hundred ul cell purification of two more bispecific antibodies, an anti-CEA suspension was plated (5000 cells per well) on a 96-well Fc:anti-CD3-Fc, an anti-Her2-Fc:anti-CD16-Fc and an anti plate. The samples were then incubated for 6 hrs before Her2-Fc:anti-CD3-Fc bispecific antibodies. The production, adding NK cells or T cells. purification and characterizations methods were the same as used in Example 1. The anti-CEA VHH sequence was SEQ (O155 NK cells or T cells were diluted to 5x10/mL, and ID NO:1. The anti-CD3 VHH sequence was SEQID NO:13. 100 ul cells were plated (50,000 cells per well) on the The anti-Her2 VHH sequence was SEQ ID NO:6. The 96-well plates, which contained the tumor cells. anti-CD16 VHH sequence was SEQ ID NO:12. The Fc 0156 The bispecific antibodies were added to the wells fragments had the same sequences as in Example 1. as Table 2 shows, and the sample was incubated for 48 hrs. 0150 FIG. 6-8 present Western blots images for obtained 20 ul CCK8 reagent was added to every well in the 96-well bispecific antibodies at each stage of purification, for the plates. Data were collected at 1, 2, 3, 4 hrs time points. TABLE 3 Plate Map

SKOV3 HT29 LS174T

2 3 4 5 6 7 8 9 O 1 12 A. umor cells + medium Tumor cells + medium Tumor cells + medium B umor cells + NKST-cells Tumor cells + NKST-cells Tumor cells + NKST-cells C umor cells + NKST-cells + Tumor cells + NKST-cells + Tumor cells + NKST-cells + BISPECIFIC ANTIBODIES BISPECIFIC ANTIBODIES BISPECIFIC ANTIBODIES Ig/ml |g/ml |g/ml D Tumor cells + NKST-cells + Tumor cells + NKST-cells + Tumor cells + NKST-cells + BISPECIFIC ANTIBODIES BISPECIFIC ANTIBODIES BISPECIFIC ANTIBODIES 0 g/ml Olg?ml Olg/ml E. Tumor cells - BISPECIFIC Tumor cells - BISPECIFIC Tumor cells - BISPECIFIC ANTIBODIES 1 g/ml ANTIBODIES 1 g/ml ANTIBODIES 1 g/ml F Tumor cells + NKST-cells + Tumor cells + NKST-cells + Tumor cells + NKST-cells + BISPECIFIC ANTIBODIES BISPECIFIC ANTIBODIES BISPECIFIC ANTIBODIES Ig/ml |g/ml |g/ml G. Tumor cells - BISPECIFIC Tumor cells - BISPECIFIC Tumor cells - BISPECIFIC ANTIBODIES 10 pg/ml ANTIBODIES 10 pg/ml ANTIBODIES 10 pg/ml H Tumor cells + NKST-cells + Tumor cells + NKST-cells + Tumor cells + NKST-cells + BISPECIFIC ANTIBODIES BISPECIFIC ANTIBODIES BISPECIFIC ANTIBODIES 0 g/ml Olg?ml Olg/ml anti-CEA-Fc:anti-CD3-Fc, the anti-Her2-Fc:anti-CD16-Fc Results and the anti-Her2-Fc:anti-CD3-Fc bispecific antibodies, 0157. The cytotoxicity assays used monospecific anti respectively. It was also observed that about 20% of the bodies as controls and included both tumor cells that expressed bispecific antibodies were soluble. The yield of expressed the targeted tumor antigen and those that did not each of these antibodies was similar to the one tested in express the targeted antigen. The bispecific antibodies Example 1. abilities to destroy tumor cells were pronounced. 0151. The above two examples, therefore, demonstrate 0158 Compared to the monospecific antibodies, the anti that soluble products of the bispecific antibodies of the CEA-Fc:anti-CD16-Fc bispecific antibody was about twice present discourse can be efficiently prepared from bacterial as effective at CEA-positive cells (FIG.9, right panel) in the cells. Further, such prepared antibodies were stable. presence of NK cells. Such a difference was not observed for CEA-positive cells (FIG.9, left panel). Example 3 0159. The advantage of the anti-CEA-Fc:anti-CD16-Fc bispecific antibody was even more dramatic over the corre In Vitro Cell-Based Assays sponding monospecific antibodies. Only about 30% of CEA 0152 This example demonstrates that the bispecific anti positive tumor cells (in the presence of T cells) survived bodies of the present disclosure are effective in targeting when treated with the bispecific antibody, as compared to the tumor cells in an in vitro cytotoxicity assay. The data, monospecific counterpart (FIG. 10). Likewise, the anti therefore, shows that such antibodies can be suitably used CEA-Fc:anti-CD3-Fc bispecific antibody exhibited greatly clinically to treat cancer. improved cytotoxicity at Her2-positive tumor cells in the presence of NK cells (FIG. 11). Materials and Methods (0160 FIG. 12 further shows that the cytotoxicity of these bispecific antibodies were dose-dependent (compare the 0153 Cell lines used included HT29 (a CEA positive cell fourth bar and the sixth bar in the right panel) and immune line), SKOV3 (a CEA negative cell line), LS174T (a CEA cell-dependent (compare the fourth bar and the fifth bar in positive cell line), and human NK cells. the right panel). US 2016/0280795 A1 Sep. 29, 2016

