CN119816521A - Molecules grafted to immunoglobulins with multiple functions or binding specificities - Google Patents

Abstract

The present invention relates to multispecific molecular complexes. In particular, the present disclosure relates to multi-specific protein complexes, such as molecular grafted immunoglobulins, platforms for preparing such immunoglobulin-based multi-specific complexes, and related uses.

Description

Molecular grafted immunoglobulins with multiple functions or binding specificities

Cross reference

The present application is based on 35U.S. C. ≡119 (e) claiming priority from U.S. provisional patent application No. 63/376,836 filed on 9/23 2022. The entire contents of the foregoing application are incorporated herein by reference.

Reference to electronic sequence Listing

The contents of the electronic sequence Listing (SeqList _190913-00401.Xml; size: 104,229 bytes; and date of creation: 2023, month 9, 18) are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to multifunctional molecular complexes. In particular, the disclosure relates to multifunctional or multispecific protein complexes, such as multispecific immunoglobulins, and platforms for preparing different forms of such complexes.

Background

Specific protein complexes, in which two or more target-specific moieties are designed as a single molecule or complex, have evolved rapidly in recent years and offer attractive solutions for a wide range of clinical and diagnostic applications. For example, bispecific antibodies and fusion proteins have been developed for binding to more than one antigen or more than one epitope on the same antigen. Multispecific antibodies have multifunctional, more precise targeting capabilities and higher potency than traditional antibodies, and thus show potential in the treatment of various diseases, for example, as mediators to relocate effector mechanisms to disease-related sites, and become attractive for next-generation antibody therapies. One of the major obstacles to the development of multispecific antibodies is the difficulty in producing materials of sufficient quality and quantity by conventional techniques (e.g., hybridomas and chemical conjugation methods). Although alternative bispecific antibody platforms, such as Knob-in-Hole, can be used, they also face challenges such as reduced antibody activity, poor stability, and difficulty in purifying or removing impurities. The inherent complexity of multi-specific recombinant protein molecules and antibodies can present challenges to process development and manufacture from a cost-effective and quality control perspective.

Of the various FDA approved drugs, bispecific T cell cement (BiTE) Blinatumomab represents a unique therapeutic prospect due to its engineering structure and clinical efficacy against relapsed or refractory B-lineage leukemia or lymphoma. However, the BiTE bispecific antibody represented by Blinatumomab has a short half-life in vivo, and requires continuous administration for a long period of time, which causes great inconvenience in treatment. Such antibodies may also cause Cytokine Release Syndrome (CRS), a series of symptoms that may occur as a side effect of certain types of immunotherapy (Klinger M, blood 2012; 119:6226-33.), but not all bispecific forms necessarily have the same risk. Trivalent bispecific forms (X) -3s have been generated in which anti-CD 3 scFv are covalently linked to a stable dimer of a cancer targeting Fab using a docking locking method. The efficacy of the cytokine release inducer in the form of (X) -3s is much lower than that of BiTE, and even the addition of interferon-alpha in the treatment regimen is unlikely to increase this risk (Rossi EA, mol Cancer Ther.2014Oct;13 (10): 2341-51.). However, as with BiTE, the lifetime of (X) -3 in circulation may be short due to the lack of an Fc domain that binds to neonatal Fc receptors and mediates circulation of the antibody to the plasma membrane and subsequent release back into serum. By this mechanism, the half-life of the IgG molecule is significantly prolonged. Thus, there is a need for methods and compositions for generating novel immunoglobulin-form-based multispecific complexes and related platforms.

Disclosure of Invention

The present disclosure addresses the above-described needs in several aspects.

In one aspect, the present disclosure provides a protein complex comprising:

(a) A first portion comprising two immunoglobulin light chains and two immunoglobulin heavy chains, wherein the two light chains or the two heavy chains are linked to two dimerization/docking domain (DDD) portions, respectively, and

(B) A second portion comprising (i) an Anchoring Domain (AD) portion and (ii) an agent linked to the AD portion, the AD portion comprising a sequence that is at least 70% (e.g., any number between 70% and 100%, including 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) identical to the sequence of SEQ ID No. 3 or 2 or 46. The two DDD moieties form a dimer that binds to the AD moiety. Examples of such DDD moieties may include the sequence of SEQ ID NO. 1 and other sequences described herein.

In one embodiment, (a) the first moiety is a targeting moiety that specifically binds an antigen or epitope, and (b) the second moiety is an effector moiety. The agent may be an effector agent.

In another embodiment, (a) the first moiety is an effector moiety comprising an effector agent, and (b) the second moiety is a targeting moiety. The agent may be a targeting agent that specifically binds an antigen or epitope.

In yet another embodiment, the first moiety and the second moiety are two targeting moieties that specifically bind to two antigens or epitopes. The two antigens or epitopes may be the same or different.

In a further embodiment, the first moiety and the second moiety are two effector moieties and the agent is an effector. The two effectors may be the same or different.

In a second aspect, the present disclosure provides a fusion protein comprising (i) a dimerization/docking domain (DDD) moiety and (ii) an immunoglobulin light chain fused to the DDD moiety, or an immunoglobulin light chain fragment fused to the DDD moiety, or an immunoglobulin heavy chain fragment fused to the DDD moiety. The immunoglobulin heavy chain may comprise an Fc region or fragment thereof.

In the protein complexes or fusion proteins described above, the DDD moiety or the AD moiety may be fused at any suitable position (e.g., N-terminal, C-terminal, or intermediate) of the polypeptide chain.

In some embodiments, each DDD moiety can be inserted into each immunoglobulin heavy chain. For example, the DDD portion can be inserted into the hinge region, variant hinge region, or hybrid hinge region of the immunoglobulin heavy chain or hinge flanking regions thereof.

In some embodiments, each DDD moiety can be fused to the C-terminus of each immunoglobulin light chain. In some examples, the DDD moiety can be fused to the C-terminus of the immunoglobulin light chain via a linker sequence. The linker sequence may comprise at least one cysteine and the protein complex may comprise a disulfide bond between two linker sequences. Disulfide bonds between the two linker sequences help form a stable linker sequence structure.

In the protein complexes or fusion proteins described above, each DDD moiety may be fused to the C-terminus of each immunoglobulin heavy chain.

The targeting moiety in the protein complex may specifically bind to a tumor-associated antigen or a disease-associated antigen. Examples of the tumor-associated antigen or the disease-associated antigen include, but are not limited to Trop2、EpCAM、GPRC5、FcRH5、RORl、BCMA、CD15、CD16、CD19、CD20、CD22、CD27、CD30、CD33、CD40、CD47、CD40L、CD66、CD70、CD74、CD79b、CD80、CD95、CD133、CD160、CD166、CD229、MUC1、MUC5、MUC16、IGF-1R、EGFR、HER2、HER3、EGP2、HLA-DR、TNF-a、TRAIL receptor, ICOS, ICOSL, VEGF, VEGFR, hypoxia-inducible factor (HIF), flt-3, folate receptor, TDGF1, tfR, mesothelin 、PSMA、CEACAM5、CEACAM6、B7、IFN-α、IFN-β、IFN-γ、IFN-λ、IL-1β、IL2、IL6、IL-6R、IL-15、IL-15R、IL-17、IL-17R、IL-12、C1r、C1s、C2、C3、C5、C5a、C5aR1、C6、MASP、MSAP2、MASP3、FB、FD、 properdin 、Lag-3、CTLA-4、PD-1、PD-L1、TIM3、SIRPa、TIGIT、OX40、OX40L、4-1BB、NKG2A、NKG2B、BTLA、GITR、GITRL、TCR、Nectin-4、c-Met、LIV1、 mesothelin, DLL3, DLL4, tissue factor, TGF-beta receptor, DKK1, and CLDN18.2.

In some embodiments of the protein complex, the immunoglobulin light chain may comprise a light chain variable region that may comprise LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the sequences of SEQ ID NOS: 21-23. The immunoglobulin heavy chain may comprise a heavy chain variable region that may comprise HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the sequences of SEQ ID NOS 24-26. The immunoglobulin light chain may comprise the sequence of SEQ ID NO. 13. The immunoglobulin heavy chain may comprise the sequence of SEQ ID NO. 14.

In some embodiments of the protein complex, the immunoglobulin light chain may comprise a light chain variable region that may comprise LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the sequences of SEQ ID NOS: 40-42. The immunoglobulin heavy chain may comprise a heavy chain variable region that may comprise HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the sequences of SEQ ID NOS: 43-45. The immunoglobulin light chain may comprise the sequence of SEQ ID NO. 10 or 47. The immunoglobulin heavy chain may comprise the sequence of SEQ ID NO. 12 or 48 or 49.

In a protein complex, the effector agent may include an antibody or antigen-binding fragment thereof, an aptamer, a ligand, a cytotoxin, a chemotherapeutic agent, a detectable label or tag, a drug, a prodrug, a toxin, an enzyme, an immunomodulator, a checkpoint inhibitor, an anti-angiogenic agent, a pro-apoptotic agent, a cytokine, a growth factor, a hormone, a cytokine, a radioisotope, a protein, a peptide, a peptidomimetic, a polynucleotide, an RNAi oligosaccharide, a natural or synthetic polymeric substance, a nanoparticle, a quantum dot, an organic compound or an inorganic compound. The antibody or antigen binding fragment thereof may specifically bind to a marker on an immune cell. In one embodiment, the antibody specifically binds to a T cell specific marker, such as CD3. The antibody or antigen binding fragment thereof may comprise (A) the sequence of SEQ ID NOS 15-20, or (B) the sequences of SEQ ID NOS 4 and 5, or (C) one or more sequences selected from the group consisting of SEQ ID NOS 8, 9, 29 and 31-38.

