WO2015197582A1 - Monomeric multispecific antigen binding proteins - Google Patents

Abstract

Monomeric multispecific proteins that bind two target antigens and have an FcRn-binding Fc domain that does not undergo CH3-CH3 dimerization are provided. The proteins have advantages in production and in the treatment of disease, notably cancer or infectious disease.

Description

MONOMERIC MULTISPECIFIC ANTIGEN BINDING PROTEINS

CROSS-REFERENCE To RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos. 62/017,898, filed June 27, 2014, which is incorporated herein by reference in its entirety; including any drawings.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled "BISP1_PCT_ST25", created June 19, 2015, which is 61 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Multispecific proteins that bind and specifically redirect NK cells to lyse a target cell of interest are provided. The proteins formats have utility in the treatment of disease.

BACKGROUND

Bispecific antibodies binding two different epitopes offer opportunities for increasing specificity, broadening potency, and utilizing novel mechanisms of action that cannot be achieved with a traditional monoclonal antibody. A variety of formats for bispecific antibodies that bind to two targets simultaneously have been reported. Cross-linking two different receptors using a bispecific antibody to inhibit a signaling pathway has shown utility in a number of applications (see, e.g., Jackman, et al., (2010) J. Biol. Chem. 285:20850-20859). Bispecific antibodies have also been used to neutralize two different receptors. In other approaches, bispecific antibodies have been used to recruit immune effector cells, where T- cell activation is achieved in proximity to tumor cells by the bispecific antibody which binds receptors simultaneously on the two different cell types (see Baeuerle, P. A., et al, (2009) Cancer Res 69(12):4941 -4). Most such approaches involve bispecific antibodies that link the CD3 complex on T cells to a tumor-associated antigen. The most well-studied bispecific antibody formats are "BiTe" antibodies and "DART" antibodies which do not comprise Fc domains. However these antibodies are known to be difficult to produce, require length cell development, have low productions yields and/or cannot be produced (based on published literature) as a homogenous protein composition. In another example, a bispecific antibody having one arm which bound FcyRIII and another which bound to the HER2 receptor was developed for therapy of ovarian and breast tumors that overexpress the HER2 antigen.

However, despite the existence of a variety of formats for bispecific antibodies, there is therefore a need in the art for proteins with new and well-defined mechanisms of action that can bind two or more biological targets and which have improved pharmaceutical properties.

SUMMARY OF THE INVENTION

The present invention arises from the discovery of a functional multispecific antibody that permits a wide range of antibody variable regions to be readily used, having advantages in manufacturing by being adapted to standard recombinant production techniques, and having improved in vivo pharmacological properties.

The antibodies are particularly adapted to bind a first antigen on a target cell to be eliminated and a second antigen on an immune effector cell (e.g. an NK cell and/or a T cell), where the effector cells are directed to the target cell, e.g. a cancer cell. The multispecific antibody retains FcRn binding and can be designed to lack FcyR binding such that the multispecific antibody has attractive in vivo pharmacodynamics yet is selective for the particular effector cells of interest, thereby reducing toxicity mediated by immune cells other than those expressing the particular effector cell surface antigen to which the antibody binds. The multispecific polypeptide is capable, for example, of directing antigen-of-interest- expressing effector cells to lyse a target cell expressing a target antigen, e.g. cancer antigen, viral antigen, etc. The multispecific antibody is particularly effective when binding both effector cell surface protein and a second antigen (an antigen expressed by a target cell) in monovalent fashion.

In one embodiment, provided is a monomeric multispecific polypeptide that binds a first and a second antigen, comprising: a first antigen binding domain (ABD-i) that specifically binds to a first antigen of interest, a second antigen binding domain (ABD₂) that specifically binds a second antigen of interest, and at least a portion of a human Fc domain that does not undergo interchain CH3-CH3 dimerization (does not form multimers via interactions with CH3 domains of other Fc-containing proteins). In one embodiment, the polypeptide comprises an Fc domain comprising a CH3 domain with an amino acid mutation to prevent CH3-CH3 dimerization. In one embodiment, the Fc domain is interposed between the first and second antigen binding domains. In one embodiment, the first and second antigen binding domains each comprise an immunoglobulin variable heavy domain and a variable light domain. In one embodiment, the first and second antigen binding domains are each an scFv.

The monomeric Fc domain can retain at least partial binding to the human neonatal Fc receptor (FcRn), yet not substantially bind human CD16 and/or other human Fey receptors. Consequently, the bispecific protein will not induce Fcy-mediated (e.g. CD16- mediated) target cell lysis, nor will it bind substantially to inhibitory Fey receptors. Accordingly, in one embodiment, provided is a monomeric multispecific polypeptide that binds a first and a second antigen in monovalent fashion, comprising: (a) a first antigen binding domain (ABD-i) that binds to a first antigen of interest; (b) a second antigen binding domain (ABD₂) that binds a second antigen of interest; and (c) at least a portion of a human Fc domain, wherein the multispecific polypeptide (and/or its Fc domain) is capable of binding to human neonatal Fc receptor (FcRn). In one embodiment, the monomeric multispecific polypeptide does not form a dimer with another Fc-derived polypeptide (e.g. does not form a homodimer with another identical polypeptide). In one embodiment, the Fc domain comprises a CH3 domain with an amino acid mutation to prevent CH3-CH3 dimerization. Optionally, the multispecific polypeptide (and/or its Fc domain) has decreased binding to a human Fey receptor, e.g., compared to a full length wild type human lgG1 antibody. In one embodiment, the Fc domain is interposed between the first and second antigen binding domains.

In one embodiment, one of the antigens of interest is an activating receptor present on an effector cell, the other is a target cell antigen (e.g. a tumor antigen, a viral antigen, a microbial antigen), and the multispecific protein is bound by FcRn and has decreased or substantially lack FcyR binding, and the multi-specific protein can, in the presence of the effector cells targeted and the target cells (e.g. tumor cells), induce signaling in and/or activation of the effector cells through the effector cell polypeptide (the protein acts as an agonist), thereby promoting activation of the effector cells and/or lysis of target cells in a directed manner. Notably, the multi-specific proteins can direct an immune effector response (cytotoxic response) toward a target cell that is substantially limited to the effector cells of interest (the effector cell receptor-expressing cells), and without activating FcyR-mediated toxicity or inhibitor FcyR-mediated inhibition.

In one aspect of any embodiment herein, a multi-specific protein that binds an activating receptor on an effector cells can for example be characterized by:

(a) ability to activate effector cells that express the activating receptor, when incubated with such effector cells in the presence of target cells; and/or (b) lack of ability to activate such effector cells when incubated with such effector cells, in the absence of target cells. Optionally, the effector cells are purified NK or T cells.

In one aspect of any embodiment herein, a multi-specific protein described herein can for example be characterized by:

(a) ability to induce effector cells that express the activating receptor to lyse target cells, when incubated such effector cells in the presence of target cells; and/or

(b) lack of ability to activate such effector cells when incubated with such effector cells, in the absence of target cells.

In one aspect of any embodiment herein, a multi-specific protein described herein can for example be characterized by:

(a) ability to induce effector cells that express the activating receptor to lyse target cells, when incubated such effector cells in the presence of target cells; and/or

(b) lack of ability to activate effector cells that express CD16 but do not express the activating receptor, when incubated with such effector cells in the presence of target cells.

In one embodiment, provided is a monomeric multispecific polypeptide that binds a first and a second antigen, comprising: (a) a first antigen binding domain (ABD-i) that binds to a first antigen of interest, (b) at least a portion of a human Fc domain, wherein the Fc domain is operably linked to the C-terminus of the first antigen binding domain, and (c) a second antigen binding domain (ABD₂) that binds a second antigen of interest operably linked to the C-terminus of the Fc domain, wherein the multispecific polypeptide (and/or its Fc domain) is capable of binding to human neonatal Fc receptor (FcRn). In one embodiment, the monomeric multispecific polypeptide does not form a dimer with another Fc-derived polypeptide (e.g. does not form a homodimer with another identical polypeptide). In one embodiment, the Fc domain comprises a CH3 domain having one or more amino acid mutations (e.g. substitutions) in the CH3 dimer interface to prevent formation of dimers. Optionally, the multispecific polypeptide (and/or its Fc domain) has decreased binding to a human Fey receptor, e.g., compared to a full length wild type human lgG1 antibody. The polypeptide can have a domain arrangement:

(ABD₁) - CH2 - CH3 - (ABD₂)

Optionally, the polypeptide further comprises linking amino acids between the aforementioned domains. In one embodiment, a CH1 domain or fragment thereof, a hinge region or fragment thereof, and/or a linker peptide can be placed between ABD-i and CH2. In one embodiment, a linker peptide can be placed between CH3 and ABD₂. In one embodiment, provided in a monomeric polypeptide comprising two antigen binding domains (ABDs) capable respectively of specifically binding a first and second antigen of interest, a human CH2 domain and a human CH3 domain, the polypeptide having a domain arrangement as follows:

(ABD₁) - CH2 - CH3 - (ABD₂)

or

(scFv - CH2 - CH3 - (scFv₂)

wherein the CH3 domain comprises an amino acid mutation to prevent CH3-CH3 dimerization.

In one aspect of any embodiment herein, the multispecific polypeptide is a bispecific antibody.

In one aspect of any embodiment herein, ABDi and/or ABD₂ each independently comprise a scFv, an affibody, a V_H domain, a V_L domain, a single domain antibody (nanobody) such as a V-NAR domain or a V_HH domain. Optionally, ABDi and ABD₂ each comprise a scFv, an affibody, a V_H domain, a V_L domain, or a single domain antibody (nanobody) such as a V-NAR domain or a V_HH domain. Optionally, each ABDi and ABD₂ are each a human or humanized scFv, an affibody, a V_H domain, a V_L domain, a single domain antibody (nanobody) such as a V-NAR domain or a V_HH domain.

In one aspect of any embodiment herein, the polypeptide may (or may not) further comprise additional protein domains at the C- and/or N-terminus, e.g. a further ABD, variable domain, scFv, etc.

In one embodiment, the polypeptide (and/or its Fc domain) has decreased binding to a human Fey receptor (e.g. CD16, CD32A, CD32B and/or CD64), e.g., compared to a full length wild type human lgG1 antibody. In one embodiment, the polypeptide has decreased (e.g. partial or complete loss of) antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP), FcR- mediated cellular activation (e.g. cytokine release through FcR cross-linking), and/or FcR- mediated platelet activation/depletion, as mediated by immune effector cells that do not express an antigen of interest bound by the antigen binding domains of the polypeptide (i.e. in the absence of cells that express an antigen of interest bound by the antigen binding domains), compared, e.g., to the same polypeptide having a wild-type Fc domain of human lgG1 isotype. In one embodiment, the first antigen binding domain comprises an antibody heavy chain variable domain and a light chain variable domain, optionally the first antigen binding domain comprises an scFv.

In one embodiment, the second antigen binding domain comprises an antibody heavy chain variable domain and a light chain variable domain, optionally the first antigen binding domain comprises an scFv.

In one embodiment, the Fc domain comprises at least a portion of a CH2 domain and at least a portion of a CH3 domain.

In one embodiment, the CH3 domain does not form a CH3-CH3 dimer with another Fc-derived polypeptide (e.g. does not form a dimer with another identical polypeptide). In one embodiment, the CH3 domain comprises amino acid mutations (e.g. substitutions) in the CH3 dimer interface to prevent formation of dimers.