0161 To the best knowledge of the inventors, these data limitation removing any subject matter from the genus, show that the potency of these bispecific antibodies is regardless of whether or not the excised material is specifi similar to other bispecific antibodies in the literature. Given cally recited herein. the much improved stability and production efficiency of 0164. In addition, where features or aspects of the dis these bispecific antibodies, especially the ability to be pro closure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also duced from bacterial cells, these bispecific antibodies show thereby described in terms of any individual member or great potential for further development and clinical use for Subgroup of members of the Markush group. Cancer treatment. 0.165 All publications, patent applications, patents, and 0162. It should be understood that although the present other references mentioned herein are expressly incorpo disclosure has been specifically disclosed by preferred rated by reference in their entirety, to the same extent as if embodiments and optional features, modification, improve each were incorporated by reference individually. In case of ment and variation of the disclosures embodied therein conflict, the present specification, including definitions, will control. herein disclosed may be resorted to by those skilled in the 0166 The disclosures illustratively described herein may art, and that Such modifications, improvements and varia suitably be practiced in the absence of any element or tions are considered to be within the scope of this disclosure. elements, limitation or limitations, not specifically disclosed The materials, methods, and examples provided here are herein. Thus, for example, the terms “comprising,” “includ representative of preferred embodiments, are exemplary, ing.” containing, etc. shall be read expansively and without and are not intended as limitations on the scope of the limitation. Additionally, the terms and expressions disclosure. employed herein have been used as terms of description and 0163 The disclosure has been described broadly and not of limitation, and there is no intention in the use of Such generically herein. Each of the narrower species and Sub terms and expressions of excluding any equivalents of the generic groupings falling within the generic disclosure also features shown and described or portions thereof, but it is form part of the disclosure. This includes the generic recognized that various modifications are possible within the description of the disclosure with a proviso or negative Scope of the disclosure claimed.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 15

<21 Os SEQ ID NO 1 &211s LENGTH: 12 O 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Anti- CEA. WHH

<4 OOs SEQUENCE: 1 Glu Val Glin Leu Val Glu Ser Gly Gly Gly Phe Val Glin Ala Gly Glu 1. 5 1O 15

Ser Lieu. Thir Lieu Ser Cys Thr Ser Ser Thr Lieu. Thr Phe Thir Pro 2O 25 3 O

Arg Met Ala Trp Tyr Arg Glin Ala Pro Gly Lys Glin Arg Asp Lell Wall 35 4 O 45

Ala Asp Ile Ser Ser Gly Asp Gly Arg Thir Thr Asn Tyr Ala Asp Phe SO 55 60

Ala Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ile Llys ASn Thir Wall 65 70 7s