The protein complex or fusion protein or antibody or antigen binding fragment described above may further comprise a variant Fc constant region.

In another aspect, the present disclosure provides an isolated nucleic acid sequence or sequences encoding the protein complexes or fusion proteins described above. Thus, expression vectors comprising the one or more nucleic acid sequences, as well as host cells comprising the vector or the one or more nucleic acid sequences, are within the scope of the present disclosure.

The present disclosure provides a method of preparing the above protein complex or fusion protein. The method may comprise:

culturing said cell under conditions permitting expression of (i) said fusion protein or (ii) said protein complex, and assembling said protein complex either intra-or extracellular, and purifying said protein complex or fusion protein from said cultured cell or said cell culture medium. In a preferred embodiment, the assembly is intracellular.

The present disclosure further provides a pharmaceutical composition comprising the above protein complex or fusion protein or antibody or antigen binding fragment and a pharmaceutically acceptable carrier.

Also provided are methods of treating cancer or a disease in a subject in need thereof. The method comprises administering to the subject an effective amount of the protein complex or fusion protein or pharmaceutical composition described above. Examples of diseases include cancer diseases (e.g., breast cancer, lung cancer, stomach cancer, colorectal cancer, bladder cancer, liver cancer, prostate cancer, pancreatic cancer, melanoma, leukemia, lymphoma, multiple myeloma), immune diseases (e.g., autoimmune diseases), and pathogen (e.g., viral, bacterial, fungal, or parasitic) infections.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and claims.

Drawings

FIG. 1 is a schematic diagram of the single chain grafting of an IgG antibody (A) to another antibody (B) to form a bispecific antibody. AD2 of antibody B binds to DDD2 dimer inserted into the hinge region of IgG antibody a.

FIG. 2 is a schematic representation of the single chain grafting of IgG antibody (A) to another antibody (B) to form a bispecific antibody. AD7 of antibody B binds to DDD2 dimer inserted into the hinge region of IgG antibody a.

FIG. 3 is a schematic representation of the grafting of an IgG antibody (A) with the Fab of another antibody (B) to form a bispecific antibody. In the Fab of antibody B, the domains of V _L and V _H or C _H1 and C _L are interchanged, and AD2 or AD7 is fused to the C-terminus of C _L and binds to the DDD2 dimer inserted into the hinge region of IgG antibody A.

FIG. 4 is a schematic representation of the grafting of an IgG antibody (A) with the Fab of another antibody (B) to form a bispecific antibody. In the Fab of antibody B, the domains of V _L and V _H or C _H1 and C _L are interchanged, and AD2 or AD7 is fused to the C-terminus of C _H1 and binds to the DDD2 dimer inserted into the hinge region of IgG antibody A.

FIG. 5 is a photograph showing bispecific antibodies and their modules in SDS-PAGE gels. Three bispecific antibodies against CD3 (huα3sc) and Trop2 (hL 0125-Cm) or HER2 (T-Cm) were constructed and generated according to the design in fig. 1 and 2. Lanes M, protein molecular weight markers, 1 and 4, huα3sc-AD7 XhL 0125-Cm, 2 and 5, huα3sc-AD2 XhL 0125-Cm, 3 and 6, huα3sc-AD2 XT-Cm. R is a reducing condition, NR is a non-reducing condition.

FIG. 6 shows high performance liquid chromatography analysis of bispecific antibodies.

FIGS. 7A and 7B are graphs showing binding of antibodies to CD3 on the surface of Jurkat cells. Cells were dispensed into 96-well plates at 2 x 10 ⁵/well and incubated with the indicated reagents for 45 min at 4 ℃. Fig. 7A shows incubation of cells with AF 488-labeled goat anti-mouse IgG Fc after washing with PBS. Fig. 7B shows incubation of cells with AF 488-labeled goat anti-human IgG Fc after washing with PBS. Binding was analyzed by Attune NxT flow cytometer.

FIGS. 8A, 8B and 8C are graphs showing binding of antibodies to MDA-MB-468, HCC1806 and BT-474 cell surface Trop2 or HER2, respectively. FIG. 8A shows binding of antibodies to MDA-MB-468 cell surface Trop2 or HER 2. Fig. 8B shows binding of antibodies to HCC1806 cell surface Trop2 or HER 2. FIG. 8C shows binding of antibodies to BT-474 cell surface Trop2 or HER 2. Cells were dispensed into 96-well plates at 2 x 10 ⁵/well and incubated with the indicated reagents for 45min at 4 ℃. After washing with PBS, cells were incubated with AF 488-labeled goat anti-human IgG Fc. Binding was analyzed by Attune NxT flow cytometer.

FIGS. 9A, 9B, 9C and 9D are graphs showing in vitro cytotoxicity of bispecific antibodies and their component monoclonal antibodies (mAbs). FIG. 9A shows in vitro cytotoxicity of bispecific antibodies and their constituent monoclonal antibodies against MDA-MB-468 cells. Fig. 9B shows in vitro cytotoxicity of bispecific antibodies and their component monoclonal antibodies against HCC1806 cells. FIG. 9C shows in vitro cytotoxicity of bispecific antibodies and their constituent monoclonal antibodies against HCT-116 cells. FIG. 9D shows in vitro cytotoxicity of bispecific antibodies and their component monoclonal antibodies against BT-474 cells. Cancer cells were mixed with human Peripheral Blood Mononuclear Cells (PBMCs) and dispensed into 96-well plates at 200 μl/well, each well containing 1X 10 ⁴ tumor cells and 1X 10 ⁵ PBMC cells (ratio of PBMC to target cells 8:1). Bispecific antibodies and their component monoclonal antibodies were serially diluted 4-fold starting at 20nmol/L to treat mixed cells. After 60 hours incubation, the medium was removed and replaced with fresh medium to flush out PBMC cells and dead cancer cells. Cell viability was determined by MTS reagent. The experiment was repeated three times. The efficacy of each agent on cancer cells is shown in IC ₅₀ values, as detailed in Table 3.

FIGS. 10A, 10B, 10C and 10D are schematic diagrams of structural models of four bispecific antibodies.

Fig. 10A shows that scFv of antibody b and IgG of antibody a are site-specifically assembled by AD2 fused at the C-terminus in scFv of antibody b and DDD2 in the two light chains of IgG of antibody a, respectively.

Fig. 10B shows that scFv of antibody B and IgG of antibody a are site-specifically assembled by AD2 fused at the C-terminus in scFv of antibody B and DDD2 in the two light chains of IgG of antibody a, respectively, and form and stabilize intramolecular DDD2 dimer by adding disulfide bonds between the two linkers.

Fig. 10C shows that AD2 in scFv of antibody b binds to DDD2 dimer inserted into IgG hinge region of antibody a.

Figure 10D shows that two DDD2 peptides fused to the C-terminus of the IgG heavy chain of antibody a were dimerized and bound to AD2 in the scFv of antibody b.

FIGS. 11A, 11B, 11C and 11D are photographs showing bispecific antibodies and their modules in SDS-PAGE gels. Four forms of bispecific antibodies against CD3 (3 scFv) and Trop2 (hL 0125) were constructed and produced as designed in the schematic models a-D in fig. 10. (A) 3scFv XhL 0125-A, (B) 3scFv XhL 0125-B, (C) 3scFv XhL 0125-C, and (D) 3scFv XhL 0125-D. Lanes M, protein molecular weight markers, bispecific conjugates of 1 and 4,3scFv-AD2, 2 and 5, hL0125-DDD2, 3 and 6,3scFv-AD2 and hL0125-DDD 2. R, reducing conditions, NR, non-reducing conditions.

Detailed Description

The present disclosure relates to multispecific molecular complexes, e.g., multispecific protein complexes, e.g., multispecific or bispecific antibodies, and platforms for preparing different forms of such complexes. Certain aspects of the invention are based, at least in part, on the unexpected discovery that heterologous protein-protein interaction domains (e.g., DDD and AD) can be incorporated into or linked to proteins or antibodies at various unexpected locations to produce functional multispecific protein complexes or multispecific antibodies.

In contrast to conventional bispecific antibody platforms, the platforms disclosed herein represent a unique novel design that differs from conventional heavy chain heteromerized bispecific antibodies, but also does not require the use of cross mab-like domain recombination. This design is not a simple fusion expression of the protein. In contrast, multi-specific platforms offer a number of advantages over conventional platforms, including ease of production and purification, due to their significant optimization in terms of structure and biological activity. Indeed, as disclosed herein, the multispecific or bispecific molecules can be expressed and assembled in a single cell, and thus conventional standard IgG separation and purification methods can be applied to obtain the multispecific or bispecific molecules.

In certain embodiments, the platform allows for the retention of intact IgG, fc, and/or Fc molecular domain structures and the realization of a "1+2" valence pattern for two different targets. In the context of immunooncology, this enables targeting cancer cells and redirecting closely contacted T cells. This "1+2" pattern can meet the requirement of different affinities of two cells or two targets.