In one embodiment, the CH2 domain is a wild-type CH2 domain. In another embodiment, a CH2 domain comprises an amino acid modification that abolishes binding to a human Fey receptor. In one embodiment, a CH3 domain comprises an amino acid modification, compared to a wild-type CH3 domain. In one embodiment, the multispecific polypeptide lacks N-linked glycosylation or has modified N-linked glycosylation. In one embodiment, the multispecific polypeptide comprises an N297X mutation, wherein X is any amino acid other than asparagine.

In one aspect of any of the embodiments herein, the bispecific polypeptide has a great binding affinity (monovalent) for a cancer antigen (or a viral or bacterial antigen) than for an antigen expressed by an immune effector cell. Such antibodies will provide for advantageous pharmacological properties. In one aspect of any of the embodiments of the invention, the polypeptide has a Kd for binding (monovalent) to an antigen expressed by immune effector cell of less than 10^"7 M, preferably less than 10^"8 M, or preferably less than 10^"9 M for binding to the antigen expressed by an immune effector cell; optionally the polypeptide has a Kd for binding (monovalent) to a cancer, viral or bacterial antigen that is less than (i.e. has better binding affinity than) the Kd for binding (monovalent) to the antigen expressed by immune effector cell.

In one embodiment of any of the polypeptides herein, the multispecific polypeptide is capable of directing effector cells (e.g. a T cell, an NK cell) expressing one of first or second antigen of interest to lyse a target cell expressing the other of said first of second antigen of interest (e.g. a cancer cell). Any of the methods can further be characterized as comprising any step described in the application, including notably in the "Detailed Description of the Invention"). The invention further relates to methods of screening, selecting and/or producing an antibody obtainable by any of present methods. The disclosure further relates to pharmaceutical or diagnostic formulations of the antibodies of the present invention. The disclosure further relates to methods of using antibodies in methods of treatment or diagnosis.

These and additional advantageous aspects and features of the invention may be further described elsewhere herein. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows two examples of multispecific polypeptides.

Figure 2 shows a schematic of an anti-CD19-Fcmono-Anti-CD3. The star in the CH2 domain indicates an optional N297S mutation to abolish remaining FcRy binding.

Figure 3 shows a schematic of an anti-CD19-Anti-CD3-Fcmono. For the scFv tandem construct, the Anti-CD3 VK domain (C-terminal) was linked to the CH2 domain (N-terminal) using a linker peptide (RTVA) that mimics the regular VK-CK elbow junction. Figure 4 shows that Anti-CD19-Fcmono-Anti-CD3 protein binds to the CD3 cell lines (HUT78 and JURKAT cell lines) (Figure 4A) and the CD19 cell line (B221 cell line) (see Figure 4B), but not to the CHO cell line used as a negative control (Figure 6B).

Figure 5 shows that the Anti-CD19-Fcmono-Anti-CD3 protein and the Anti-CD19-

Anti-CD3-lgG1 -Fcmono bind to the CD3 cell lines (JURKAT cell lines), while the Anti-CD19- lgG1 -Fcmono does not. Each of the Anti-CD19-Fcmono-Anti-CD3 protein, Anti-CD19-Anti- CD3-lgG1 -Fcmono and Anti-CD19-lgG1 -Fcmono bind to the CD19 cell lines (B221 cell lines).

Figure 6 shows that Anti-CD19-Fcmono-Anti-CD3 does not cause T/B cell aggregation in the presence of either B221 (CD19) or JURKAT (CD3) cell lines, but it does cause aggregation of cells when both B221 and JURKAT cells are co-incubated.

Figure 7 shows affinity for human FcRn using SPR, comparing Anti-CD19-Fcmono- Anti-CD3 in comparison to a chimeric full length antibody having human lgG1 constant regions. The Anti-CD19-Fcmono-Anti-CD3 retained FcRn binding with a model suggesting 1 :1 ratio (1 FcRn for each monomeric Fc).

Figure 8 shows Anti-CD19-Fcmono-Anti-CD3 retains binding to FcRn, with a 1 :1 ratio (1 FcRn for each monomeric Fc) (KD = 194 nM), in comparison to a chimeric full length antibody having human lgG1 constant regions (KD = 15.4 nM) which binds to FcRn with a 2:1 ratio (2 FcRn for each antibody).

Figure 9 shows superimposed sensorgrams showing the binding of Macaca fascicularis recombinant FcgRs (upper panels ; CyCD64, CyCD32a, CYCD32b, CyCD16) and of human recombinant FcgRs (lower panels ; HuCD64, HuCD32a, HuCD32b, HUCD16a ) to the immobilized human lgG1 control (grey) and CD19/NKp46-1 bi-specific antibody (black). While full length wild type human lgG1 bound to all cynomolgus and human Fey receptors, the CD19/NKp46-1 bi-specific antibodies did not bind to any of the receptors.

Figures 10 show anti-CD19-Fcmono-anti-Nkp46 having NKp46 binding region based on NKp46-1 , NKp46-2, NKp46-3 or NKp46-4 are able to direct resting NK cells to their CD19-positive Daudi tumor target cells, while isotype control antibody did not lead to elimination of the Daudi cells. Rituximab (RTX) served as positive control of ADCC, where the maximal response obtained with RTX (at 10 μg ml in this assay) was 21.6% specific lysis.

Figure 1 1A shows results of purification of anti-CD19-Fcmono-anti-Nkp46 compared with DART and BITE. BITE and DART showed a very low production yield compared to anti- CD19-Fcmono-anti-Nkp46, and have a very complex SEC profile. Figure 1 1 B shows SDS- PAGE after Coomassie staining in the expected SEC fractions (3 and 4 for BITE and 4 and 5 for DART), whereas anti-CD19-Fcmono-anti-Nkp46 showed clear and simple SEC and SDS- PAGE profiles with a major peak containing the monomeric bispecific proteins.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the specification, "a" or "an" may mean one or more. As used in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

Where "comprising" is used, this can optionally be replaced by "consisting essentially of", more optionally by "consisting of".

As used herein, the term "antigen binding domain" refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope. Thus, in one embodiment, said domain can comprise a hypervariable region, optionally a VH and/or VL domain of an antibody chain, optionally at least a VH domain. In another embodiment, the binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain. In another embodiment, the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.

The term "antibody" herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). An "antibody fragment" comprises a portion of a full-length antibody, e.g. antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab)₂, F(ab')₂, F(ab)₃, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997;10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23, 1 126-1 136; WO2005040219, and published U.S. Patent Applications 20050238646 and 20020161201 .

The term "antibody derivative", as used herein, comprises a full-length antibody or a fragment of an antibody, e.g. comprising at least antigen-binding or variable regions thereof, wherein one or more of the amino acids are chemically modified, e.g., by alkylation, PEGylation, acylation, ester formation or amide formation or the like. This includes, but is not limited to, PEGylated antibodies, cysteine-PEGylated antibodies, and variants thereof.

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity-determining region" or "CDR" (e.g. residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31 -35 (H1 ), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991 ) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1 ), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering as in Kabat" and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

By "framework" or "FR" residues as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1 , FR2, FR3 and FR4).

By "constant region" as defined herein is meant an antibody-derived constant region that is encoded by one of the light or heavy chain immunoglobulin constant region genes. By "constant light chain" or "light chain constant region" as used herein is meant the region of an antibody encoded by the kappa (Ckappa) or lambda (Clambda) light chains. The constant light chain typically comprises a single domain, and as defined herein refers to positions 108-214 of Ckappa, or Clambda, wherein numbering is according to the EU index (Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). By "constant heavy chain" or "heavy chain constant region" as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full length IgG antibodies, the constant heavy chain, as defined herein, refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 1 18-447, wherein numbering is according to the EU index.

By "Fab" or "Fab region" as used herein is meant the polypeptide that comprises the VH, CH1 , VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a polypeptide, multispecific polypeptide or ABD, or any other embodiments as outlined herein.

By "single-chain Fv" or "scFv" as used herein are meant antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

By "Fv" or "Fv fragment" or "Fv region" as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody.

By "Fc" or "Fc region", as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cv2 (CH2) and Cv3 (CH3) and the hinge between Cy1 and Cv2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226, P230 or A231 to its carboxyl-terminus, wherein the numbering is according to the EU index. Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below. By "Fc polypeptide" or "Fc-derived polypeptide" as used herein is meant a polypeptide that comprises all or part of an Fc region. Fc polypeptides include but is not limited to antibodies, Fc fusions and Fc fragments.

By "variable region" as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including Vkappa and Vlambda) and/or VH genes that make up the light chain (including kappa and lambda) and heavy chain immunoglobulin genetic loci respectively. A light or heavy chain variable region (VL and VH) consists of a "framework" or "FR" region interrupted by three hypervariable regions referred to as "complementarity determining regions" or "CDRs". The extent of the framework region and CDRs have been precisely defined, for example as in Kabat (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, (1983)), and as in Chothia. The framework regions of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen.

The term "specifically binds to" means that an antibody or polypeptide can bind preferably in a competitive binding assay to the binding partner, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

The term "affinity", as used herein, means the strength of the binding of an antibody or polypeptide to an epitope. The affinity of an antibody is given by the dissociation constant K_D, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant K_A is defined by 1/K_D. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device).

By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. An example of amino acid modification herein is a substitution. By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution Y50W refers to a variant of a parent polypeptide, in which the tyrosine at position 50 is replaced with tryptophan. A "variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or "parent" polypeptide. The polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.

"Conservative" amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The term "identity" or "identical", when used in a relationship between the sequences of two or more polypeptides, refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1 , Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991 ; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity.

An "isolated" molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition). Commonly, a composition of a polypeptide will exhibit 98%, 98%, or 99% homogeneity for polypeptides in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use.

In the context herein, "treatment" or "treating" refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context. For example, "treatment" of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas "treatment" of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.

As used herein, "NK cells" refers to a sub-population of lymphocytes that is involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD56 and/or NKp46 for human NK cells, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express "self" MHC/HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells. Within the context herein "active" NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells. For instance, an "active" NK cell can be able to kill cells that express a ligand for an activating NK receptor and/or fail to express MHC/HLA antigens recognized by a KIR on the NK cell. NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Human Press, pp. 219-238 (2000).

As used herein, "T cells" refers to a sub-population of lymphocytes that mature in the thymus, and which display, among other molecules T cell receptors on their surface. T cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including the TCR, CD4 or CD8, the ability of certain T cells to kill tumor or infected cells, the ability of certain T cells to activate other cells of the immune system, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify T cells, using methods well known in the art. Within the context herein, "active" or "activated" T cells designate biologically active T cells, more particularly T cells having the capacity of cytolysis or of stimulating an immune response by, e.g., secreting cytokines. Active cells can be detected in any of a number of well-known methods, including functional assays and expression-based assays such as the expression of cytokines such as TNF- alpha.

Producing polypeptides

The antigen binding domains described herein can be readily derived a variety of immunoglobulin or non-immunoglobulin scaffolds, for example affibodies based on the Z- domain of staphylococcal protein A, engineered Kunitz domains, monobodies or adnectins based on the 10th extracellular domain of human fibronectin III, anticalins derived from lipocalins, DARPins (desiged ankyrin repeat domains, multimerized LDLR-A module, avimers or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference.

Antigen binding domains are commonly derived from antibodies (immunoglobulin chains), for example in the form of associated VL and VH domains found on two polypeptide chains, or single chain antigen binding domains such as scFv, a V_H domain, a V_L domain, a dAb, a V-NAR domain or a V_HH domain. The an antigen binding domain (e.g., ABD-i and ABD₂) can also be readily derived from antibodies as a Fab.