Phe Lieu. Arg Met Thr Asn Lieu Lys Pro Glu Asp Thir Ala Wall Tyr 85 90 95

Cys Asn Thr Phe Val Ser Phe Val Gly Ile Ala Arg Ser Trp Gly Glin 1OO 105 110 Gly Thr Glin Val Thr Val Ser Ser 115 12O

<21 Os SEQ ID NO 2 &211s LENGTH: 102 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: OTHER INFORMATION: Anti-CD16 WHH US 2016/0280795 A1 Sep. 29, 2016 15

- Continued

<4 OOs, SEQUENCE: 2 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Ser Phe Pro Gly Ser Ile Phe Ser Lieu. Thr 2O 25 3O Met Gly Trp Tyr Arg Glin Ala Pro Gly Lys Glu Arg Glu Lieu Val Thr 35 4 O 45 Ser Ala Thr Pro Gly Gly Asp Thr Asn Tyr Ala Asp Phe Wall Lys Gly SO 55 6 O Arg Phe Thir Ile Ser Arg Asp Asn Ala Arg Ser Ile Ile Tyr Lieu. Glin 65 70 7s 8O Met Asin Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 85 90 95 Arg Thr Arg Asn Trp Gly 1OO

<210s, SEQ ID NO 3 &211s LENGTH: 102 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Anti-CD16 WHH

<4 OOs, SEQUENCE: 3 Glu Val Glin Lieu Val Glu Ser Gly Gly Glu Lieu Val Glin Ala Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Lieu. Thir Phe Ser Ser Tyr 2O 25 3O Asn Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val 35 4 O 45 Ala Ser Ile Thr Trp Ser Gly Arg Asp Thr Phe Tyr Ala Asp Ser Val SO 55 6 O Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 7s 8O Lieu Gln Met Ser Ser Lieu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn. Pro Trp Pro 1OO

<210s, SEQ ID NO 4 &211s LENGTH: 102 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Anti-CD16 WHH

<4 OOs, SEQUENCE: 4 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Glu 1. 5 1O 15

Ser Lieu. Thir Lieu Ser Cys Val Val Ala Gly Ser Ile Phe Ser Phe Ala 2O 25 3O

Met Ser Trp Tyr Arg Glin Ala Pro Gly Lys Glu Arg Glu Lieu Val Ala 35 4 O 45 Arg Ile Gly Ser Asp Asp Arg Val Thir Tyr Ala Asp Ser Val Lys Gly SO 55 6 O US 2016/0280795 A1 Sep. 29, 2016 16

- Continued Arg Phe Thir Ile Ser Arg Asp Asn. Ile Lys Arg Thr Ala Gly Lieu. Glin 65 70 7s 8O Met Asn. Ser Lieu Lys Pro Glu Asp Thir Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Glin Thr Asp Lieu. Arg Asp 1OO

<210s, SEQ ID NO 5 &211s LENGTH: 102 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Anti-CD16 WHH

<4 OOs, SEQUENCE: 5 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Thir Lieu Ser Cys Val Ala Ala Gly Ser Ile Phe Thr Phe Ala 2O 25 3O Met Ser Trp Tyr Arg Glin Ala Pro Arg Lys Glu Arg Glu Lieu Val Ala 35 4 O 45 Arg Ile Gly Thr Asp Asp Glu Thir Met Tyr Lys Asp Ser Val Lys Gly SO 55 6 O Arg Phe Thir Ile Ser Arg Asp Asn. Wall Lys Arg Thr Ala Gly Lieu. Glin 65 70 7s 8O Met Asn. Asn Lieu Lys Pro Glu Asp Thir Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Arg Thr Asp Tyr Arg Asp 1OO

<210s, SEQ ID NO 6 &211s LENGTH: 123 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Anti-Her2 scAb

<4 OOs, SEQUENCE: 6 Glin Val Glin Lieu Val Glin Ser Gly Gly Gly Lieu Val Glin Ala Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 2O 25 3O Ala Met Ala Trp Phe Arg Glin Ala Pro Gly Lys Glu Arg Glu Phe Val 35 4 O 45 Ala Ala Ile Ser Trp Ser Gly Ala Asn. Ile Tyr Val Ala Asp Ser Val SO 55 6 O Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asp Thr Val Tyr 65 70 7s 8O