Multispecific molecular complexes

One aspect of the disclosure provides a platform for a multi-specific protein complex. In one embodiment, the protein complex may generally have, inter alia, two functional components or portions (1) a first portion comprising two immunoglobulin light chains and two immunoglobulin heavy chains, wherein the two light chains or the two heavy chains are linked to two dimerization/docking domain (DDD) portions, respectively, and (2) a second portion comprising (i) an Anchor Domain (AD) portion and (ii) an agent linked to the AD portion. Two copies of the DDD moiety form dimers that bind to the AD moiety. Preferably, one of the two immunoglobulin heavy chains may have an Fc region or fragment thereof. In some embodiments, both immunoglobulin heavy chains may have an Fc region or Fc fragment.

The first moiety or the second moiety may comprise or be a targeting moiety or an effector moiety. For example, in one embodiment, the first moiety is a targeting moiety that specifically binds an antigen or epitope, and the second moiety is an effector moiety and the agent is an effector. In another embodiment, the first moiety is an effector moiety comprising an effector agent, the second moiety is a targeting moiety, and the agent is a targeting agent that specifically binds an antigen or epitope. In further embodiments, the first moiety and the second moiety are two targeting moieties that specifically bind to two antigens or epitopes. In yet another embodiment, the first moiety and the second moiety are two effector moieties and the agent is an effector.

The first and second portions may together provide a plurality of binding sites, and the close proximity of binding sites may result in the formation of new complexes (target cells and effectors) and trigger new cell contacts. For example, as disclosed herein, with two or more sites of interaction with target cells (e.g., cancer cells), more targeted binding can be achieved and additional immune responses involving immune effector cells (e.g., T cells and natural killer cells) can be activated, resulting in greater targeted cytotoxic effects.

Immunoglobulin protein

Immunoglobulin refers to a naturally occurring or recombinantly produced antibody molecule that serves as a critical part of an immune response by specifically recognizing and binding to an antigen. There are five main classes of immunoglobulins, igA, igD, igE, igG and IgM. Based on additional minor differences in heavy chain amino acid sequences, igG and IgA are further divided into subclasses (e.g., human IgG1, igG2, igG3, igG4, igA1, and IgA 2). The various immunoglobulin classes and subclasses vary in biological characteristics, structure, and target specificity.

Immunoglobulins are heterodimeric proteins consisting of two heavy chains and two light chains, which may consist of kappa chains or lambda chains. Either the heavy or light chain can be functionally divided into a variable domain (Fv) that binds antigen and a constant domain (Fc) that specifies effector functions (e.g., activation of complement or binding to Fc receptors). The light chain comprises only one constant domain (Cκ or Cλ), whereas the heavy chain generally comprises three such domains (CH 1, CH2, CH 3) and a hinge region between the first domain (CH 1) and the second domain (CH 2) (Schroeder et al, J ALLERGY CLIN immunol.2010,125 (2 02): S41-S52).

"Hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to a domain that connects a CH1 domain to the heavy chain constant region of a CH2 domain, and includes the upper, middle and lower portions of the hinge (Roux et al, 1998J Immunol161:4083). The hinge provides a different level of flexibility between the binding and effector regions of the antibody and provides an intermolecular disulfide bonding site between the two heavy chain constant regions. As used herein, for all IgG isotypes, the hinge starts at E216 and ends at Gly237 (Roux et al, 1998JImmunol 161:4083). The sequences of wild-type IgG1, igG2, igG3 and IgG4 hinges are shown in table a. The term "hinge" includes wild-type hinges (e.g., those listed in tables A, B and C), variant hinges, and hybrid hinges thereof (e.g., non-naturally occurring hinges or modified hinges). For example, the term "IgG1 hinge" includes wild-type IgG1 hinges (E216-G237, SEQ ID NO: 65), as shown in Table B, as well as variants having 1,2, 3, 4, 5, 1-3, 1-5, 3-5 and/or up to 5, 4, 3, 2, or 1 mutations, such as substitutions, deletions, or additions. In certain embodiments, the hinge is a hybrid hinge comprising sequences from at least two isoforms. For example, the hinges may include an upper hinge, a middle hinge, or a lower hinge from one isoform and the remainder of the hinge from one or more other isoforms. For example, the hinge may be an IgG2/IgG1 hinge and may include, for example, an upper hinge and a middle hinge of IgG2 and a lower hinge of IgGl. The hinge may have an effector function or be deprived of an effector function. For example, the lower hinge of wild-type IgG1 provides effector function.

IgG hinge region amino acids (Roux et al 1998J Immunol 161:4083)

* The C-terminal amino acid sequence of the CH1 domain.

The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge in the heavy chain constant domain. As used herein, an IgG CH1 domain starts at a118 and ends at V215 (table B), and an IgA CH1 domain starts at a120 and ends at P221 (table C). The term "CH1 domain" includes wild-type CH1 domains (e.g., having the sequence of SEQ ID NO:65 for IgG1 and SEQ ID NO:66 for IgG2, table B), and variants thereof (e.g., non-naturally occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes wild-type CH1 domains and variants thereof having 1,2,3, 4, 5, 1-3, 1-5, 3-5 and/or up to 5, 4, 3, 2 or 1 mutations, such as substitutions, deletions or additions. Exemplary CH1 domains include CH1 domains with mutations that alter the biological activity of the antibody, such as ADCC, CDC, or half-life.

TABLE B IgG heavy chain constant region amino acids

The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain in the heavy chain constant domain. As used herein, an IgG CH2 domain starts at P238 and ends at K340 (table B), and an IgA CH2 domain starts at C241 and ends at S341 (table C). The term "CH2 domain" includes wild-type CH2 domains (e.g., domains with IgG 1; table B), and variants thereof (e.g., non-naturally occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wild-type CH2 domains and variants thereof having 1, 2,3, 4, 5, 1-3, 1-5, 3-5 and/or up to 5, 4, 3, 2 or 1 mutation, e.g., substitution, deletion or addition. Exemplary CH2 domains include CH2 domains with mutations that alter the biological activity of the antibody, such as ADCC, CDC, or half-life. In certain embodiments, provided herein are CH2 domains comprising modifications that affect the biological activity of an antibody.

The term "CH3 domain" refers to the heavy chain constant region at the C-terminus of the CH2 domain in the heavy chain constant domain. As used herein, an IgG CH3 domain starts at G341 and ends at K447 (table B), and an IgA CH3 domain starts at G342 and ends at Y472 (table C). The term "CH3 domain" includes wild-type CH3 domains (e.g., domains with IgG 1; table B), and variants thereof (e.g., non-naturally occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wild-type CH3 domains and variants thereof having 1,2, 3, 4, 5, 1-3, 1-5, 3-5 and/or up to 5, 4, 3, 2 or 1 mutation, e.g., substitution, deletion or addition. Exemplary CH3 domains include CH3 domains with mutations that alter the biological activity of the antibody, such as ADCC, CDC, or half-life.

IgA heavy chain constant region amino acids (patent US10822399B 2)

Amino acid numbering above human IgA1 is according to the protocol commonly used for IgA1 Bur (Liu et al, science,1976, vol. 193:1017-1020).

Targeting moiety

One component of the protein complex described above may be a targeting moiety that specifically binds to a target. A targeting moiety or targeting domain or targeting agent, as used interchangeably herein, refers to an entity (e.g., a molecule) that facilitates interaction (e.g., binding of a protein to a target) and/or directs a protein to a target. The targeting moiety or domain or agent may be a polypeptide, an antibody or an antigen binding portion thereof.

As used herein, a "target" may be a cell, pathogen, metabolite, polypeptide complex, or any molecule or structure present in the tissue of a subject or circulating in the circulatory system or lymphatic system, such as an immune cell or cancer cell. The target may be any such aspect that readily interacts with a targeting moiety or targeting domain. In some embodiments, the term refers to a moiety, e.g., an antibody molecule, that preferentially localizes a therapeutic compound to a target tissue or cell as a component of the therapeutic compound.

In some embodiments, the targeting moiety may act in the protein complex by delivering the protein complex and/or the effector moiety to the local environment of a pathogen, disease cell, or cancer cell, thereby effecting a local therapeutic strategy. In certain embodiments, the targeting moiety targets a cancer cell by specifically binding to a pathogen, a disease cell, or a cancer cell. As disclosed herein, the targeting moiety or targeting domain or targeting agent described above may specifically bind to a disease-associated antigen, such as a tumor-associated antigen.

"Disease-associated antigen" refers to an antigen that is expressed concurrently with a particular disease process, wherein antigen expression is associated with or predicts the progression of the disease. The disease-associated antigen may be an antigen recognized by T cells or B cells. Some disease-associated antigens may also be tissue specific. Tissue specific antigens are expressed in a limited number of tissues. The disease-associated antigen may be, for example, a tumor-associated antigen, a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen.