Typically, antibodies are initially obtained by immunization of a non-human animal, e.g., a mouse, with an immunogen comprising a polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, for which it is desired to obtain antibodies (e.g. a human polypeptide). The step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is herein incorporated by reference). Other protocols may also be used as long as they result in the production of B cells expressing an antibody directed to the antigen used in immunization. Lymphocytes from a non-immunized non-human mammal may also be isolated, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out. For exemplary monoclonal antibodies, the next step is the isolation of splenocytes from the immunized non- human mammal and the subsequent fusion of those splenocytes with an immortalized cell in order to form an antibody-producing hybridoma. The hybridoma colonies are then assayed for the production of antibodies that specifically bind to the polypeptide against which antibodies are desired. The assay is typically a colorimetric ELISA-type assay, although any assay may be employed that can be adapted to the wells that the hybridomas are grown in. Other assays include radioimmunoassays or fluorescence activated cell sorting. The wells positive for the desired antibody production are examined to determine if one or more distinct colonies are present. If more than one colony is present, the cells may be re-cloned and grown to ensure that only a single cell has given rise to the colony producing the desired antibody. After sufficient growth to produce the desired monoclonal antibody, the growth media containing monoclonal antibody (or the ascites fluid) is separated away from the cells and the monoclonal antibody present therein is purified. Purification is typically achieved by gel electrophoresis, dialysis, chromatography using protein A or protein G-Sepharose, or an anti-mouse Ig linked to a solid support such as agarose or Sepharose beads (all described, for example, in the Antibody Purification Handbook, Biosciences, publication No. 18-1037- 46, Edition AC, the disclosure of which is hereby incorporated by reference).

Human antibodies may also be produced by using, for immunization, transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods. For example, a XenoMouse (Abgenix, Fremont, CA) can be used for immunization. A XenoMouse is a murine host that has had its immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by this mouse or in hybridomas made from the B cells of this mouse, are already humanized. The XenoMouse is described in United States Patent No. 6,162,963, which is herein incorporated in its entirety by reference.

Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in (Ward et al. Nature, 341 (1989) p. 544, the entire disclosure of which is herein incorporated by reference). Phage display technology (McCafferty et al (1990) Nature 348:552-553) can be used to produce antibodies from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. See, e.g., Griffith et al (1993) EMBO J. 12:725- 734; US 5565332; US 5573905; US 5567610; US 5229275). When combinatorial libraries comprise variable (V) domain gene repertoires of human origin, selection from combinatorial libraries will yield human antibodies.

Additionally, a wide range of antibodies are available in the scientific and patent literature, including DNA and/or amino acid sequences, or from commercial suppliers. Antibodies will typically be directed to a pre-determined antigen. Examples of antibodies include antibodies that recognize an antigen expressed by a target cell that is to be eliminated, for example a proliferating cell or a cell contributing to pathology. Examples include antibodies that recognize tumor antigens, microbial (e.g. bacterial) antigens or viral antigens.

Antigen binding domains (ABDs) can be selected based on the desired cellular target, and may include for example cancer antigens, bacterial or viral antigens, etc. As used herein, the term "bacterial antigen" includes, but is not limited to, intact, attenuated or killed bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Examples include gram-positive bacterial antigens and gram-negative bacterial antigens. In some embodiments the bacterial antigen is derived from a bacterium selected from the group consisting of Helicobacter species, in particular Helicobacter pyloris; Borelia species, in particular Borelia burgdorferi; Legionella species, in particular Legionella pneumophilia; Mycobacteria s species, in particular M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae; Staphylococcus species, in particular Staphylococcus aureus; Neisseria species, in particular N. gonorrhoeae, N. meningitidis; Listeria species, in particular Listeria monocytogenes; Streptococcus species, in particular S. pyogenes, S. agalactiae; S. faecalis; S. bovis, S. pneumonias; anaerobic Streptococcus species; pathogenic Campylobacter species; Enterococcus species; Haemophilus species, in particular Haemophilus influenzue; Bacillus species, in particular Bacillus anthracis; Corynebacterium species, in particular Corynebacterium diphtheriae; Erysipelothrix species, in particular Erysipelothrix rhusiopathiae; Clostridium species, in particular C. perfringens, C. tetani; Enterobacter species, in particular Enterobacter aerogenes, Klebsiella species, in particular Klebsiella 1 S pneumoniae, Pasturella species, in particular Pasturella multocida, Bacteroides species; Fusobacterium species, in particular Fusobacterium nucleatum; Streptobacillus species, in particular Streptobacillus moniliformis; Treponema species, in particular Treponema pertenue; Leptospira; pathogenic Escherichia species; and Actinomyces species, in particular Actinomyces israelii.

As used herein, the term "viral antigen" includes, but is not limited to, intact, attenuated or killed whole virus, any structural or functional viral protein, or any peptide portion of a viral protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Sources of a viral antigen include, but are not limited to viruses from the families: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses); Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), Hepatitis C; Norwalk and related viruses, and astroviruses). Alternatively, a viral antigen may be produced recombinantly.

As used herein, the terms "cancer antigen" and "tumor antigen" are used interchangeably and refer to antigens that are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.

The cancer antigens are usually normal cell surface antigens which are either over- expressed or expressed at abnormal times. Ideally the target antigen is expressed only on proliferative cells (e.g., tumour cells), however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between proliferative and healthy tissue. Antibodies have been raised to target specific tumour related antigens including: Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1 ), Cripto, CD4, CD20, CD30, CD19, CD33, CD38, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD22 (Siglec2), CD33 (Siglec3), CD79, CD138, CD171 , PSCA, L1 -CAM, PSMA (prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin, EphB2, Melanotransferin, Mud 6 and TMEFF2. Examples of cancer antigens also include B7-H3, B7-H4, B7-H6, PD-L1 , MAGE, MART-1/Melan-A, gp100, major histocompatibility complex class l-related chain A and B polypeptides (MICA and MICB), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, Killer- Ig Like Receptor 3DL2 (KIR3DL2), protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), nectins (e.g. nectin-4), major histocompatibility complex class l-related chain A and B polypeptides (MICA and MICB), proteins of the UL16-binding protein (ULBP) family, proteins of the retinoic acid early transcript-1 (RAET1 ) family, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1 , prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens, GAGE-family of tumor antigens, anti-Mullerian hormone Type II receptor, delta-like ligand 4 (DLL4), DR5, BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, MUC family, VEGF, VEGF receptors, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor, a member of the human EGF-like receptor family such as HER-2/neu, HER-3, HER-4 or a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, Muc-1 , CA125, ανβ3 integrins, α5β1 integrins, αΙ^β3 -integrins, PDGF beta receptor, SVE- cadherin, IL-8, hCG, IL-6, IL-6 receptor, IL-15, ofetoprotein, E-cadherin, ocatenin, β- catenin and γ-catenin, p120ctn, PRAME, NY-ESO-1 , cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, imp-1 , P1A, EBV-encoded nuclear antigen (EBNA)-1 , brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM-MEL-40), SSX-1 , SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is not intended to be exhaustive.

In one embodiment, an ABD binds to a cancer antigen, a viral antigen, a microbial antigen, or an antigen present on an infected cell (e.g. virally infected) or on a proinflammatory immune cell. In one embodiment, said antigen is a polypeptide selectively expressed or overexpressed on a tumor cell, and infected cell or a pro-inflammatory cell. In one embodiment, said antigen is a polypeptide that when inhibited, decreases the proliferation and/or survival of a tumor cell, an infected cell or a pro-inflammatory cell. For example, a first and/or second antibody or fragment can respectively bind anti-Herl and anti- Her2. Anti-Her2 can be for example an antibody comprising the CDRs derived from Herceptin® (trastuzumab) or 2C4 (pertuzumab). Anti-Her2 and anti-Herl (antibodies D1-5 and C3-101 ) amino acid sequences are shown in WO201 1/069104.

In one embodiment, ABD-i and/or ABD₂ inhibits (neutralizes) the function of a polypeptide to which it specifically binds. In one embodiment, the polypeptide is a polypeptide selectively expressed or overexpressed on a tumor cell. In one embodiment, the polypeptide is a polypeptide selectively expressed or overexpressed on an infected (e.g. virally or bacterially infected) cell or a pro-inflammatory cell. In one embodiment, the polypeptide is a polypeptide that when inhibited, decreases the proliferation and/or survival of a tumor cell, an infected cell or a pro-inflammatory cell. For example bispecific antibodies that bind ErbB2 and ErbB3 and blocks ligand-induced receptor activation have been reported to be effective in ErbB2-amplified tumors (MacDonagh et al. (2012) Mol. Cancer Ther. 1 1 :582).

In exemplary embodiments, one of ABD-i or ABD₂will bind an antigen expressed by a target cell that is to be eliminated (e.g., a tumor antigen, microbial (e.g. bacterial) antigen, viral antigen, or antigen expressed on an immune cell that is contributing to inflammatory or autoimmune disease, and the other of ABD-i or ABD₂ will bind to an antigen expressed on an immune cell, for example an immune effector cell, e.g. a cell surface receptor of an effector cells such as a T or NK cell. Examples of antigens expressed on immune cells, optionally immune effector cells, include antigens expressed on a member of the human lymphoid cell lineage, e.g. a human T cell, a human B cell or a human natural killer (NK) cell, a human monocyte, a human neutrophilic granulocyte or a human dendritic cell. Advantageously, such cells will have either a cytotoxic or an apoptotic effect on a target cell that is to be eliminated (e.g., that expresses a tumor antigen, microbial antigen, viral antigen, or antigen expressed on an immune cell that is contributing to inflammatory or autoimmune disease). Especially advantageously, the human lymphoid cell is a cytotoxic T cell or NK cell which, when activated, exerts a cytotoxic effect on the target cell. According to this embodiment, then, the cytotoxic activity of the human effector cells are recruited. According to another embodiment, the human effector cell is a member of the human myeloid lineage.

Antigens expressed on an immune cell to which antibodies of fragments that make up multispecific antibodies can bind also include NK and/or T cell receptors, e.g. any molecule on the surface of NK cells or T cells, respectively, that can serve to direct the NK or T cell to the intended target cell to be eliminated. Examples include, e.g., members of the immunoglobulin superfamily, members of the killer-cell immunoglobulin-like receptor (KIR) family, the leukocyte immunoglobulin-like receptors (LILR) family, or the lectin family or the NK cell lectin-like receptor family. Activity can be measured for example by bringing target cells and effector cells into contact in presence of the multispecific polypeptide. Optionally the immune cell receptor is an activating receptor, e.g. an activating NK cell or T cell receptor. As used herein, the terms "activating NK cell receptor" and "activating T cell receptor" refers to any molecule on the surface of NK cells or T cells, respectively, that, when stimulated, causes a measurable increase in any property or activity known in the art as associated with NK cell or T cell activity, respectively, such as cytokine (for example IFN- Y or TNF-a) production, increases in intracellular free calcium levels, the ability to lyse target cells in a redirected killing assay as described, e.g. elsewhere in the present specification, or the ability to stimulate NK cell or T cell proliferation, respectively. The term "activating NK receptor" includes but is not limited to DNAX accessory molecule-1 (DNAM-1 ), 2B4, activating forms of KIR proteins (for example KIR2DS receptors, KIR2DS2, KIR2DS4), NKG2D, NKp30, CD69, NKp80, NKp44, NKp46, IL-2R, IL-12R, IL-15R, IL-18R and IL-21 R. In one embodiment, the activating NK cell receptor is a receptor other than an Fey receptor. In one embodiment, the activating NK cell receptor is a receptor other than NKp46.