Lieu Gln Met Asn Ser Lieu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Wall Lys Lieu. Gly Phe Ala Pro Val Glu Glu Arg Glin Tyr Asp Tyr 1OO 105 11 O

Trp Gly Glin Gly Thr Glin Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 7 US 2016/0280795 A1 Sep. 29, 2016 17

- Continued

&211s LENGTH: 128 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Anti-EGFR1 WHH

<4 OO > SEQUENCE: 7 Met Ala Glu Val Glin Glin Ala Ser Gly Gly Gly Lieu Val Glin Ala Gly 1. 5 1O 15 Gly Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Arg Thr Glu Thir Thr 2O 25 3O Ser Ala Ile Ala Trp Phe Arg Glin Ala Pro Gly Lys Glu Arg Glu Phe

Val Ala Glin Ile Ser Ala Ser Gly Lieu. Gly Ile Asn Tyr Ser Gly Thr SO 55 6 O Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Ala Asp Llys Thir Thr Val 65 70 7s 8O Tyr Lieu Gln Met Asn Ser Lieu. Thr Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Gly Phe His Tyr Ile Ala Ala Ile Arg Arg Thir Thr Asp 1OO 105 11 O

Phe His Ph. Trp Gly Pro Gly Thr Lieu Val Thr Val Ser Ser Gly Arg 1. 5 12 O 125

<210 SEQ ID NO 8 &211s LENGTH: 121 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Anti-F4+ETEC bacteria

<4 OOs, SEQUENCE: 8 Glin Val Glin Lieu. Glin Glu Ser Gly Gly Gly Lieu Val Glin Ala Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Glu Ala Ser Gly Asn Val Asp Arg Ile Asp 2O 25 3O Ala Met Gly Trp Phe Arg Glin Ala Pro Gly Lys Glin Arg Glu Phe Val 35 4 O 45 Gly Tyr Ile Ser Glu Gly Gly Ile Lieu. Asn Tyr Gly Asp Phe Wall Lys SO 55 6 O Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Lieu. 65 70 7s 8O Gln Met Ser Asn Lieu Lys Ser Glu Asp Thr Gly Val Tyr Phe Cys Ala 85 90 95 Ala Ser His Trp Gly. Thir Lieu. Lieu. Ile Lys Gly Ile Glu. His Trp Gly 1OO 105 11 O

Lys Gly Thr Glin Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 9 &211s LENGTH: 127 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Anti-PS2- 8 WHH

<4 OOs, SEQUENCE: 9 US 2016/0280795 A1 Sep. 29, 2016 18

- Continued Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Ala Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Arg Asp 2O 25 3O Ala Met Gly Trp Phe Arg Glin Ala Pro Gly Lys Glu Arg Asp Val Val 35 4 O 45 Ala Ala Ile Asn Lieu. Asn Gly Gly Arg Thr Tyr Ser Ala Asp Ser Val SO 55 6 O Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr 65 70 7s 8O Lieu Gln Met Ser Asn Lieu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Glu Gly Asp Val Gly Lieu Val Ser Tyr Lys Arg Ser Ser 1OO 105 11 O Asn Tyr Pro Tyr Trp Gly Glin Gly Thr Glin Val Thr Val Ser Ser 115 12 O 125

<210s, SEQ ID NO 10 &211s LENGTH: 121 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Anti-Huntavirus WHH

<4 OOs, SEQUENCE: 10 Met Ala Glu Val Glin Lieu. Glin Ala Ser Gly Gly Gly Lieu Val Glin Ala 1. 5 1O 15 Gly Gly Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Arg Thir Ser Ser 2O 25 3O Met Tyr Ser Met Val Trp Phe Arg Glin Ala Pro Gly Lys Glu Arg Glu 35 4 O 45 Phe Val Ala Gly Ile Ile Trp Thr Ser Ser Lieu. Thr Tyr Tyr Ala Asp SO 55 6 O Ser Lieu Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 7s 8O Val Tyr Lieu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Ala Asp Thr Lys Thr Gly Gly Gly Gly Tyr Glu Tyr Trp 1OO 105 11 O Gly Glin Val Thr Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 11 &211s LENGTH: 119 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Anti-Huntavirus WHH