"Tumor-associated antigen" refers to an antigen that is predominantly present on, in, or in the microenvironment of a tumor, and that can be used to treat one or more tumors. Tumor-associated antigens differ from normal cellular proteins by their unique characteristics of expression levels, localization, or major histocompatibility processing, which enable them to target malignant tumors effectively. Tumor-associated antigens can be broadly divided into three classes, aberrantly expressed autoantigens, mutated autoantigens and tumor-specific antigens. Tumor-associated antigens as used herein include antigens that are useful as targets for the treatment of one or more tumors, wherein up-regulation/activation or down-regulation/inhibition thereof is associated with tumorigenesis or tumor progression. Tumor-associated antigens as used herein also refer to peptides that have been isolated and identified from tumor material, which undergo antigen processing in antigen presenting cells, and thus are recognized by immune effector cells of the host. In particular, it refers to antigens that are specifically expressed on, associated with, or overexpressed in tumor tissue. TAA peptides may include or may consist of 5 to 20, 8 to 14, 8 to 12, e.g. 9 to 11 amino acids. In one aspect, TAA peptides that can be used with the methods and embodiments described herein include, for example, those TAA peptides described in U.S. patent publication No. 20160187351, U.S. patent publication No. 20170165335, U.S. patent publication No. 20170035807, U.S. patent publication No. 20160280759, U.S. patent publication No. 20160287687, U.S. patent publication No. 20160287687, U.S. patent publication No. 20160346371, U.S. patent publication No. 20160368965, U.S. patent publication No. 20170022251, U.S. patent publication No. 20170002055, U.S. patent publication No. 20170029486, U.S. patent publication No. 20170037089, U.S. patent publication No. 20170136108, U.S. patent publication No. 20170101473, U.S. patent publication No. 20170096461, U.S. patent publication No. 20170165337, U.S. patent publication No. 20170189505, U.S. patent publication No. 20170173132, U.S. patent publication No. 20170296640, U.S. patent publication No. 20170253633, U.S. patent publication No. 20170260249, U.S. patent publication No. 20180051080, and U.S. patent publication No. 20180164315, the contents of each of these publications and the sequence listing therein are incorporated herein by reference in their entirety.

The term "viral antigen" refers to an antigen derived from any pathogenic virus associated with a disease. Exemplary disease-associated viral antigens include, but are not limited to, those derived from adenovirus, coxsackie virus, criomitic-congo hemorrhagic fever virus, cytomegalovirus ("CMV"), dengue virus, ebola virus, epstein barr virus ("EBV"), melon rito virus, herpes simplex virus type 1 ("HSV-1"), herpes simplex virus type 2 ("HSV-2"), human herpes virus type 8 ("HHV-8"), hepatitis a virus ("HAV"), hepatitis b virus ("HBV"), hepatitis c virus ("HCV"), hepatitis delta virus ("HDV"), hepatitis e virus ("HEV"), human immunodeficiency virus ("HIV"), influenza virus, hoof virus, lassa virus, ma Qiubo virus, marburg virus, measles virus, human metapneumovirus, mumps virus, norwalk virus, human papilloma virus ("HPV"), parainfluenza virus, parvovirus, polio virus, rabies virus, respiratory syncytial virus ("RSV"), rhinovirus, rotavirus, rubella virus, sarcandid virus, severe respiratory syndrome virus ("HEV"), varicella virus, and west nile virus.

The term "bacterial antigen" refers to an antigen derived from any disease-associated pathogenic virus. Exemplary bacterial antigens include, but are not limited to, antigens derived from bacillus anthracis, bordetella pertussis, borrelia burgdorferi, brucella abortus, brucella canis, brucella ovis, brucella suis, campylobacter jejuni, chlamydia pneumoniae, chlamydia trachomatis, chlamydia psittaci, clostridium botulinum, clostridium difficile, clostridium aerogenes, clostridium tetani, diphtheria, enterococcus faecalis, enterococcus faecium, escherichia coli, enterotoxigenic escherichia coli, pathogenic escherichia coli, escherichia coli) 157:h7, franciscens, haemophilus influenzae, helicobacter pylori, legionella pneumophila, leptospira question, listeria monocytogenes, mycobacterium leprosy, mycobacterium tuberculosis, mycoplasma pneumoniae, neisseria meningitidis, pseudomonas aeruginosa, rickettsia, typhoid, typhus, murine, shigella, staphylococcus epidermidis, staphylococcus, streptococcus agaricus, streptococcus mutans, streptococcus mutans, and streptococcus mutans.

The term "fungal antigen" refers to an antigen derived from any disease-associated pathogenic fungus. Exemplary fungal antigens include, but are not limited to, antigens derived from aspergillus clavatus, aspergillus flavus, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus terreus, blastomycosis dermatitis, candida albicans, candida dubli, candida glabrata, candida parapsilosis, candida tropicalis, cryptococcus albus, cryptococcus laurentii, cryptococcus neoformans, histoplasma capsulatum, microsporobacteria canis, pneumosporium carinii, pneumosporium yersii, trichosporon sinkii, staphylococcus epidermidis, trichophyton mentagrophytes, trichophyton, tinea capitis, tinea corporis, tinea cruris, facial tinea, cryptomycosis, trichophyton rubrum and trichophyton mentagrophytes. The term "parasite antigen" refers to an antigen derived from any pathogenic parasite associated with a disease. Exemplary parasite antigens include, but are not limited to, antigens derived from the genera of Aphelenchoides, babesia, begonia, cryptosporidium, cyclosporia, echinococcus, meindian, endomenta, giardia duodenum, giardia intestinalis, giardia lamblia, leishmania falciparum, schistosoma mansoni, egyptia, schistosoma japonicum, taenia, toxoplasma, trichinella and trypanosoma cruzi.

Antibodies to

In certain embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. An antigen binding fragment refers to any antibody fragment, e.g., scFv or other functional fragment, that retains its binding activity to a target on a cancer cell, including an immunoglobulin lacking a light chain, VHH, VNAR, fab, fab ', F (ab') 2, fv, antibody fragment, diabody, scAB, single domain heavy chain antibody, single domain light chain antibody, fd, CDR region, or any portion or peptide sequence of an antibody capable of binding an antigen or epitope. VHH and VNAR are alternatives to traditional antibodies, although they are produced in different species (camelid and shark, respectively). Unless specifically noted as a "full length antibody," when the disclosure relates to an antibody, it inherently includes reference to antigen-binding fragments thereof.

Some targets for antibodies, antigen binding fragments or proteins (examples of cancer cell types in brackets) may include Her2/Neu (epithelial malignancy), CD22 (B cells, autoimmune or malignancy), epCAM (CD 326) (epithelial malignancy), EGFR (epithelial malignancy), PSMA (prostate cancer), CD30 (B cell malignancy), CD20 (cells, autoimmune, allergic or malignancy), CD33 (bone marrow malignancy), membranlgE (allergic B cells), lgE receptor (CD 23) (mast cells or B cells in allergic diseases), and, CD80 (B cell, autoimmune, allergic or malignant), CD86 (B cell, autoimmune, allergic or malignant), CD2 (T cell or NK cell lymphoma), CA125 (various cancers including ovarian cancer), carbonic anhydrase IX (various cancers including renal cell carcinoma), CD70 (B cell, autoimmune, allergic or malignant), CD74 (B cell, autoimmune, allergic or malignant), CD56 (T cell or NK cell lymphoma), CD40 (B cell, autoimmune, allergic or malignant), CD19 (B cell, autoimmune, Allergic or malignant), c-met/HGFR (gastrointestinal and hepatic malignancy), TRAIL-R1 (various malignancies including ovarian and colorectal cancers), DRS (various malignancies including ovarian and colorectal cancers), PD-1 (B cells, autoimmune, allergic or malignant), PDL1 (various malignancies including epithelial adenocarcinoma), IGF-1R (most malignancies including epithelial adenocarcinoma), VEGF and VEGFR (solid tumor and ocular AMD), VEGF-R2 (vasculature associated with most malignancies including epithelial adenocarcinoma), prostate Stem Cell Antigen (PSCA) (prostate adenocarcinoma), MUC1 (epithelial malignancy), canag (tumors such as colon cancer and pancreatic cancer), mesothelin (many tumors including mesothelioma), Ovarian cancer and pancreatic cancer), P-cadherin (epithelial malignancy, including breast cancer), myostatin (GDF 8) (many tumors including sarcomas, ovarian cancer, and pancreatic cancer), cripto (TDGF 1) (epithelial malignancy, including colon cancer, breast cancer, lung cancer, ovarian cancer, and pancreatic cancer), ACVRL1/ALK1 (various malignancies including leukemia and lymphoma), MUC5AC (epithelial malignancy, including breast cancer), CEACAM (epithelial malignancy, including breast cancer), CD137 (B or T cells, autoimmune, allergic, or malignancy), CXCR4 (B or T cells, autoimmune, Allergic or malignant), neuropilin 1 (epithelial malignancy, including lung cancer), glypican (a variety of cancers, including liver, brain and breast cancers), HER3/EGFR (epithelial malignancy), PDGFRa (epithelial malignancy), ephA2 (a variety of cancers, including neuroblastoma, melanoma, breast and small cell lung cancers), CD38 (myeloma), CD138 (myeloma), alpha 4-integrin (AML, myeloma, CLL and most lymphomas), C5 complement (PNH, aHUS, gMG and NMOSD), C3 complement (PNH), MASP-2 (IgA nephropathy), C5aR (solid tumor), CR1 (eye wAMD disease), C3b and CFH (PNH, aHUS, gMG and NMOSD).

The U.S. Food and Drug Administration (FDA) maintains a list of approved antibody drugs or therapeutic antibodies for the treatment of cancer, see drugs@fda on the online orange book or FDA website. The FDA also maintains a list of ongoing clinical trials of therapeutic antibodies in the clinical trims. These antibody drugs or therapeutic antibodies, or antigen binding portions thereof, are specific for various disease-associated antigens or tumor-associated antigens and can be used as targeting or effector moieties in the protein complexes, related compositions, and related methods of treatment disclosed herein.