Activation of cytotoxic T cells may occur via binding of the CD3 antigen as effector antigen on the surface of the cytotoxic T cell by a multispecific (e.g. bispecific) polypeptide of this embodiment. The human CD3 antigen is present on both helper T cells and cytotoxic T cells. Human CD3 denotes an antigen which is expressed on T cells as part of the multimolecular T cell complex and which comprises three different chains: CD3-epsilon, CD3-delta and CD3-gamma.

Other effector cell antigens bound by a multispecific polypeptide are the human CD16 antigen, the human CD64, the human CD2 antigen, the human CD28 antigen or the human CD25 antigen. In one embodiment, the effector cell antigen is CD16; such a polypeptide, when having an Fc domain that does not substantially bind inhibitory FcyR, will have CD16 agonist activity without contribution of inhibition from inhibitory FcyR. In other embodiments, the effector cell activating receptor is a receptor other than CD16.

The ABD which are incorporated into the polypeptides can be tested for any desired activity prior to inclusion in a polypeptide. Once appropriate antigen binding domains having desired specificity and/or activity are identified, DNA encoding each of the or ABD can be placed, in suitable arrangements, in an appropriate expression vector(s), together with DNA encoding any elements such as an enzymatic recognition tag, or CH2 and CH3 domains and any other optional elements (e.g. DNA encoding a linker or hinge region) for transfection into an appropriate host. The host is then used for the recombinant production of the multispecific polypeptide.

An ABD derived from an antibody will generally comprise at minimum a hypervariable region sufficient to confer binding activity. It will be appreciated that an ABD may comprise other amino acids or functional domains as may be desired, including but not limited to linker elements (e.g. linker peptides, CH1 , CK or CA domains, hinges, or fragments thereof, each of which can be placed between an ABD and a CH2 or CH3 domain, or between other domains as needed). In one example an ABD comprises an scFv, a V_H domain and a V_L domain, or a single domain antibody (nanobody or dAb) such as a V-NAR domain or a V_HH domain. Exemplary antibody formats are further described herein and an ABD can be selected based on the desired format.

In any embodiment, an antigen binding domain can be obtained from a humanized antibody in which residues from a complementary-determining region (CDR) of a human antibody are replaced by residues from a CDR of the original antibody (the parent or donor antibody, e.g. a murine or rat antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. The CDRs of the parent antibody, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted in whole or in part into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies is described in, e.g., WO 92/1 1018, Jones, 1986, Nature 321 :522-525, Verhoeyen et al., 1988, Science 239:1534-1536. An antigen binding domain can thus have non-human hypervariable regions or CDRs and human frameworks region sequences (optionally with backmutations).

ABDs will be arranged in an expression vector so as to produce the Fc-polypeptides having the desired domains operably linked to one another. The multispecific polypeptide can then be produced in an appropriate host cell or by any suitable synthetic process. The host cell may be of mammalian origin or may be selected from COS-1 , COS-7, HEK293, BHK21 , CHO, BSC-1 , Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of generating mammalian glycosylation on antibodies, e.g. a prokaryotic cell or organism, such as natural or engineered E. coli spp., Klebsiella spp., or Pseudomonas spp.

Monomeric bispecific Fc-derived polypeptides having advantageous properties can be constructed that comprise: (a) a first antigen binding domain (ABD-i ) that binds to a first antigen of interest; (b) a second antigen binding domain (ABD₂) that binds a second antigen of interest; and (c) at least a portion of a human Fc domain, wherein the Fc domain (i) does not dimerize with another Fc-derived polypeptide, (ii) is capable of binding to human FcRn and (iii) has decreased binding to a human Fey receptor compared to a wild type human lgG1 Fc domain.

Various domain arrangements can be envisaged. For example the Fc domain or portion thereof can be fused to the C-terminus of a tandem scFv. In one example, the ABDs are each scFv and arranged as tandem scFvs, for example having the following domain arrangement (from N-terminus to C-terminus):

(ABD₁) - (ABD₂) - CH2 - CH3

CH3 - CH2 - (ABD-i) - (ABD₂) - (scFv - (scFv₂) - CH2 - CH3

CH2 - CH3 - (scFv - (scFv₂)

A CH1 domain may be present between an ABD (or scFV) and a CH2 domain, or the CH1 domain may be absent between the an ABD (or scFV) and a CH2 domain. In one embodiment, a peptide linker is present between an ABD (or scFV) and a CH2 domain (or between and ABD and a CH1 domain. In one embodiment, a hinge region or fragment thereof is present between a CH1 and a CH2 domain, or between an ABD and an adjacent domain (e.g. a CH1 , a CH2, etc.). The first and second ABDs can be linked together by a linker of sufficient length to enable the ABDs to fold in such a way as to permit binding to the respective antigen for which the ABD is intended to bind. Suitable peptide linkers for use in linking ABD-i to ABD₂, or for use in linking an ABD to a CH2 or CH3 are known in the art, see, e.g. WO2007/073499, the disclosure of which is incorporated herein by reference. Examples of linker sequences include (G₄S)_X wherein x is an integer (e.g. 1 , 2, 3, 4, or more). The tandem antigen binding domain can thus for example have the structure (ABD-i - peptide linker - ABD₂ - peptide linker - (monomeric CH2-CH3 domain-containing polypeptide)). For example, the polypeptide may comprise, as a fusion product, the structure (scFv-i - peptide linker - scFv₂- peptide linker - CH2 - CH3), wherein each element is fused to the following element.

Alternatively, a monomeric multispecific antibody can be prepared in which the two antigen binding domains are positioned on opposite termini of an Fc domain. This protein can provide for conformations that can provide better binding to target antigens on different cells. This protein also permits a wider range of antibody variable regions to be used; some antibody binding domains that do not remain functional in tandem scFv format will remain functional in single scFv form. Thus, in one aspect the polypeptide can comprise: (a) a first antigen binding domain (ABDi) that binds to a first antigen of interest, (b) at least a portion of a human Fc domain, wherein the Fc domain is fused (optionally via intervening amino acid sequences) to the C-terminus of the first antigen binding domain, and (c) a second antigen binding domain (ABD₂) that binds a second antigen of interest is fused (optionally via intervening amino acid sequences) to the C-terminus of the Fc domain, wherein the multispecific polypeptide (and/or its Fc domain) is capable of binding to human neonatal Fc receptor (FcRn). In one embodiment, the monomeric multispecific polypeptide does not form a dimer with another Fc-derived polypeptide (e.g. does not form a homodimer with another identical polypeptide). In one embodiment, the Fc domain comprises a CH3 domain having one or more amino acid mutations (e.g. substitutions) in the CH3 dimer interface to prevent formation of dimers. Optionally, the multispecific polypeptide (and/or its Fc domain) has decreased binding to a human Fey receptor, e.g., compared to a full length wild type human lgG1 antibody. The polypeptide can have a domain arrangement:

(ABD₁) - CH2 - CH3 - (ABD₂)

Optionally, the polypeptide further comprises linking amino acids between the aforementioned domains. In one embodiment, a CH1 domain or fragment thereof, a hinge region or fragment thereof, and/or a linker peptide can be placed between ABDi and CH2. In one embodiment, a hinge region or fragment thereof, and/or a linker peptide can be placed between CH3 and ABD₂.

In another embodiment, provided is a monomeric multispecific polypeptide that binds a first and a second antigen in monovalent fashion, comprising: (a) a first antigen binding domain (ABDi) that binds to a first antigen of interest, (b) a human Fc domain (full or partial) comprising a CH3 domain comprising an amino acid mutation to prevent CH3-CH3 dimerization, wherein the Fc domain is fused (optionally via intervening amino acid sequences) to the C-terminus of the first antigen binding domain, and (c) a second antigen binding domain (ABD₂) that binds a second antigen of interest is fused (optionally via intervening amino acid sequences) to the C-terminus of the Fc domain, wherein the multispecific polypeptide (and/or its Fc domain) is capable of binding to human neonatal Fc receptor (FcRn). In one embodiment, the monomeric multispecific polypeptide does not form a dimer with another Fc-derived polypeptide (e.g. does not form a homodimer with another identical polypeptide). In one embodiment, the Fc domain comprises a CH3 domain having one or more amino acid mutations (e.g. substitutions) in the CH3 dimer interface to prevent formation of dimers. Optionally, the multispecific polypeptide (and/or its Fc domain) has decreased binding to a human Fey receptor, e.g., compared to a full length wild type human lgG1 antibody.

In one aspect of any embodiment, the first antigen binding domain and/or the second antigen binding domain comprise a scFv, optionally where the scFv comprises human framework amino acid sequences.

In one embodiment the monomeric polypeptide will bind to FcRn, with a 1 :1 ratio (1 FcRn for each monomeric polypeptide).

The CH3 domains may comprise amino acid modification to prevent homodimerization. Such monomers will retain partial FcRn binding (compared, e.g., to a wild type full length human lgG1 antibody). The examples of CH2-CH3 domains provided herein retain partial FcRn binding but have decreased human Fey receptor binding. Optionally the monomeric polypeptide is capable of binding to human FcRn with intermediate affinity, e.g. retains binding to FcRn but has decreased binding to a human FcRn receptor compared to a full-length wild type human lgG1 antibody. The Fc moiety may further comprise one or more amino acid modifications, e.g. in the CH2 domain, that increases or decreases binding to one or more Fey receptors.

Optionally in any of the embodiments, the Fc domain comprises a human CH2 domain and a human CH3 domain comprising one or more amino acid modifications such that the Fc domain which does not dimerize with another Fc-derived polypeptide (e.g. does not dimerize via interactions with another CH3 domain).

The monomeric Fc-derived polypeptide does not substantially bind to an FcylllA polypeptide (CD16) can comprise a wild-type or modified CH2 domain, and a CH3 domain, wherein said CH3 domain comprises a modified CH3 dimer interface (e.g. a mutations in the CH3 dimer interface) to prevent dimerization with another Fc-derived polypeptide, optionally further wherein the CH2-CH3 domain comprises an amino acid sequence of SEQ ID NO 18, or a sequence at least 90, 95% or 98% identical thereto:

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18), optionally comprising a substitution at 1 , 2, 3, 4, 5, 6 of residues 121 , 136, 165, 175, 177 or 179 of SEQ ID NO: 18.

In one embodiment of any of the polypeptides or methods herein, the CH3 domain comprises an amino acid substitution at 1 , 2, 3, 4, 5, 6 or 7 of the positions L351 , T366, L368, P395, F405, T407 (or Y407) and/or K409 (EU numbering as in Kabat). In one embodiment, a peptide linker used to link an ABD (e.g. an scFv, a VH or VL domain) to a CH2 or CH3 comprises a fragment of a CH1 domain. For example, a N- terminal amino acid sequence of CH1 can be fused to an ABD (e.g. an scFv, a VH or VL domain, etc.) in order to mimic as closely as possible the natural structure of an antibody. In one embodiment, the linker comprises a N-terminal CH1 amino acid sequence of between 2- 4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2- 10 residues, between 2-12 residues, between 2-14 residues, between 2-16 residues, between 2-18 residues, between 2- 20 residues, between 2-22 residues, between 2-24 residues, between 2-26 residues, between 2-28 residues, or between 2-30 residues. In one embodiment linker comprises or consists of the amino acid sequence RTVA.

When an ABD is an scFv, the VH domain and VL domains (VL or VH domains or fragments thereof that retain binding specificity) that form a scFv are linked together by a linker of sufficient length to enable the ABD to fold in such a way as to permit binding to the antigen for which the ABD is intended to bind. Examples of linkers include, for example, linkers comprising glycine and serine residues, e.g., the amino acid sequence GEGTSTGS(G₂S)₂GGAD. In another specific embodiment, the VH domain and VL domains of an svFv are linked together by the amino acid sequence (G₄S)₃.