<4 OOs, SEQUENCE: 11 Met Ala Glu Val Glin Lieu. Glin Ala Ser Gly Gly Gly Lieu Val Glin Pro 1. 5 1O 15

Gly Gly Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser 2O 25 3O

Ser Asp Wal Met Gly Trp Phe Arg Glin Ala Pro Gly Lys Glu Arg Glu 35 4 O 45 US 2016/0280795 A1 Sep. 29, 2016 19

- Continued

Lieu Val Ala Phe Ile Thr Asp Asp Gly Gly Thr Asn Tyr Ala Asp Ser SO 55 6 O Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Glu Asn Thr Val 65 70 7s 8O Ser Leu Gln Met Asn Ser Lieu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Asn Ala Arg Tyr Tyr Ser Gly Gly Tyr Arg Asn Tyr Trp Gly Glin 1OO 105 11 O

Wall. Thir Wall. Thir Wal Ser Ser 115

<210s, SEQ ID NO 12 &211s LENGTH: 115 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Anti-CD16 WHH

<4 OOs, SEQUENCE: 12 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Ser Phe Pro Gly Ser Ile Phe Ser Lieu. Thr 2O 25 3O Met Gly Trp Tyr Arg Glin Ala Pro Gly Lys Glu Arg Glu Lieu Val Thr 35 4 O 45 Ser Ala Thr Pro Gly Gly Asp Thr Asn Tyr Ala Asp Phe Wall Lys Gly SO 55 6 O Arg Phe Thir Ile Ser Arg Asp Asn Ala Arg Ser Ile Ile Tyr Lieu. Glin 65 70 7s 8O Met Asin Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 85 90 95 Arg Thr Arg Asn Trp Gly Thr Val Trp Gly Glin Gly Thr Glin Val Thr 1OO 105 11 O

Wall Ser Ser 115

<210s, SEQ ID NO 13 &211s LENGTH: 129 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Anti-CD3 WHH

<4 OOs, SEQUENCE: 13 Glin Val Glin Lieu. Glin Glu Ser Gly Gly Gly Lieu Val Glin Ala Gly Gly 1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr 2O 25 3O His Met Gly Trp Phe Arg Glin Ala Pro Gly Lys Glu Arg Glu Lieu Val 35 4 O 45

Ala Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Thr Asp Ser Val SO 55 6 O

Lys Gly Arg Phe Thir Ile Ser Arg Asn. Asn Ala Lys Asn. Thir Met Ser 65 70 7s 8O

Lieu Gln Met Ser Asn Lieu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys US 2016/0280795 A1 Sep. 29, 2016 20

- Continued

85 90 95 Thir Thr Pro Thr Glu Lys Gly Ser Ser Ile Asp Tyr Trp Gly Glin Gly 1OO 105 11 O Thr Glin Val Thr Val Ser Ser Gly Arg Tyr Pro Tyr Asp Val Pro Asp 115 12 O 125 Tyr

<210s, SEQ ID NO 14 &211s LENGTH: 227 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 14 Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly 1. 5 1O 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met 2O 25 3O Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 4 O 45 Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val SO 55 6 O His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 8O Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile 1OO 105 11 O Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val 115 12 O 125 Tyr Thr Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val Ser 13 O 135 14 O Lieu. Trp Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp. Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 1.65 17O 17s Val Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val 18O 185 19 O Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met 195 2 OO 2O5 His Glu Ala Lieu. His Asn His Tyr Thr Glin Llys Ser Lieu. Ser Lieu. Ser 21 O 215 22O Pro Gly Lys 225

<210s, SEQ ID NO 15 &211s LENGTH: 227 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OOs, SEQUENCE: 15 Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly US 2016/0280795 A1 Sep. 29, 2016 21