Examples of such antibody drugs or therapeutic antibodies include, but are not limited to, 3F8, 8H9, ab Fu Shan antibody (Abagovomab), acximab (Abciximab), abituzumab (Abituzumab), ab Li Lushan antibody (Abrilumab), abituzumab Shu Shan antibody (Actoxumab), abitumumab (Abciximab), abituzumab (Abituzumab), ab Li Lushan antibody (Abrilumab), abitumumab Shu Shan antibody (Actoxumab), adalimumab (Adalimumab), Adalimumab (Adecatumumab), adalimumab (Aducanumab), atozumab (Afutuzumab), pego-Alemtuzumab (Alacizumab pegol), ALD518, alemtuzumab (Alemtuzumab), alemtuzumab (Alirocumab), alemtuzumab pentetate (Altumomab pentetate), amanitab (Amatuximab), ma Anna momab (Anatumomab mafenatox), Lei Xing-anetuzumab (Anetumab ravtansine), anilurumab (Anifrolumab), an Lu group mab (Anrukinzumab) (=ima-638), aprepitant (Apolizumab), aximomab (Arcitumomab), atorvastatin Su Shan antibody (Ascrinvacumab), amolizumab (Aselizumab), atrazumab (Atezolizumab), atino mab (Atinumab), tolizumab (Atlizumab) (= tocilizumab), tolizumab (Atlizumab), Atropumab (Atorolimumab), bazomib (Bapineuzumab), bazomib (Basiliximab), bazomib (Bavituximab), bei Tuo momab (Bectumomab), bei Geluo mab (Begelomab), belimumab (Belimumab), benzoin (Benralizumab), bai Ti mab (Bertilimumab), bei Suoshan mab (Besilesomab), bevacizumab (Bevacizumab), Bei Luotuo Shu Shan antibodies (Bezlotoxumab), biximab (Biciromab), bima Lu Shankang (Bimagrumab), bimetazumab (Bimekizumab), bivalizumab maytansine (Bivatuzumab mertansine), rituximab (Blinatumomab), busulfameb (Blosozumab), primary cooperizumab (Bococizumab), rituximab (Brentuximab vedotin), plainomab (Briakinumab), and, Budadizumab (Brodalumab), budesoximab (Brolucizumab), budesoximab Long Tuozhu (Brontictuzumab), canakinumab (Canakinumab), mo Kantuo bead mab (Cantuzumab mertansine), lei Kantuo group mab (Cantuzumab ravtansine), carasiemab (Caplacizumab), carlizumab (Capromab pendetide), carlizumab (Carlumab), Katuxostat (Catumaxomab), cBR-doxorubicin immunoconjugate (cBR-doxorubicin immunoconjugate), seldelizumab (Cedelizumab), cetuximab (Cetuximab), ch.14.18, posituzumab (Citatuzumab bogatox), cetuximab (Cixutumumab), clazazumab (Clazakizumab), celecoxib (Clenoliximab), Tataman-clerituximab (Clivatuzumab tetraxetan), costuzumab (Codrituzumab), lei Xing-Cotuximab (Coltuximab ravtansine), coronamumab (Conatumumab), kang Saizhu mab (Concizumab), crigthenab (Crenezumab), CR6261, daclizumab (Dacetuzumab), daclizumab (Daclizumab), daclizumab Luo Tuo (Dalotuzumab), pego-dapirinotecan (Dapirolizumab pegol), daragon Mu Shan (Daratumuma), de Qu Kushan (Dectrekumab), denciclizumab (Demcizumab), martin-dim Ning Tuo bead mab (Denintuzumab mafodotin), dino Su Shan mab (Denosumab), rituximab (Derlotuximab biotin), delumomab Detumomab, denonootuximab (Dinutuximab), utilize vomumab (Diridavumab), and, Rituximab (Dorlimomab aritox), qu Jituo mab (Drozitumab), du Lige mab (Duligotumab), dipirumab (Dupilumab), dewaruzumab (Durvalumab), desituzumab (Dusigitumab), exemestane (Ecromeximab), eculizumab (Eculizumab), epstein (Edobacomab), ibritumomab (Edrecolomab), efalizumab (Efalizumab), valvulumab (Efalizumab), Ifenacin (Efungumab), eddie, ibrutinab (Eldelumab), ibritumomab (Elgemtumab), ibritumomab (Elotuzumab), islimab (Elsilimomab), ibritumomab (Emactuzumab), ibritumomab (Emibetuzumab), ibritumomab (Enavatuzumab), retinaculum-enframomab (Enfortumab vedotin), en Li Moshan, pegol (Enlimomab pegol), Enoxazumab (Enoblituzumab), enoxazumab (Enokizumab), enoxazumab Su Shan (Enoticumab), antuximab (Ensituximab), epimomab (Epitumomab cituxetan), epaizumab (Epratuzumab), erlizumab (Erlizumab), ertuzumab (Ertumaxomab), ada mab (Etaracizumab), itrarinab (Etrolizumab), Ewei Su Shan antibody (Evinacumab), elol You Shan antibody (Evolocumab), ai Weishan antibody (Exbivirumab), fanooxomab (Fanolesomab), farimumab (Faralimomab), farituximab (Farletuzumab), fasinumab, FBTA05, panwei-zuizumab (Felvizumab), non-zanomab (Fezakinumab), non-trastuzumab (Ficlatuzumab), phenytoin (Figitumumab), festuzumab (Firivumab), rituximab (Flanvotumab), frekuzumab (Fletikumab), rituximab (Fontolizumab), fu Lei Lushan antibody (Foralumab), fula Wei Shankang (Foravirumab), non-sappan mab (Fresolimumab), fula MANCB (Fulrauab), fuluramab (Futuximab), gancicumab (Galiximab), gancicumab (Ganitumab), more Tinoscapizumab (Gantenerumab), Gammamizumab (Gavilimomab), gemtuzumab ozogamicin (Gemtuzumab ozogamicin), gemtuzumab (Gevokizumab), ji Tuo ximab (Girentuximab), glembatumumab vedotin, gomiliximab, gu Seku mab (Guselkumab), ibamzumab (Ibalizumab), ibritumomab (Ibritumomab tiuxetan), ai Luku mab (Icrucumab), and, Aidamascizumab (Idarucizumab), igovacizumab (Igovomab), IMAB362, imalurumab (Imalumab), incemumab (Imciromab), I Ma Qushan antibody (Imgatuzumab), inclacumab, lei Ying toximab (Indatuximab ravtansine), vitin-INGYTOMAb (Indusatumab vedotin), inttumumab, indomab (Inolimomab), almomab (Inolimomab), oxtuzumab (Inotuzumab ozogamicin), ipituzumab (Ipilimumab), itumomab (Iratumumab), ai Satuo ximab (Isatuximab), illicitab (Itolizumab), ixekizumab, iximab Bei Shan, keliximab (Keliximab), la Bei Zhushan anti (Labetuzumab), pembrolizumab (Lambrolizumab), lanpalizumab (Lampalizumab), letromab (Lebrikizumab), leitikizumab (Lebrikizumab), Ma Suoshan antibodies (Lemalesomab), lorentzumab (Lenzilumab), le Demu antibodies (Lerdelimumab), cissamumab (Lexatumumab), li Weishan antibodies (Libivirumab), statin-rituximab (Lifastuzumab vedotin), li Geli bead monoclonal antibodies (Ligelizumab), lilotomab satetraxetan, rituximab (Lintuzumab), li Ruilu monoclonal antibodies (Lirilumab), Lomab (Lodelcizumab), lomab Ji Weishan (Lokivetmab), moxidec-lomab Wo Tuozhu (Lorvotuzumab mertansine), mab Lu Kamu (Lucatumumab), pego-Lu Lizhu mab (Lulizumab pegol), lu Xishan (Lumiliximab), lu Tuozhu mab (Lumretuzumab), ma Pamu mab (Mapatumumab), moxidec mab (Margetuximab), ma Simo mab (Maslimomab), and pharmaceutical compositions, MAVERIMIMAZOMAIN (Mavrilimumab), MATUUZOMAIN (Matuzumab), mepolizumab (Mepolizumab), metemumab (Metelimumab), milauximab (Milatuzumab) Minremimumab (Minretumomab), sorrel-rituximab (Mirvetuximab soravtansine), mi Tuomo mab (Mitumomab), mo Geli mab (Mogamulizumab), moromimumab (Morolimumab), Movezumab (Motavizumab), moxetumomab pasudotox, movezumab-CD 3 (Muromonab-CD 3), tanatalizumab (Nacolomab tafenatox), nalmefene mab (Namilumab), eto-Natalizumab (Naptumomab estafenatox), naratomab (Narnatumab), natalizumab (Natalizumab), ne Baku mab (Nebacumab), cetuximab (Nerituximab), Ne Mo Lizhu mab (Nemolizumab), neval Su Shan mab (Nesvacumab), nituzumab (Nimotuzumab), nawuzumab (Nivolumab), nofetumomab merpentan, otussah mab (Obiltoxaximab), atozumab (Obinutuzumab), okatuzumab (Ocaratuzumab), ocreelizumab (Ocreelizumab), ondimomab (Odulimomab), ofatuzumab (Ofatumumab), Olamab (Olaratumab), olob (Olokizumab), omalizumab (Omalizumab), onarituximab (Onartuzumab), ondtuzumab (Ontuxizumab), ompartuzumab (Opicinumab), motuzumab (Oportuzumab monatox), ago Fu Shan anti (Oregovomab), octreotide Su Shan anti (Orticumab), oxybuzumab (Otelixizumab), ox Le Tuozhu mab (Otlertuzumab), Oxepizumab (Oxelumab), ozagruzumab (Ozanezumab), parecoxib mab (Pagibaximab), palivizumab (Palivizumab), panitumumab (Panitumumab), pankomab, pam Baku mab (Panobacumab), paspaltuzumab (Parsatuzumab), pecobuzumab (Pascolizumab), pertuzumab (Pasotuxizumab), pertuzumab (Pateclizumab), panitumumab (Pateclizumab), pa Qu Tuoshan antibody (Patritumab), pembrolizumab (Pembrolizumab), pemtumomab, perrakazumab (Perakizumab), pertuzumab (Pertuzumab), pegzhuzumab (Pexelizumab), pilizumab (Pidilizumab), statin-pinatuzumab (Pinatuzumab vedotin), smooth and proper mab (Pintumomab), praluruzumab (Placulumab), polatuzumab vedotin, Ponesoniumab (Ponezumab), prizetimab (Priliximab), rituximab (Pritoxaximab), prizetimab (Pritumumab), PRO 140, quinizumab (Quilizumab), tetulomab, lei Tuomo mab (Racotumomab), lei Qu tomab (Radretumab), lei Weishan antibody (Rafivirumab), lei saibulab (Ralpancizumab), ramucirumab (Ramucirumab), Ranitizumab (Ranibizumab), lei Xiku mab (Raxibacumab), repaonelizumab (Refanezumab), regasifi Wei Shankang (Regavirumab), rayleigh beadmab (Reslizumab), rituximab (Rilotumumab), li Nusu mab (Rinucumab), rituximab (Rituximab), luo Tuomu mab (Robatumumab), roteizumab (Roledumab), lox Mo Suozhu mab (Romosozumab), and, Long Li sets of mab (Rontalizumab), luo Weizhu mab (Rovelizumab), lu Lizhu mab (Ruplizumab), goretinide-Sha Xituo bead mab (Sacituzumab govitecan), sar Ma Zushan mab (Samalizumab), sarilumab (Sarilumab), satumomab pendetide, questor numab (Secukinumab), sirtuin mab (Seribantumab), sirtuin mab (Setoxaximab), sarsashiximab, Se Wei Shankang (Sevirumab), cetrimab (Sibrotuzumab), SGN-CD19A, SGN-CD33A, cetrimab (Sifalimumab), cetuximab (Siltuximab), xin Tuozhu mab (Simtuzumab), cetrimab (Siplizumab), cet Lu Kushan-anti (Sirukumab), vitamin-Sorafizumab (Sofituzumab vedotin), solanesol mab (Solanezumab), sorafizumab (Solitomab), Sonepcizumab, soxhlet mab (Sontuzumab), statuzumab (Stamulumab), thioxomab (Sulesomab), shu Weizu mab (Suvizumab), he Bei Lushan mab (Tabalumab), timezumab (Tacatuzumab tetraxetan), tabanitumumab (Tadocizumab), talizumab (Talizumab), tanezumab (tanumab), tarituximab (Taplitumomab paptox), tabanitumomab (Tabalumab), Tarituximab (Tarextumab), tilapril mab (Tefibazumab), atisimab (Telimomab aritox), titamoxifen (Tenatumomab), tinesuximab (Teneliximab), tilapril mab (Teplizumab), tituzumab (Teprotumumab), terstuzumab (Tesidolumab), TGN1412, CTLA-4 mab (Ticilimumab) (=tremelimiumab), Ti Qu Jizhu mab (Tildrakizumab), tigegroup mab (Tigatuzumab), TNX-650, tobalizumab (Tocilizumab) (=atlizumab), toralizumab, toxotrop Shu Shan mab Tosatoxumab, tositumomab (Tositumomab), toxituzumab (Tovetumab), qu Luolu mab (Tralokinumab), trastuzumab (Trastuzumab), trastuzumab-maytansinoid conjugate (Trastuzumab emtansine), TRBS, trilizumab (Tregalizumab), CTLA-4 mab (Tremelimumab), qu Gelu mab (Trevogrumab), cetuximab (Tucotuzumab celmoleukin), to Wei Shankang (Tuvirumab), rituximab (Ublituximab), wu Luolu mab (Ulocuplumab), wu Ruilu mab (Urelumab), wu Zhushan mab (Urtoxazumab), wu Sinu mab (Ustekinumab), cetuximab (Ulocuplumab), Vitin-vanadoxolizumab (Vandortuzumab vedotin), vantuzumab (Vantictumab), valdecoxelizumab (Vanucizumab), valdecoxib (Vapaliximab), valdecomab (Varlilumab), valdecomab (Vatelizumab), vedolizumab (Vedolizumab), veltuzumab (Veltuzumab), velpamizumab (Vepalimomab), velsen kuizumab (Vesencumab), valdecomab (Vesencumab), wikipedia mab (Visilizumab), fu Luoxi mab (Volociximab), martin-Wo Setuo mab (Vorsetuzumab mafodotin), votamab (Votumumab), zafimbrukinumab (Zalutumumab), zafimbrizumab (Zanolimumab), zatuximab (Zatuximab), ziralimumab, and arzomomab (Zolimomab aritox). other examples include those described in U.S. patent No. 11,119,096, U.S. patent No. 11,091,562, and U.S. patent No. 20210269547, the disclosures of which are incorporated herein by reference.