Any of the peptide linkers may comprise a length of at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, at least 25 residues, at least 30 residues or more. In other embodiments, the linkers comprises a length of between 2-4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2-10 residues, between 2-12 residues, between 2-14 residues, between 2-16 residues, between 2-18 residues, between 2- 20 residues, between 2-22 residues, between 2-24 residues, between 2-26 residues, between 2-28 residues, or between 2-30 residues.

In one embodiment, the hinge region will be a fragment of a hinge region (e.g. a truncated hinge region without cysteine residues) or may comprise one or amino acid modifications to remove (e.g. substitute by another amino acid, or delete) a cysteine residue, optionally both cysteine residues in a hinge region. Removing cysteines can be useful to prevent formation of disulfide bridges in a monomeric polypeptide.

Constant region domains can be derived from any suitable antibody. Of particular interest are the heavy chain domains, including, the constant heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, "CH" domains in the context of IgG are as follows: "CH1 " refers to positions 1 18-220 according to the EU index as in Kabat. "CH2" refers to positions 237-340 according to the EU index as in Kabat, and "CH3" refers to positions 341 -447 according to the EU index as in Kabat. By "hinge" or "hinge region" or "antibody hinge region" is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Thus for IgG the hinge is herein defined to include positions 221 (D221 in lgG1 ) to 236 (G236 in lgG1 ), wherein the numbering is according to the EU index as in Kabat. References to amino acid residue within constant region domains found within the polypeptides shall be, unless otherwise indicated or as otherwise dictated by context, with reference to Kabat, in the context of an IgG antibody.

CH3 domains that can serve in the present antibodies can be derived from any suitable antibody. Such CH3 domains can serve as the basis for a modified CH3 domain. Optionally the CH3 domain is of human origin.

In certain embodiments herein the CH3 domain will comprise one or more amino acid modifications (e.g. amino acid substitutions) to disrupt the CH3 dimerization interface. Optionally the CH3 domain modifications will prevent protein aggregation caused by the exposure of hydrophobic residues when the CH2-CH3 domains are in monomeric form. Optionally, the CH3 domain modifications will additionally not interfere with the ability of the Fc-derived polypeptide to bind to neonatal Fc receptor (FcRn), e.g. human FcRn.

CH3 domains that can be used to prevent homodimer formation have been described in various publications. See, e.g. US 2006/0074225, WO2006/031994, WO201 1/063348 and Ying et al. (2012) J. Biol. Chem. 287(23):19399-19407, the disclosures of each of which are incorporated herein by reference. In order to discourage the homodimer formation, one or more residues that make up the CH3-CH3 interface are replaced with a charged amino acid such that the interaction becomes electrostatically unfavorable. For example, WO201 1/063348 provides that a positive-charged amino acid in the interface, such as lysine, arginine, or histidine, is replaced with a different (e.g. negative-charged amino acid, such as aspartic acid or glutamic acid), and/or a negative-charged amino acid in the interface is replaced with a different (e.g. positive charged) amino acid. Using human IgG as an example, charged residues within the interface that may be changed to the opposing charge include R355, D356, E357, K370, K392, D399, K409, and K439. In certain embodiments, two or more charged residues within the interface are changed to an opposite charge. Exemplary molecules include those comprising K392D and K409D mutations and those comprising D399K and D356K mutations. In order to maintain stability of the polypeptide in monomeric form, one or more large hydrophobic residues that make up the CH3-CH3 interface are replaced with a small polar amino acid. Using human IgG as an example, large hydrophobic residues of the CH3-CH3 interface include Y349, L351 , L368, L398, V397, F405, and Y407. Small polar amino acid residues include asparagine, cysteine, glutamine, serine, and threonine. Thus in one embodiment, a CH3 domain will comprise an amino acid modification (e.g. substitution) at 1 , 2, 3, 4, 5, 6, 7 or 8 of the positions R355, D356, E357, K370, K392, D399, K409, and K439. In WO201 1/063348, two of the positively charged Lys residues that are closely located at the CH3 domain interface were mutated to Asp. Threonine scanning mutagenesis was then carried out on the structurally conserved large hydrophobic residues in the background of these two Lys to Asp mutations. Fc molecules comprising K392D and K409D mutations along with the various substitutions with threonine were analyzed for monomer formation. Exemplary monomeric Fc molecules include those having K392D, K409D and Y349T substitutions and those having K392D, K409D and F405T substitutions.

In Ying et al. (2012) J. Biol. Chem. 287(23):19399-19407, amino acid substitutions were made within the CH3 domain at residues L351 , T366, L368, P395, F405, T407 and K409. Combinations of different mutations resulted in the disruption of the CH3 dimerization interface, without causing protein aggregation. Thus in one embodiment, a CH3 domain will comprise an amino acid modification (e.g. substitution) at 1 , 2, 3, 4, 5, 6 or 7 of the positions L351 , T366, L368, P395, F405, T407 and/or K409. In one embodiment, a CH3 domain will comprise amino acid modifications L351Y, T366Y, L368A, P395R, F405R, T407M and K409A. In one embodiment, a CH3 domain will comprise amino acid modifications L351 S, T366R, L368H, P395K, F405E, T407K and K409A. In one embodiment, a CH3 domain will comprise amino acid modifications L351 K, T366S, P395V, F405R, T407A and K409Y.

CH2 domains can be readily obtained from any suitable antibody. Optionally the CH2 domain is of human origin. A CH2 may or may not be linked (e.g. at its N-terminus) to a hinge of linker amino acid sequence. In one embodiment, a CH2 domain is a naturally occurring human CH2 domain of lgG1 , 2, 4 or 4 subtype. In one embodiment, a CH2 domain is a fragment of a CH2 domain (e.g. at least 10, 20, 30, 40 or 50 amino acids).

In one embodiment, a CH2 domain, when present in a polypeptide described herein, will retain binding to a neonatal Fc receptor (FcRn), particularly human FcRn.

In one embodiment, a CH2 domain, when present in a polypeptide described herein, and the polypeptides described herein, will confer decreased or lack of binding to a Fey receptor, notably FcyRIIIA (CD16). Polypeptides that comprise a CH2 domain that are not bound by CD16 will not be capable of activating or mediating ADCC by cells (e.g. NK cells, T cells) that do not express the effector cell antigen of interest (e.g. NKp46, CD3, etc.).

In one embodiment, the polypeptides described herein and their Fc domain(s) and/or a CH2 domain thereof, will have decreased or will substantially lack antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP), FcR-mediated cellular activation (e.g. cytokine release through FcR cross-linking), and/or FcR-mediated platelet activation/depletion.

In one embodiment, a CH2 domain in a polypeptide will have substantial loss of binding to activating Fey receptors, e.g., FCYRI I IA (CD16), FcyRIIA (CD32A) or CD64, or to an inhibitory Fc receptor, e.g., FcyRIIB (CD32B). In one embodiment, a CH2 domain in a polypeptide will furthermore have substantial loss of binding to the first component of complement (C1 q).

For example, Substitutions into human lgG1 of lgG2 residues at positions 233-236 and lgG4 residues at positions 327, 330 and 331 were shown to greatly reduce binding to Fey receptors and thus ADCC and CDC. Furthermore, Idusogie et al. (2000) J Immunol.

164(8):4178-84 demonstrated that alanine substitution at different positions, including K322, significantly reduced complement activation.

In one embodiment, a CH2 domain that retains binding to a Fey receptor but has reduction of binding to Fey receptors will lack or have modified N-linked glycosylation, e.g. at residue N297 (Kabat EU). For example the polypeptide is expressed in a cell line which naturally has a high enzyme activity for adding fucosyl to the N-acetylglucosamine that binds to the Fc region of the polypeptides, or which does not yield glycosylation at N297 (e.g. bacterial host cells). In another embodiment, a polypeptide may have one or more substitution that result in lack of the canonical Asn-X-Ser/Thr N-linked glycosylation motif at residues 297-299, which can also thus also result in reduction of binding to Fey receptors.

Thus, a CH2 domain may have a substitution at N297 and/or at neighboring residues (e.g.

298, 299).

In one embodiment, an Fc domain or a CH2 domain therefrom is derived from an lgG2 Fc mutant exhibiting diminished FcyR binding capacity but having conserved FcRn binding. In one aspect, the lgG2 Fc mutant or the derived multispecific polypeptide, Fc domain or CH2 domain comprises the mutations V234A, G237A, P238S according to the EU numbering system. In another aspect, the lgG2 Fc mutant or the derived multispecific polypeptide or Fc domain comprises mutations V234A, G237A, H268Q or H268A, V309L, A330S, P331 S according to the EU numbering system. In a particular aspect, the lgG2 Fc mutant or the derived multispecific polypeptide or Fc domain comprises mutations V234A, G237A, P238S, H268A, V309L, A330S, P331 S, and, optionally, P233S according to the EU numbering system. Optionally, a CH2 domain with loss of binding to Fey receptors may comprises residues 233, 234, 235, 237, and 238 (EU numbering system) that comprise a sequence selected from PAAAP, PAAAS, and SAAAS; optionally an Fc domain having such mutations can further comprise mutations H268A or H268Q, V309L, A330S and P331 S (see WO201 1/066501 , the disclosure of which is incorporated herein by reference).

In one embodiment, a CH2 domain that loses binding to a Fey receptor will comprise at least one amino acid modification (for example, possessing 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain of the Fc region, optionally further in combination with one or more amino acid modification in other domains (e.g. in a hinge domain or a CH3 domain). Any combination of Fc modifications can be made, for example any combination of different modifications disclosed in Armour KL. et al., (1999) Eur J Immunol. 29(8):2613-24; Presta, L.G. et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields, R.L. et al. (2002) J. Biol. Chem. 26; 277(30):26733-26740 and Shields, R.L. et al. (2001 ) J. Biol. Chem. 276(9):6591 -6604). In one embodiment, a polypeptide of the invention that has decreased binding to a human Fey receptor will comprise at least one amino acid modification (for example, possessing 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type CH2 domain within amino acid residues 237-340 (EU numbering), such that the polypeptide comprising such CH2 domain has decreased affinity for a human Fey receptor of interest relative to an equivalent polypeptide comprising a wild- type CH2 domain, optionally wherein the variant CH2 domain comprises a substitution at any one or more of positions 233, 234, 235, 236, 237, 238, 268, 297, 238, 299, 309, 327, 330, 331 (EU numbering).

In one aspect of any of the embodiments herein, provided is a recombinant nucleic acid encoding a polypeptide of the disclosure. In one aspect of any of the embodiments herein, provided is a recombinant host cell comprising a nucleic acid encoding a polypeptide of the disclosure, optionally wherein the host cell produces a protein of the disclosure with a yield (final productivity, after purification) of at least 1 , 2, 3 or 4 mg/L. Also provided is a kit or set of nucleic acids comprising a recombinant nucleic acid encoding a polypeptide of the disclosure. Also provided are methods of making proteins of the disclosure.

In one embodiment, provided are methods of making (or, e.g., testing, selecting, screening) a polypeptide described herein comprising: (a) providing a nucleic acid encoding a monomeric multispecific polypeptide described herein (e.g., a polypeptide that binds a first and a second antigen, comprising: a first antigen binding domain (ABDi) that specifically binds to a first antigen of interest, a human Fc domain or portion thereof that is incapable of interchain CH3-CH3 dimerization, and a second antigen binding domain (ABD₂) that specifically binds a second antigen of interest, wherein the multispecific polypeptide is capable of binding to human FcRn); and

(b) expressing said nucleic acid in a host cell to produce said polypeptide, respectively; and recovering said polypeptide. Optionally, the polypeptide produced represents at least 20%, 25% or 30% of the total proteins obtained from the host cell prior to purification. Optionally step (b) comprises loading the polypeptide produced onto an affinity purification support, optionally an affinity exchange column, optionally a Protein-A support or column, and collecting the polypeptide. Optionally the method further comprises a step (c): evaluating the plurality of heterodimeric proteins produced for a biological activity of interest, e.g., an activity disclosed herein.