- Continued

15

Gly Pro Ser Wall Phe Lell Phe Pro Pro Lys Pro Asp Thir Luell Met 2O 25

Ile Ser Arg Thir Pro Glu Wall Thr Wall Wall Wall Asp Wall Ser His 35 4 O 45

Glu Asp Pro Glu Wall Lys Phe Asn Trp Tyr Wall Asp Gly Wall Glu Wall SO 55 6 O

His Asn Ala Thir Lys Pro Arg Glu Glu Glin Tyr Asn Ser Thr Tyr 65 70

Arg Wall Wall Ser Wall Lell Thir Wall Luell His Glin Asp Trp Luell Asn Gly 85 90 95

Glu Tyr Lys Lys Wall Ser Asn Lys Ala Lieu. Pro Ala Pro Ile 105 11 O

Glu Thir Ile Ser Ala Lys Gly Gln Pro Arg Glu Pro Glin Wall 115 12 O 125

Thir Leu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Wall Ser 13 O 135 14 O

Luell Ser Ala Wall Lys Gly Phe Pro Ser Asp Ile Ala Wall Glu 145 150 155 160

Trp Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Thir Thir Pro Pro 1.65 17s

Wall Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu Wal Ser Luell Thir Wall 18O 185 19 O

Asp Ser Arg Trp Glin Glin Gly Asn. Wall Phe Ser Cys Ser Wal Met 195 2O5

His Glu Ala Lieu. His Asn His Thr Gin Lys Ser Luell Ser Luell Ser 21 O 215 22O

Pro Gly 225

1. A bivalent bispecific antibody comprising (a) a first 7. The antibody of claim 1, wherein the second VHH polypeptide comprising a first Fc fragment and a first fragment has specificity to a mammalian T cell or a mam single-domain antigen-binding (VHH) fragment and (b) a malian NK cell. second polypeptide comprising a second Fc fragment and a 8. The antibody of claim 7, wherein the second VHH second VHH fragment, wherein the first VHH fragment has fragment has specificity to an antigen selected from the specificity to a tumor cell and the second VHH fragment has group consisting of CD3, CD16, CD19, CD28 and CD64. specificity to an immune cell. 9. The antibody of claim 7, wherein the antigen is CD16 2. The antibody of claim 1, containing no antibody light or CD3. chains. 10. The antibody of claim 7, wherein the second VHH 3. The antibody of claim 1, wherein the first VHH fragment comprises the amino acid sequence of one of SEQ fragment has specificity to a tumor antigen. ID NO:2-5 or 12-13, or an amino acid having at least about 4. The antibody of claim 3, wherein the tumor antigen is 95% sequence identity thereto. selected from the group consisting of CEA, EGFR. Her2. 11. The antibody of claim 1, wherein the first VHH EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, fragment and/or the second VHH fragment does not contain gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding pro Val, Gly, Leu, and Trp residues at Kabat positions 37, 44, 45, tein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, CVB3, and 47, respectively. c5|B1, ERBB2, ERBB3, MET, IGFIR, EPHA3, TRAILR1, 12. The antibody of claim 1, wherein each of the Fc TRAILR2, RANKL, FAP and Tenascin. fragments comprises a CH2 domain and a CH3 domain. 5. The antibody of claim 3, wherein the tumor antigen is 13. The antibody of claim 1, wherein the first Fc fragment CEA or Her2. and the second Fc fragment each comprises a different 6. The antibody of claim 1, wherein the first VHH amino acid sequence selected from SEQID NO:14 or SEQ fragment comprises the amino acid sequence of SEQ ID ID NO:15. NO:1 or 6, or an amino acid having at least about 95% 14. A polypeptide comprising the amino acid sequence of sequence identity thereto. SEQID NO:13 or having at least 95% sequence identity to US 2016/0280795 A1 Sep. 29, 2016 22

SEQID NO:13, wherein the polypeptide has binding speci ficity to a mammalian CD3 protein. 15. The polypeptide of claim 1, wherein the mammalian CD3 protein is a human CD3 protein. 16. A bivalent antibody comprising (a) a first polypeptide comprising a first Fc fragment and a first single-domain antigen-binding (VHH) fragment and (b) a second polypep tide comprising a second Fc fragment and a second VHH fragment.