Ligand

In some other embodiments, the targeting moiety may be or may include one member of a binding pair, while the other member of the binding pair is located on the target of interest. Examples of such binding pairs include ligand-receptor pairs. In certain embodiments, the targeting moiety may be a binding partner for a protein known to be expressed on cancer cells. Such expression levels may include overexpression.

Examples of such ligands may include IL-2, IL-4, IL-6, alpha-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), CD40 or CD47. In some embodiments, the targeting moiety contains the full length sequence of IL-2, IL-4, IL-6, alpha-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, a stem cell factor, an insulin-like growth factor (IGF), or CD 40. In some embodiments, the targeting moiety comprises a truncated form, analog, variant, or derivative of IL-2, IL-4, IL-6, alpha-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, a stem cell factor, an insulin-like growth factor (IGF), or CD 40. In some embodiments, the targeting moiety binds to a target on cancer, including IL-2 receptor, IL-4, IL-6, melanocyte stimulating hormone receptor (MSH receptor), transferrin Receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH 1), EGF receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR, or CD40L.

The binding partner need not include the full length or wild type sequence of the binding partner. All that is required is that the binding partner is capable of binding to a target on a cancer cell and thus may include truncated forms, analogues, variants and derivatives as are well known in the art.

Others

In some embodiments, the targeting moiety capable of targeting a target (e.g., cancer) is not an antibody, but is another type of targeting moiety. A variety of targeting moieties capable of targeting cancer are known, including DNA aptamers, RNA aptamers, albumin, lipocalins, fibronectin, ankyrin, CH1/2/3 scaffolds (including abdurins (IgG CH2 scaffold), fynomers, obodies, DARPins, desmin (knotins), avimers, atrimers, anticallins, affilis, affibodies, bicyclic peptides, cys-knots, FN3 (ADNECTINS, CENTRYRINS, PRONECTINS, TN 3), and Kunitz domains these and other non-antibody scaffold structures can be used to target cancer cells. 1271-1283 (2015) many non-antibody scaffolds against cancer are already in the clinical development stage, as are other drug candidates in the preclinical stage see Lombardi et al, drug Discovery Today 20 (1): 1271-1283 (2015) table 1.

In addition, in some embodiments, the binding partner may be an aptamer capable of binding to a protein known to be expressed on cancer cells. Aptamers that bind to cancer cells, such as cancer cells, are well known and methods of designing them are also known.

Effector moieties

Effector moiety or effector domain or effector agent is used interchangeably herein to refer to an entity (e.g., an atom, molecule, compound or cell) that mediates a biological activity or response (e.g., an immune response) or for diagnostic or therapeutic applications. The effector agent may be a diagnostic agent or a therapeutic agent.

The diagnostic effector moiety or domain or agent may be any entity useful for diagnosing a disease. Useful diagnostic agents include, but are not limited to, enzymes, DNA, RNA, peptides, substrates, chemiluminescent agents, radioisotopes, dyes, contrast agents, fluorescent compounds or molecules, enhancers (e.g., paramagnetic ions), or beads, or other conjugates for collection. It should be understood that the magnetic beads may be any suitable magnetic beads for standard purification or isolation. Thus, the magnetic beads may be ferromagnetic or paramagnetic or superparamagnetic, such as permanent magnets or materials attracted to magnetic materials.