By virtue of their ability to be produced in standard cell lines and standardized methods with high yields, unlike BITE, DART and other bispecific formats, the proteins of the disclosure also provide a convenient tool for screening for the most effective variable regions to incorporated into a multispecific protein.

In one aspect, the present disclosure provides a method for identifying or evaluating a polypeptide, comprising the steps of:

(a) providing a nucleic acid encoding a polypeptide described herein ;

(b) expressing said nucleic acid in a host cell to produce said polypeptide, respectively; and recovering said polypeptide; and

(c) evaluating the polypeptide produced for a biological activity of interest, e.g., an activity disclosed herein. In one embodiment, a plurality of different polypeptides are produced and evaluated.

In one embodiment, the polypeptide binds an activating receptor on an effector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the polypeptide to activate effector cells that express the activating receptor, when incubated with such effector cells in the presence of target cells (that express antigen of interest). Optionally, step (i) is followed by a step comprising: selecting a polypeptide (e.g., for further development, for use as a medicament) that activates said effector cells. In one embodiment, the polypeptide binds an activating receptor on an effector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the polypeptide to activate effector cells that express the activating receptor, when incubated with such effector cells in the absence of target cells (that express antigen of interest). Optionally, step (i) is followed by a step comprising: selecting a polypeptide (e.g., for further development, for use as a medicament) that does not substantially activate said effector cells.

In one embodiment, the polypeptide binds an activating receptor on an effector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the polypeptide to activate effector cells that express the activating receptor, when incubated with such effector cells in the presence of target cells (that express antigen of interest); and

(ii) testing the ability of the polypeptide to activate effector cells that express the activating receptor, when incubated with such effector cells in the absence of target cells (that express antigen of interest). Optionally, the method further comprises: selecting a polypeptide (e.g., for further development, for use as a medicament) that does not substantially activate said effector cells when incubated in the absence of target cells, and that activates said effector cells when incubated in the presence of target cells.

In one embodiment, the polypeptide binds an activating receptor on an effector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the polypeptide to induce effector cells that express the activating receptor to lyse target cells (that express antigen of interest), when incubated such effector cells in the presence of target cells. Optionally, step (i) is followed by a step comprising: selecting a polypeptide (e.g., for further development, for use as a medicament) that induces effector cells that express the activating receptor to lyse the target cells, when incubated such effector cells in the presence of the target cells.

In one embodiment, the polypeptide binds an activating receptor on an effector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the polypeptide to activate effector cells that express CD16 but do not express the activating receptor, when incubated with such effector cells in the presence of target cells. Optionally, step (i) is followed by a step comprising: selecting a polypeptide (e.g., for further development, for use as a medicament) that do not substantially activate said effector cells, when incubated with such effector cells in the presence of target cells. Uses of compounds

In one aspect, provided are the use of any of the compounds defined herein for the manufacture of a pharmaceutical preparation for the treatment or diagnosis of a mammal being in need thereof. Provided also are the use any of the compounds defined above as a medicament or an active component or active substance in a medicament. In a further aspect provided is a method for preparing a pharmaceutical composition containing a compound as defined above, to provide a solid or a liquid formulation for administration orally, topically, or by injection. Such a method or process at least comprises the step of mixing the compound with a pharmaceutically acceptable carrier.

In one aspect, provided is a method to treat, prevent or more generally affect a predefined condition by exerting a certain effect, or detect a certain condition using a compound herein, or a (pharmaceutical) composition comprising a compound disclosed herein.

The polypeptides described herein can be used to prevent or treat disorders that can be treated with antibodies, such as cancers, solid and non-solid tumors, hematological malignancies, infections such as viral infections, and inflammatory or autoimmune disorders.

In one embodiment, the antigen of interest is expressed on the surface of a malignant cell of a type of cancer selected from the group consisting of: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezary syndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1 +) large cell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL).

In one embodiment, polypeptides described herein can be used to prevent or treat a cancer selected from the group consisting of: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B- cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma. Other exemplary disorders that can be treated according to the invention include hematopoietic tumors of lymphoid lineage, for example T- cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezary syndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1 +) large cell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL).

In one aspect, the methods of treatment comprise administering to an individual a multispecific polypeptide in a therapeutically effective amount. A therapeutically effective amount may be any amount that has a therapeutic effect in a patient having a disease or disorder (or promotes, enhances, and/or induces such an effect in at least a substantial proportion of patients with the disease or disorder and substantially similar characteristics as the patient). The multispecific polypeptides can be included in kits. The kits may optionally further contain any number of polypeptides and/or other compounds, e.g., 1 , 2, 3, 4, or any other number of multispecific polypeptide and/or other compounds. It will be appreciated that this description of the contents of the kits is not limiting in any way. For example, the kit may contain other types of therapeutic compounds. Optionally, the kits also include instructions for using the polypeptides, e.g., detailing the herein-described methods.

Also provided are pharmaceutical compositions comprising the compounds as defined above. A compound may be administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition. The form depends on the intended mode of administration and therapeutic or diagnostic application. The pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the compounds to the patient. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as (sterile) water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters, alcohol, fats, waxes, and inert solids A pharmaceutically acceptable carrier may further contain physiologically acceptable compounds that act for example to stabilize or to increase the absorption of the compounds Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical compositions.

The compounds can be administered parenterally. Preparations of the compounds for parenteral administration must be sterile. Sterilization is readily accomplished by filtration through sterile filtration membranes, optionally prior to or following lyophilization and reconstitution. The parenteral route for administration of compounds is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, or intralesional routes. The compounds may be administered continuously by infusion or by bolus injection. A typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile 0.9% NaCI or 5% glucose optionally supplemented with a 20% albumin solution and 1 mg to 10 g of the compound, depending on the particular type of compound and its required dosing regimen. Methods for preparing parenterally administrable compositions are well known in the art.

EXAMPLES

Example 1 : Construction of Anti-CD19 x anti-CD3 bispecific monomeric CH 3 -mutated Fc polypeptides

Materials and Methods

Different constructs were made for use in the preparation of a bispecific Fc-based on a scFv specific for tumor antigen CD19 (anti-CD19 scFv) and a scFV specific for activating receptor CD3 on a T cell (anti-CD3 scFv). The CH3 domain incorporated the mutations (EU numbering) L351 K, T366S, P395V, F405R, T407A and K409Y. The CH2 domain was either a wild-type CH2 or a mutated CH2 comprising a N297S substitution.

The light chain and heavy chain DNA and amino acid sequences for the anti-CD19 and anti-CD3 scFv are shown in the corresponding SEQ ID NOS shown in the table below.

The amino acid sequences for the monomeric CH2-CH3 Fc portion are also shown below: lgG1 -Fcmono^* (^*the last K residue was removed in that construct)

APELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKT I SKAKGQPRE PQVYTKPPSREEMTKNQVSLS CLVKGFYPSDIAVEWESNGQPENNYKTTVPVLDSDGSFRLASYLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPG (SEQ ID NO: 6)

Cloning and production of the recombinant proteins

Coding sequences were generated by direct synthesis and/or by PCR. PCR were performed using the PrimeSTAR MAX DNA polymerase (Takara, #R045A) and PCR products were purified from 1 % agarose gel using the NucleoSpin gel and PCR clean-up kit (Macherey-Nagel, #740609.250). Once purified the PCR product were quantified prior to the In-Fusion ligation reaction performed as described in the manufacturer's protocol (ClonTech, #ST0345). The plasmids were obtained after a miniprep preparation run on an EVO200 (Tecan) using the Nucleospin 96 plasmid kit (Macherey-Nagel, #740625.4). Plasmids were then sequenced for sequence confirmation before to transfect the CHO cell line.

CHO cells were grown in the CD-CHO medium (Invitrogen) complemented with phenol red and 6 mM GlutaMax. The day before the transfection, cells were counted and seeded at 175.000 cells/ml. For the transfection, cells (200.000 cells/transfection) were prepared as described in the AMAXA SF cell line kit (AMAXA, #V4XC-2032) and nucleofected using the DS137 protocol with the Nucleofector 4D device. All the tranfections were performed using 300 ng of verified plasmids. After transfection, cells are seeded into 24 well plates in pre-warmed culture medium. After 24H, hygromycine B is added in the culture medium (200 g/ml). Protein expression is monitored after one week in culture. Cells expressing the proteins are then sub-cloned to obtain the best producers. Sub-cloning is performed using 96 flat-bottom well plates in which the cells are seeded at one cell per well into 200 μΙ of culture medium complemented with 200 μg ml of hygromycine B. Cells were left for three weeks before to test the clone's productivity.

Recombinant proteins which contain a lgG1 -Fc fragment were purified using Protein-

A beads (- rProteinA Sepharose fast flow, GE Healthcare, ref.: 17-1279-03). Briefly, cell culture supernatants were concentrated, clarified by centrifugation and injected onto Protein- A columns to capture the recombinant Fc containing proteins. Proteins were eluted at acidic pH (citric acid 0.1 M pH3), immediately neutralized using TRIS-HCL pH8.5 and dialyzed against 1X PBS. Recombinant scFV which contain a "six his" tag are purified by affinity chromatography using Cobalt resin. Other recombinant scFv were purified by size exclusion chromatography (SEC).

Anti-CD19-lgG1 -Fcmono-Anti-CD3 A bispecific Fc-containing polypeptide was constructed based on an scFv specific for the tumor antigen CD19 (anti-CD19 scFv) and an scFV specific for an activating receptor CD3 on a T cell (anti-CD3 scFv). The polypeptide has domains arranged as follows: anti- CD19-CH2-CH3-anti-CD3, as shown in Figure 2. DNA sequence coding for a CH3A H linker peptide having the amino acid sequence STGS was designed in order to insert a specific Sail restriction site at the CH3-VH junction.

The DNA and amino acid sequences of the bispecific polypeptide are shown in SEQ ID NOS: 1 1 and 12, respectively. Anti-CD19-lgG1 -Fcmono-Anti-CD3 Complete sequence (mature protein)

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSG SGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSST AYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGSSAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTKPPSREEMTKNQVSLSCLVKGFYPSDIAVEWES NGQPENNYKTTVPVLDSDGSFRLASYLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSD IKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVD DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGT

SYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

(SEQ ID NO: 12)

Anti-CD19-Anti-CD3-lqG1 -Fcmono

A bispecific Fc-containing polypeptide was constructed based on a scFv specific for tumor antigen CD19 (anti-CD19 scFv) and an scFV specific for activating receptor CD3 on a T cell (anti-CD3 scFv). The polypeptide has domains arranged as shown in Figure 3, and as follows:

(anti-CD19 scFv) - (anti-CD3scFv) - CH2 - CH3.

The DNA and amino acid sequences of the bispecific polypeptide are shown in SEQ

ID NOS: 13 and 14, respectively. The lgG1-Fcmono as encoded by SEQ ID NO: 5 was modified to include a terminal lysine.