A therapeutic effector moiety or domain or agent refers to any entity that can exert a therapeutic effect. Examples include immune checkpoint inhibitors, immune co-stimulators/agonists (antibodies, ligands or chemicals), immune co-suppressors/antagonists (antibodies, proteins or chemicals), cytokines, complement agents, cancer vaccines, anti-cancer agents, radioisotopes such as radioiodinated compounds, toxins, drugs that inhibit cell growth or lyse cells, and the like. Immune checkpoint inhibitors include, for example, antibodies or chemical agents directed against PD-1, PD-L1 or CTLA-4. Immune co-stimulators/agonists include, for example, antibodies, ligands or chemical agents directed against 4-1BB, ICOS, GITR, CD, CD27, OX40 or CD 40. Immune co-inhibitors/antagonists include, for example, antibodies, proteins or chemicals against VISTA, CCR4, B7-H3, TIM-3, LAG-3, KIR, IDO-1,2, TIGIT, A2aR, TGF- β, CD47, CD73, NKG2A or NKG 2B. Cytokines include, for example, IFN- α, IFN- β, IFN- γ, IFN- λ, IL-1β, IL2, IL6, IL-15, IL-17, or IL-12. Complement agents include, for example, antibodies, proteins, or chemical agents directed against C1r, C1s, C2, C3, C5a, C5aR1, C6, MASP, MSAP2, MASP3, FB, FD, or properdin. cancer vaccines include any cancer specific antigen that can induce a body immune response to attack cancer. Anticancer agents include, for example, aminoglutethimide (aminoglutethimide), azathioprine (azathioprine), bleomycin sulfate (bleomycin sulfate), busulfan (busulfan), carmustine (carmustine), chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabine (cytarabidine), dacarbazine (dacarbazine), actinomycin (dactinomycin), daunorubicin (daunorubin), doxorubicin, and, Paclitaxel, etoposide, fluorouracil, interferon-alpha, lomustine (lomustine), mercaptopurine (mercaptopurine), methotrexate (methotrexa), mitotane (mitotane), procarbazine hydrochloride (procarbazine HCl), thioguanine (thioguanine), vinblastine sulfate (vinblastine sulfate) and vincristine sulfate (VINCRISTINE SULFATE). Other anticancer agents are described, for example, in Goodman and Gilmanl,"The Pharmacological Basis of Therapeutics",8th Edition,1990,McGraw-Hill,Inc.,in particular Chapter 52(Antineoplastic Agents(Paul Calabresi and Bruce A.Chabner). toxins, which may be proteins, such as pokeweed antiviral protein (pokeweed antiviral protein), cholera toxin, pertussis toxin, ricin, gelonin (gelonin), abrin (abrin), diphtheria exotoxin (DIPHTHERIA EXOTOXIN), ranpirnase (Onconase) (RANPIRNASE) or pseudomonas exotoxin (Pseudomonas exotoxin). The toxin residue may also be a high energy radionuclide, such as cobalt 60.

Other examples include cytotoxins or cytotoxic agents. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells and in particular kills cells. Useful classes of cytotoxic agents include, for example, oncolytic peptides, anti-tubulin agents, DNA minor groove binders (e.g., enediyne and lexitropsins), DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono (platinum), bis (platinum) and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, polycarbomycin, etoposide, fluorinated pyrimidines, ionophores, nitrosoureas, platinum alcohols, preformed compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes (e.g., paclitaxel and docetaxel), topoisomerase inhibitors, vinca alkaloids, and the like.

Individual cytotoxic agents include, for example, androgens, anthracycline (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, thioflavine sulfimide (buthionine sulfoximine), camptothecins, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine (cytidine arabinoside), cytochalasin B, dacarbazine (dacarbazine), actinomycin (dactinomycin) (procyanidins), daunorubicin, dicarbazine (decarbazine), docetaxel, doxorubicin, estrogens, 5-fluorodeoxyuridine 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin (idarubicin), ifosfamide (ifosfamide), irinotecan, lomustine (lomustine) (CCNU), nitrogen mustard, melphalan, 6-mercaptopurine, methotrexate, mithramycin (mithramycin), mitomycin C, mitoxantrone (mitoxantrone), nitroimidazole, paclitaxel, plicamycin (plicamycin), procarbazine (procarbizine), streptozotocin, tenopuiposide (tenoside), 6-thioguanine, thiotepa (thioTEPA), topotecan, vinca, vincristine, vinorelbine (vinorelbine), VP-16, VM-26, and anti-tubulin agents. Examples of anti-tubulin agents include, but are not limited to, dolastatin (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB), maytansinoids, taxanes (e.g., paclitaxel, docetaxel), T67 (Tularik), vinca alkaloids (vinca alkyloid) (e.g., vincristine, vinblastine, vindesine (vindesine), and vinorelbine), baccatin derivatives (baccatin derivative), taxane analogs (e.g., epothilones a and B), nocodazole (nocodazole), colchicine and colchicine, estramustine, cryptosporine (cryptosporin), cimadodine (cemadotin), combretastatin (combretastatin), discodermolide (discodermolide), and acanthopanaxase (elehereunder). Radioisotopes that produce cytotoxic radiopharmaceuticals include, for example, iodine 131, yttrium 90, or indium 111.

Additional exemplary therapeutic agents that may be used as therapeutic effector moieties are described in the other therapeutic agent moieties below.

Techniques for coupling such therapeutic effector moieties (drugs) to antibodies, proteins or peptides are well known. The production of antibody/protein/peptide-drug conjugates can be accomplished by any technique known to those skilled in the art. The peptide and the drug may be directly bound to each other through their own linking groups or indirectly bound to each other through linking groups or other substances.

Immune cell binding domains

In certain embodiments, the effector moiety may be or may include an immune cell binding domain that may bind to or recruit one or more immune cells. In some embodiments, the immune cell is a T cell, natural killer cell, macrophage, neutrophil, eosinophil, basophil, γδ T cell, NKT cell, or engineered immune cell.

T cell

For binding to T cells, the effector moiety may bind to CD3 antigen and/or T cell receptor or any specific binding marker on the surface of T cells. CD3 is present on all T cells and consists of subunits designated gamma, delta, epsilon, zeta and eta. In the absence of other components of the TCR receptor complex, the cytoplasmic tail of CD3 is sufficient to transduce signals required for T cell activation. In general, activation of T cytotoxicity depends primarily on the binding of TCRs to Major Histocompatibility Complex (MHC) proteins, which themselves bind to foreign antigens located on individual cells. Under normal circumstances, only when this initial TCR-MHC binding occurs, the CD 3-dependent signaling cascade can lead to clonal expansion of T cells and ultimately to T cell cytotoxicity. However, in some embodiments of the invention, when the multi-specific protein complex binds to CD3 and/or TCR, activation of cytotoxic T cells can occur by virtue of cross-linking of CD3 and/or TCR mimicking immune synapse formation in the absence of independent TCR-MHC. This means that T cells can be activated by cytotoxicity in a clone-independent manner, i.e. in a manner independent of the particular TCR clone carried by the T cell. This allows activation of the whole T cell compartment, rather than activating only specific T cells of a specific clonal identity.

In some embodiments, the T cell binding domain may comprise an scFv specific for an antigen expressed on the surface of a T cell (e.g., CD3 or TCR). If the antigen is CD3, one potential T cell binding domain may be derived from Moruzumab (muromonab) (Moruzumab-CD 3 or OKT 3), oletlizumab (otelixizumab), teprilizumab (teplizumab), wikiuzumab (visilizumab), fulauzumab (foralumab), 20G6 or SP34. Those skilled in the art will recognize a variety of anti-CD 3 antibodies, some of which are approved therapies or have been clinically tested in human patients (see Kuhn and Weiner Immunotherapy (8): 889-906 (2016).

Natural killer cells

In some embodiments, the immune cell binding domain may be or may include a Natural Killer (NK) cell binding domain that specifically binds an antigen on NK cells. The antigen on the surface of the NK cell may be NKG2D, CD, NKp30, NKp44, NKp46 or DNAM.

In some embodiments, the effector moiety is bound to a surface protein on a natural killer cell and the targeting moiety is bound to a target cell (e.g., a pathogen, a disease cell, or a cancer cell) to achieve specific binding of the natural killer cell. Natural killer cell binding can lead to its activation and induction of natural killer cell mediated cytotoxicity and cytokine release.

The natural killer cells can specifically lyse target cells bound by the protein complex. Killing of target cells can be mediated by the perforin/granzyme system or by FasL-Fas binding. In addition to this potential cytotoxic function, natural killer cells can also secrete pro-inflammatory cytokines, including interferon gamma and tumor necrosis factor alpha, which can activate nearby macrophages and dendritic cells to enhance anti-target (e.g., anti-cancer) immune responses. The natural killer cell binding domain may comprise scFv, fab or antigen binding fragments specific for antigens expressed on the surface of natural killer cells (e.g., NKG2D, CD, NKp30, NKp44, NKp46 and DNAM).

Macrophages with a function of promoting the growth of human body

In some embodiments, the immune cell binding domain may be or may include a macrophage binding domain. As used herein, "macrophage" may refer to any cell of the mononuclear phagocytic system, such as, for example, grouped lineage committed bone marrow precursor cells, circulating monocytes, resident macrophages, and Dendritic Cells (DCs). Examples of resident macrophages may include Kupffer cells and microglia. The macrophage binding domain specifically binds to macrophage surface antigen to bind to these cells. In some embodiments, the macrophage surface antigen can be CD89 (fcα receptor 1), CD64 (fcγ receptor 1), CD32 (fcγ receptor 2A), or CD16a (fcγ receptor 3A).

Binding of the effector moiety to a surface protein on the macrophage and binding of the targeting moiety to a target cell (e.g., pathogen, disease cell, or cancer cell) to achieve specific binding of the macrophage. Binding of macrophages can cause the macrophages to phagocytose target cells.

In some embodiments, the induction of macrophage phagocytosis by binding to macrophage surface antigen is not dependent on Fc receptor binding, which has previously been demonstrated to be a method of macrophages killing target (e.g., tumor) cells. Normally, cancer cells bind to intact antibodies, the Fc portion of which binds to Fc receptors and induces phagocytosis. In some embodiments, the binding of Toll-like receptors on the surface of macrophages (see patent application US20150125397 A1) results in the binding of macrophages. The macrophage binding domain can include an scFv, fab, or antigen binding fragment specific for an antigen expressed on the surface of a macrophage (e.g., CD89, CD64, CD32, CD16a, or toll-like receptor).