Anti-CD19-Anti-CD3-lgG1 -Fcmono: (complete sequence :mature protein)

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSG SGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSST AYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGAS VKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSS LTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPG EKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAAT YYCQQWSSNPLTFGAGTKLELKRTVAAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTKPPSREEMTKNQVSLSCLVKGFYPSDIAVEWESNGQPENNYKTTVPVLDSDGSFRLAS YLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 14) Anti-CD19-lgG1 -Fcmono with N297S mutation

A monospecific Fc-containing polypeptide was constructed based on an scFv specific for the tumor antigen CD19 (anti-CD19 scFv). The polypeptide has domains arranged as shown in Figure 3, and as follows:

(anti-CD19 scFv) - CH2 - CH3.

The CH2 domain of the polypeptide has a N297S mutation which abrogates N-linked glycosylation and renders the glutamine at residue 295 (Kabat EU) reactive with TGase. The DNA sequence of the lgG1 -Fcmono-N297S portion is shown in SEQ ID NO: 15. The DNA and amino acid sequence of the anti-CD19-lgG1 -Fcmono Mut N297S (complete DNA sequence coding for mature protein) are shown in SEQ ID NOS: 16 and 17, respectively.

Anti-CD19-lgG1 -Fcmono Mut N297S: Complete sequence

(mature protein)

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSG SGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSST AYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTKPPSREEMTKNQVSLSCLVKGFYPSDIAVEWES NGQPENNYKTTVPVLDSDGSFRLASYLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17)

Example 2: Binding analysis of Anti-CD19-lgG1 -Fcmono-Anti-CD3 to B221 , JURKAT, HUT78 and CHO cell lines

Cells were harvested and stained with the cell supernatant of the anti-CD19-lgG1 -

Fcmono-anti-CD3 producing cells during 1 H at 4°C. After two washes in staining buffer (PBS1 X / BSA 0.2% / EDTA 2mM), cells were stained for 30 min at 4°C with goat anti- human (Fc)-PE antibody (IM0550 Beckman Coulter - 1/200). After two washes, stainings were acquired on a BD FACS Canto II and analyzed using the FlowJo software.

CD3 and CD19 expression were also controlled by flow cytometry: Cells were harvested and stained in PBS1X / BSA 0.2% / EDTA 2mM buffer during 30 min at 4°C using 5μΙ of the anti-CD3-APC and 5μΙ of the anti-CD19-FITC antibodies. After two washes, stainings were acquired on a BD FACS Canto II and analyzed using the FlowJo software.

Results are shown in Figure 4. The Anti-CD19-lgG1 -Fcmono-Anti-CD3 protein binds to the CD3 cell lines (HUT78 and JURKAT cell lines) (see Figure 4A) and the CD19 cell line (B221 cell line) (see Figure 4B) but not to the CHO cell line used as a negative control (Figure 4B).

Example 3: Titration analysis of the purified Anti-CD19-lgG1 -Fcmono-Anti-CD3, Anti- CD19-Anti-CD3-lgG1 -Fcmono and Anti-CD19-lgG1 -Fcmono proteins on B221 and JURKAT cell lines:

Cells were harvested and stained in PBS1X / BSA 0.2% / EDTA 2mM buffer during 1 H at 4°C using a range of dilutions of the proteins from 10 μg/ml to 3.6.10^"5 μg/ml. After two washes in staining buffer, cells were stained for 30 min at 4°C with goat anti-human (Fc)-PE antibody (1/200). After two washes, stainings were acquired on a BD FACS Canto II and analyzed using the FlowJo software.

Results are shown in Figure 5. The Anti-CD19-lgG1 -Fcmono-Anti-CD3 protein and the Anti-CD19-Anti-CD3-lgG1-Fcmono bind to the CD3 cell lines (JURKAT cell lines), while the Anti-CD19-lgG1 -Fcmono does not. Each of the Anti-CD19-lgG1 -Fcmono-Anti-CD3 protein, Anti-CD19-Anti-CD3-lgG1-Fcmono and Anti-CD19-lgG1 -Fcmono bind to the CD19 cell line (B221 ).

Example 4: T- and B- cell aggregation by purified Anti-CD19-lgG1 -Fcmono-Anti-CD3 Purified Anti-CD19-lgG1 -Fcmono-Anti-CD3 was tested in a T/B cell aggregation assay to evaluate whether the antibody is functional in bringing together CD19 and CD3 expressing cells.

Results are shown in Figure 6. The top panel shows that Anti-CD19-lgG1 -Fcmono- Anti-CD3 does not cause aggregation in the presence of B221 (CD19) or JURKAT (CD3) cell lines, but it does cause aggregation of cells when both B221 and JURKAT cells are co- incubated, illustrating that the bispecific antibody is functional. The lower panel shows control without antibody. Example 5: Binding of bispecific monomeric Fc polypeptide to FcRn

Affinity study by Surface Plasmon Resonance (SPR)

Biacore T100 general procedure and reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GE Healthcare) at 25°C. In all Biacore experiments Acetate Buffer (50 mM Acetate pH5.6, 150 mM NaCI, 0.1 % surfactant p20) and HBS-EP+ (Biacore GE Healthcare) served as running buffer and regeneration buffer respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Recombinant mouse FcRn was purchase from R&D Systems.

Immobilization of FcRn

Recombinant FcRn proteins were immobilized covalently to carboxyl groups in the dextran layer on a Sensor Chip CM5. The chip surface was activated with EDC/NHS (N- ethyl-N'-(3-dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). FcRn proteins were diluted to 10 μg ml in coupling buffer (10 mM acetate, pH 5.6) and injected until the appropriate immobilization level was reached (i.e. 2500 RU). Deactivation of the remaining activated groups was performed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

Affinity study

Monovalent affinity study was done following the Single Cycle Kinetic (SCK) protocol. Five serial dilutions of soluble analytes (antibodies and bi-specific molecules) ranging from 41.5 to 660 nM were injected over the FcRn (without regeneration) and allowed to dissociate for 10 min before regeneration. For each analyte, the entire sensorgram was fitted using the 1 :1 SCK binding model.

Results Anti-CD19-lgG1 -Fcmono-Anti-CD3 having its CH2-CH3 domains placed between two antigen binding domains, here two scFv, was evaluated to assess whether such bispecific monomeric Fc protein could retain binding to FcRn and thereby have improved in vivo half- lives compared to convention bispecific antibodies. Results showed that FcRn binding was retained, the model suggesting 1 :1 ratio (1 FcRn for each monomeric Fc). Results are shown in Figure 7.

Affinity was evaluated using SPR, in comparison to a chimeric full length antibody having human lgG1 constant regions. Results are shown in Figure 8. The monomeric Fc retained significant monomeric binding to FcRn (monomeric Fc: affinity of KD=194 nM; full length antibody with bivalent binding: avidity of KD=15.4 nM).

Example 6: Generation of anti-huNKp46 antibodies

Balb/c mice were immunized with a recombinant human NKp46 extracellular domain recombinant-Fc protein. Mice received one primo-immunization with an emulsion of 50 μg NKp46 protein and Complete Freund Adjuvant, intraperitoneally, a 2nd immunization with an emulsion of 50 μg NKp46 protein and Incomplete Freund Adjuvant, intraperitoneally, and finally a boost with 10 μg NKp46 protein, intravenously. Immune spleen cells were fused 3 days after the boost with X63.Ag8.653 immortalized B cells, and cultured in the presence of irradiated spleen cells.

Primary screen: Supernatant (SN) of growing clones were tested in a primary screen by flow cytometry using a cell line expressing the human NKp46 construct at the cell surface. Briefly, for FACS screening, the presence of reacting antibodies in supernanants was revealed by Goat anti-mouse polyclonal antibody (pAb) labeled with PE.

A selection of antibodies that bound NKp46 were selected, produced and their variable regions further evaluated for their activity in the context of a bispecific molecule.

Example 7: Construction of Anti-CD19-lgG1 -Fcmono-Anti-NKp46

The aim of this experiment was to develop a new bispecific protein format that has advantages in production over currently available bispecific antibodies in development. Anti- CD19- Fcmono-Anti-NKp46 having its CH2-CH3 domains placed between two antigen binding domains, here two scFv units, was constructed using the same Fcmono and CD19 components as in Example 1 , in which the anti-CD3 scFv was replaced by an anti-NKp46 scFv constructed from the anti-NKp46 antibodies generated in Example 6. The anti-NKp46 scFv binds the activating receptor NKp46 on NK cells, and the other scFv to the lymphoma tumor antigen CD19. Four different antibodies were generated in Example 6 and selected to be functional as scFv for use in the bispecific protein format. The resulting proteins produced had the domain arrangement (N- to C- terminus):

(VK-VH)^anti-^CD19 - CH2 - CH3 - (VH-VK)^anti"NKp46

A DNA sequence coding for a CH3A H linker peptide having the amino acid sequence STGS was designed in order to insert a specific Sail restriction site at the CH3-VH junction. The domain arrangement of the final polypeptide is as in Figure 2 where the anti- CD3 scFv is replaced by the anti-NKp46 scFv (star in the CH2 domain indicates an optional N297S mutation). The (VK-VH) units include a linker between the VH and VK domains. Proteins were cloned, produced and purified as in Example 1. The amino acid sequences of the bispecific polypeptides (complete sequence (mature protein)) are shown in the corresponding SEQ ID NOS listed in the table below.

Example 8: NKp46 binding affinity by Surface Plasmon Resonance (SPR) Biacore T100 general procedure and reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GE Healthcare) at 25°C. In all Biacore experiments HBS-EP+ (Biacore GE Healthcare) and NaOH 10mM served as running buffer and regeneration buffer respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Protein-A was purchase from (GE Healthcare). Human NKp46 recombinant proteins were cloned, produced and purified at Innate Pharma.

Immobilization of Protein-A

Protein-A proteins were immobilized covalently to carboxyl groups in the dextran layer on a Sensor Chip CM5. The chip surface was activated with EDC/NHS (N-ethyl-N'-(3- dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). Protein-A was diluted to 10 μg ml in coupling buffer (10 mM acetate, pH 5.6) and injected until the appropriate immobilization level was reached (i.e. 2000 RU). Deactivation of the remaining activated groups was performed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

Binding study

Bispecific proteins at 1 μg mL were captured onto Protein-A chip and recombinant human NKp46 proteins were injected at 5 μg mL over captured bispecific antibodies. For blank subtraction, cycles were performed again replacing NKp46 proteins with running buffer.

Affinity study

The monovalent affinity study was done following a regular Capture-Kinetic protocol recommended by the manufacturer (Biacore GE Healthcare kinetic wizard). Seven serial dilutions of human NKp46 recombinant proteins, ranging from 6.25 to 400 nM were sequentially injected over the captured Bi-Specific antibodies and allowed to dissociate for 10 min before regeneration. The entire sensorgram sets were fitted using the 1 :1 kinetic binding model.

Results

SPR showed that the CD19-Fcmono-Anti-NKp46 bispecific polypeptides having the NKp46-1 , 2, 3 and 4 scFv binding domains bound to NKp46 with the monovalent affinities and kinetic association and dissociation rate constants are shown below in the table below. NKp46-3 lgG1 is full-length anti-NKp46 antibody of human lgG1 isotype shown for comparison.

Example 9: Binding of Anti-CD19-lgG1 -Fcmono-Anti-NKp46 to FcyR

The Anti-CD19-Fcmono-Anti-NKp46 were evaluated to assess whether such bispecific monomeric Fc protein could retain binding to Fey receptors.

Human lgG1 antibodies and CD19/NKp46-1 bi-specific antibodies were immobilized onto a Sensor Chip CM5. Recombinant FcyRs (cynomolgus monkey and human CD64, CD32a, CD32b, and CD16) were cloned, produced and purified at Innate Pharma. Figure 9 shows superimposed sensorgrams showing the binding of Macaca fascicularis recombinant FcgRs (upper panels ; CyCD64, CyCD32a, CYCD32b, CyCD16) and of Human recombinant FcgRs (lower panels ; HuCD64, HuCD32a, HuCD32b, HUCD16a ) to the immobilized human lgG1 control (grey) and CD19/NKp46-1 bi-specific antibody (black). Sensorgrams were aligned to zero in the y and x axis at the sample injection start.

Figure 9 shows that while full length wild type human lgG1 bound to all cynomolgus and human Fey receptors, the CD19/NKp46-1 bi-specific antibodies did not bind to any of the receptors. Example 10: Engagement of NK cells against Daudi tumor target with Fc-containing NKp46 x CD19 bispecific protein

CD19-lgG1-Fcmono-Anti-NKp46 bispecific antibodies having a monomeric Fc domain and a NKp46 binding region based on the different functional anti-NKp46 variable domains (NKp46-1 , NKp46-2, NKp46-3 or NKp46-4) were tested for functional ability to direct CD16-negative NK cells that express NKp46 to lyse CD19-positive tumor target cells (Daudi, a well characterized B lymphoblast cell line).

Briefly, the cytolytic activity of each of (a) resting human NK cells, and (b) human NK cell line KHYG-1 transfected with human NKp46, was assessed in a classical 4-h ⁵¹Cr- release assay in U-bottom 96 well plates. Daudi cells were labelled with ⁵¹Cr (50 μθί (1.85 MBq)/1 x 10⁶ cells), then mixed with KHYG-1 transfected with hNKp46 at an effector/target ratio equal to 50 for KHYG-1 , and 10 for resting NK cells, in the presence of monomeric bispecific antibodies at different concentrations. After brief centrifugation and 4 hours of incubation at 37°C, samples of supernatant were removed and transferred into a LumaPlate (Perkin Elmer Life Sciences, Boston, MA), and ⁵¹Cr release was measured with a TopCount NXT beta detector (PerkinElmer Life Sciences, Boston, MA). All experimental conditions were analyzed in triplicate, and the percentage of specific lysis was determined as follows: 100 x (mean cpm experimental release - mean cpm spontaneous release)/ (mean cpm total release - mean cpm spontaneous release). Percentage of total release is obtained by lysis of target cells with 2% Triton X100 (Sigma) and spontaneous release corresponds to target cells in medium (without effectors or Abs).

Results

In the KHYG-1 hNKp46 NK experimental model, each bi-specific antibody NKp46-1 , NKp46-2, NKp46-3 or NKp46-4 induced specific lysis of Daudi cells by human KHYG-1 hNKp46 NK cell line compared to negative controls (Human lgG1 isotype control (IC) and CD19/CD3 bi-specific antibodies), thereby showing that these antibodies induce Daudi target cell lysis by KHYG-1 hNKp46 through CD19/NKp46 cross-linking.

When resting NK cells were used as effectors, each bi-specific antibody NKp46-1 , NKp46-2, NKp46-3 or NKp46-4 again induced specific lysis of Daudi cells by human NK cells compared to negative control (Human lgG1 isotype control (IC) antibody), thereby showing that these antibodies induce Daudi target cell lysis by human NK cells through CD19/NKp46 cross-linking. Rituximab (RTX, chimeric lgG1 ) was used as a positive control of ADCC (Antibody-Dependent Cell Cytotoxicity) by resting human NK cells. The maximal response obtained with RTX (at 10 μg ml in this assay) was 21.6% specific lysis illustrating that the bispecific antibodies have high target cell lysis activity. Results for experiments with resting NK cells are shown in Figure 10.

Example 11 : Improved product profile and yield of different bispecific formats compared to existing formats Blinatumomab and bispecific antibodies having NKp46 and CD19 binding regions and different anti-NKp46 variable regions from antibodies NKp46-1 , NKp46-2 and NKp46-3, and blinatumomab, respectively were cloned and produced as CD19-lgG1 -Fcmono-Anti- NKp46 structures, and as DART and BITE formats following the same protocol and using the same expression system. CD19-lgG1 -Fcmono-Anti-NKp46, DART and BITE bispecific proteins were purified from cell culture supernatant by affinity chromatography using prot-A beads for CD19-lgG1 -Fcmono-Anti-NKp46 or Ni-NTA beads for DART and BITE. Purified proteins were further analysed and purified by SEC (Figure 1 1-A). BITE and DART showed a very low production yield compared to CD19-lgG1 -Fcmono-Anti-NKp46, and also have a very complex SEC profile. As shown in Figure 1 1 -B (arrows), DART and BITE are barely detectable by SDS-PAGE after Coomassie staining in the expected SEC fractions (3 and 4 for BITE and 4 and 5 for DART), whereas CD19-lgG1 -Fcmono-Anti-NKp46 format showed clear and simple SEC and SDS-PAGE profiles with a major peak containing the monomeric bispecific proteins. Moreover, the Fc domains present in proteins CD19-lgG1 -Fcmono-Anti- NKp46 proteins have the advantage of being adapted to affinity chromatography without the need for incorporation of peptide tags that will thereafter remain present as an unwanted part of a therapeutic product, such as in the case of BiTe and DART antibodies which cannot be purified by protein A. CD19-lgG1 -Fcmono-Anti-NKp46 antibodies were all bound by protein A. The table below shows productivity of different proteins. Protein Final

productivity yield

CD19-lgG1-Fcmono-NKp46-1 13.5 mg/L

CD19-lgG1-Fcmono- NKp46-2 4.1 mg/L

CD19-lgG1-Fcmono- NKp46-3 1 .5 mg/L

CD19-lgG1-Fcmono- NKp46-4 5.3 mg/L

DART -

BITE -

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e. g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of" or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

All publications and patent applications cited in this specification are herein incorporated by reference in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1 . A monomeric multispecific polypeptide that binds a first and a second antigen, comprising:

(a) a first antigen binding domain (ABD-i ) that specifically binds to a first antigen of interest,

(b) a human Fc domain or portion thereof that does not undergo interchain CH3-CH3 dimerization, wherein the Fc domain is operably linked to the C-terminus of the first antigen binding domain, and

(c) a second antigen binding domain (ABD₂) that specifically binds a second antigen of interest, wherein ABD₂ is operably linked to the C-terminus of the Fc domain, wherein the multispecific polypeptide is capable of binding to human neonatal Fc receptor (FcRn).

2. The composition of claim 1 , wherein the polypeptide substantially lacks binding to one or more human Fey receptors selected from the group consisting of: CD16, CD32A, CD32B and CD64.

3. The composition of claims 1-2, wherein the polypeptide comprises an amino acid mutation in the CH3 domain to prevent CH3-CH3 dimerization.

4. The composition of claims 1 -3, wherein the polypeptide has a domain arrangement: (ABD-i ) - CH2 - CH3 - (ABD₂).

5. The composition of claim 4, wherein the polypeptide has a domain arrangement: (ABD-i )— linker - CH2 - CH3— linker— (ABD₂).

6. The composition of any of the above claims, wherein the Fc domain comprises (i) a CH2 domain and (ii) a CH3 domain mutated to prevent CH3-CH3 dimerization.

7. The composition of any of the above claims, wherein the CH3 domain comprises an amino acid substitution at 1 , 2, 3, 4, 5, 6 or 7 of the positions L351 , T366, L368, P395, F405, T407 and/or K409 (EU numbering as in Kabat).

8. The composition of any of the above claims, wherein the polypeptide is a bispecific polypeptide.

9. The composition of any of the above claims, wherein each antigen binding domain comprises the hypervariable regions, optionally the heavy and light chain CDRs, of an antibody.

10. The composition of any of the above claims, wherein each antigen binding domain is an scFv.

1 1. The composition of any of the above claims, wherein the Fc domain is a human lgG1 Fc domain or a portion thereof, optionally comprising one or more amino acid modifications.

12. The composition of any of the above claims, wherein the Fc domain comprises a human CH2 domain comprising an amino acid substitution to reduce binding to a human Fey receptor.

13. The composition of any of the above claims, wherein one of the antigen binding domains binds to a cancer antigen.

14. The composition of any of the above claims, wherein one of the antigen binding domains binds to a viral or bacterial antigen.

15. The composition of any of the above claims, wherein one of the antigen binding domains binds to an activating cell surface receptor on an immune effector cell.

16. The composition of any of the above claims, wherein one of the antigen binding domains binds to a cell surface receptor on an immune effector cell and the other of the antigen binding domains binds to a cancer, viral or bacterial antigen.

17. The composition of claims 15-16, wherein the cell surface receptor on an immune effector cell is an activating receptor.

18. The composition of claims 15-17, wherein the cell surface receptor on an immune effector cell is member of the immunoglobulin superfamily, a member of the natural cytotoxicity receptor family or a member of the NK cell lectin-like receptor family.

19. The composition of claim 17-18, wherein the activating cell surface receptor on an immune effector cell is selected from the group consisting of: NKG2D, NKp30, NKG2D, NKp44 and DNAM-1 .

20. The composition of claims 16-19, wherein the polypeptide activates effector cells expressing the activating receptor, when incubated with such effector cells in the presence of cells expressing the cancer, viral or bacterial antigen.

21. The composition of claims 16-20, wherein the polypeptide does not exhibit activation of effector cells expressing the activating receptor when incubated with such effector cells in the absence of cells expressing the cancer, viral or bacterial antigen.

22. The composition of claims 16-21 , wherein the polypeptide does not exhibit activation of CD16-positive cells that do not express the activating receptor, when incubated with such cells in the presence of cells expressing the cancer, viral or bacterial antigen.

23. The composition of claims 16-19, wherein the multispecific protein (a) activates effector cells, when incubated with effector cells expressing the activating receptor in the presence of target cells; and (b) does not activate a such effector cells when incubated with such effector cells in the absence of target cells.

24. The composition of claims 20-23, wherein the effector cells are NK cells.

25. The composition of claims 20-23, wherein the effector cells are T cells.

26. The composition of any of the above claims, wherein the antibody comprises framework residues from a human framework region.

27. A pharmaceutical composition comprising a compound of any one of the above claims, and a pharmaceutically acceptable carrier.

28. Use of a polypeptide or composition of any one of the above claims as a medicament for the treatment of disease.

29. A method of treating a disease in a subject comprising administering to the subject a composition of claims 1 -27.

30. The method or use of claims 28-29, wherein the disease is a cancer, infectious disease or an inflammatory or autoimmune disease.

31. A method of producing a polypeptide described herein comprising:

(a) providing a nucleic acid encoding a polypeptide of claim 1-27; and

(b) expressing said nucleic acid in a host cell to produce said polypeptide, respectively; and recovering said polypeptide.

32. The method of claim 31 , wherein recovering said polypeptide comprises loading the polypeptide produced onto an affinity purification support, optionally a Protein-A support or column, and collecting the polypeptide.

33. A method for identifying or evaluating a polypeptide, comprising the steps of:

(a) providing a nucleic acid encoding a polypeptide of claim 1 -27;

(b) expressing said nucleic acid in a host cell to produce said polypeptide, respectively; and recovering said polypeptide; and

(c) evaluating the polypeptide produced for a biological activity of interest.

34. The method of claim 33, wherein evaluating the polypeptide comprises:

(i) testing the ability of the polypeptide to activate effector cells that express the activating receptor, when incubated with such effector cells in the presence of target cells (that express antigen of interest) and/or

(i) testing the ability of the polypeptide to activate effector cells that express the activating receptor, when incubated with such effector cells in the absence of target cells (that express antigen of interest).