Neutrophils

In some embodiments, the immune cell binding domain may be or may include a neutrophil binding domain that specifically binds an antigen on a neutrophil. Examples of antigens on the surface of neutrophils include CD89 (fcαr1), fcγri (CD 64), fcγriia (CD 32), fcγriiia (CD 16 a), CD11b (CR 3, αmβ2), TLR2, TLR4, CLEC7A (Dectin 1), formyl peptide receptor 1 (FPR 1), formyl peptide receptor 2 (FPR 2) and formyl peptide receptor 3 (FPR 3).

In some embodiments, the effector moiety is allowed to bind to a surface protein on a neutrophil and the targeting moiety is allowed to bind to a target cell (e.g., pathogen, disease cell, or cancer cell) to achieve specific binding of the neutrophil. Neutrophil binding can lead to phagocytosis and target cell uptake. That is, neutrophils can phagocytose target cells. The neutrophil binding domain may comprise an scFv, fab or antigen-binding fragment specific for a neutrophil surface-expressed antigen, such as any of those described above.

Eosinophils

In some embodiments, the immune cell binding domain may be or may include an eosinophil binding domain that specifically binds to an antigen on an eosinophil. Examples of eosinophil surface antigens include CD89 (fcα receptor 1), fceri, fcyri (CD 64), fcyriia (CD 32), fcyriiib (CD 16 b), or TLR4.

In some embodiments, the effector moiety is bound to a surface protein on eosinophils and the targeting moiety is bound to a target cell (e.g., a pathogen, a disease cell, or a cancer cell) to achieve specific binding of eosinophils. Eosinophil binding can result in degranulation and release of preformed cationic proteins such as EPO, major basic protein 1 (MBP 1), and eosinophil-associated ribonuclease (EAR) (referred to as ECP and eosinophil-derived neurotoxin). In this case, eosinophils may phagocytose target cells or secrete Neutrophil Extracellular Traps (NETs), and finally they can activate the respiratory burst cascade to kill phagocytosed cells.

Eosinophil-binding domains may include scFv, fab or antigen-binding fragments specific for an antigen expressed on the surface of an eosinophil, such as any of those described above.

Basophils

In some embodiments, the immune cell binding domain may be or may include a basophil binding domain that specifically binds an antigen on a basophil. Examples of basophil surface antigens may be CD89 (fcα receptor 1) or fceri.

In some embodiments, effector moieties are bound to surface proteins on basophils and targeting moieties are bound to target cells (e.g., pathogens, disease cells, or cancer cells) to achieve specific binding of basophils. The binding of basophils can result in the release of basophil granule components such as histamine, proteoglycans, and proteolytic enzymes. They also secrete leukotriene (LTD-4) and cytokines.

In some embodiments, the basophil binding domain may include an scFv, fab, or antigen-binding fragment specific for an antigen expressed on the surface of a basophil, such as any of those described above.

Gamma delta T cells

In some embodiments, the immune cell binding domain may be or may include a γδ T cell binding domain. As used herein, γδ T cells refer to T cells having a TCR consisting of one γ chain (γ) and one δ chain (δ). The γδ T cell binding domain specifically binds to antigens on the γδ T cell surface to bind these cells. Examples of γδ T cell surface antigens include γδ TCR, NKG2D, CD3 complex (CD 3 epsilon, CD3 γ, CD3 δ, CD3z and CD3 η), 4-1BB, DNAM-1 or TLR (e.g. TLR2, TLR 6).

In some embodiments, the effector moiety is bound to a surface protein on γδ T cells and the targeting moiety is bound to a target cell (e.g., a pathogen, disease cell, or cancer cell) to achieve specific binding of γδ T cells. Binding of γδ T cells can result in cytolysis of target cells and release of pro-inflammatory cytokines, such as tnfα and ifnγ. In this case, γδ T cells are able to kill target cells.

In some embodiments, the γδ T cell binding domain can comprise a scFv, fab or antigen binding fragment specific for an antigen expressed on the γδ T cell surface, such as any one of those described above.

NKT cells

In some embodiments, the immune cell binding domain may be or may include an NKT binding domain. NKT cells refer to T cells expressing vα24 and vβ11TCR receptors. The NKT binding domain specifically binds to antigens on the NKT surface to bind to these cells. Examples of NKT surface antigens include αβTCR, NKG2D, CD3 complex (CD 3 ε, CD3 γ, CD3 δ, CD3z and CD3 n), 4-1BB or IL-12R.

In some embodiments, the effector moiety is bound to a surface protein on NKT and the targeting moiety is bound to a target cell (e.g., pathogen, disease cell, or cancer cell) to achieve specific binding of NKT. Binding of NKT can result in cytolysis of target cells. In this case, NKT is able to lyse target cells and release pro-inflammatory cytokines.

In some embodiments, the NKT binding domain may comprise a scFv, fab, or antigen binding fragment specific for an antigen expressed on the surface of NKT, such as any of those described above.

Engineered immune cells

In some embodiments, the immune cell binding domain may be or may include an engineered immune cell binding domain. The binding domain specifically binds to antigens on the surface of engineered immune cells to bind to these cells. In some embodiments, the antigen of the engineered immune cell surface may be a binding domain specific for a T cell, NK cell, NKT cell, or γδ T cell as described herein.

In some embodiments, the engineered immune cell is a Chimeric Antigen Receptor (CAR) cell. The CAR may include an extracellular domain (e.g., scFv) capable of tightly binding to a tumor antigen, fused to a signaling domain derived in part from a receptor naturally expressed by an immune cell. Exemplary CARs are described in facts about Chimeric Antigen Receptor (CAR) T cell therapies, association of leukemia and lymphoma, month 12 of 2017.

The CAR may include a scFV region, an intracellular co-stimulatory domain, and a linker and transmembrane region that are specific for a target (e.g., a tumor antigen). For example, a CAR in a CAR T cell can include an extracellular domain that targets a tumor antigen fused to a signaling domain that is partially derived from a T cell receptor. The CAR may also include a co-stimulatory domain, such as CD28, 4-1BB, or OX40. In some embodiments, binding of the immune cell-expressed CAR to the tumor target antigen results in immune cell activation, proliferation, and elimination of the target cell. Thus, a series of CARs that differ in scFV region, intracellular co-stimulatory domain, linker, and transmembrane region can be used to generate an engineered immune cell.

Exemplary engineered immune cells include CAR T cells, NK cells, NKT cells, and γδ T cells. In some embodiments, the engineered immune cells may be derived from the patient's own immune cells or from a healthy donor. In some embodiments, the tumor of the patient expresses a tumor antigen that binds to the scFV of the CAR.

Potential CAR targets studied to date include CD19, CD20, CD22, CD30, CD33, CD123, ROR1, igk light chain, BCMA, LNGFR and NKG2D. However, CAR technology can be used to develop engineered immune cells against a range of tumor antigens.

Likewise, the effector moiety is bound to a surface protein of an engineered immune cell and the targeting moiety is bound to a target cell (e.g., a pathogen, disease cell, or cancer cell) to achieve specific binding of the engineered immune cell. Binding of engineered immune cells can result in activation of effector responses of these cells, such as lysis of target cells, release of cytokines, and killing of target cells. Based on the cell type used for engineering, the engineered immune cell binding domain may include scFv specific for an antigen expressed on the surface of the engineered immune cell.

Various exemplary antibodies specific for antigens expressed on the surface of T cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, γδ T cells, NKT cells or engineered immune cells are known in the art. Examples include, for example, those described in US20210269547, the contents of which are incorporated herein by reference.

The targeting moiety and effector moiety described above may be joined together to form a protein complex using any method known in the art. In a preferred embodiment, the targeting moiety and the effector moiety may be formed by two DDD moieties and an AD moiety. As described herein, two DDD moieties form a dimer that binds to an AD moiety.

DDD and AD

This DDD/AD approach exploits specific and high affinity binding interactions that occur between the dimerization/docking domain (DDD) sequence of the cAMP-dependent Protein Kinase (PKA) regulatory (R) subunit and the Anchoring Domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al, FEBS letters 2005;579:3264.Wong and Scott,Nat.Rev.Mol.Cell Biol.2004;5:959). The DDD peptide and AD peptide may be linked to any protein, peptide or other molecule. This technique enables the formation of complexes between any selected molecule capable of linking to a DDD or AD sequence, as the DDD sequence spontaneously dimerizes and binds to the AD sequence. See, for example, U.S. patent nos. 7,521,056, 7,527,787, 7,534,866, 7,550,143, 7,666,400, 7,901,680, 7,906,118, 7,981,398, and 8,003,111. The entire contents of these patents are incorporated herein by reference.

Although standard DDD/AD complexes include trimers in which two DDD linker molecules are linked to one AD linker molecule, variations in complex structure allow for the formation of dimers, trimers, tetramers, pentamers, hexamers, and other multimers. In some embodiments, a complex described herein may include two or more antibodies, antibody fragments, or fusion proteins that bind to the same epitope or bind to two or more different antigens. The complex may also include one or more other effectors, such as proteins, peptides, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases (e.g., onconase), inhibitory oligonucleotides (e.g., siRNA), antigens or xenogenic antigens, polymers (e.g., PEG), enzymes, therapeutic agents, hormones, cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, or any other molecules or aggregates.

PKA plays a central role in one of the signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunit, which was first isolated from rabbit skeletal muscle in 1968 (Walsh et al, J.biol. Chem.1968; 243:3763). The structure of the holoenzyme consists of two catalytic subunits held in inactive form by the R subunit (Taylor, J. Biol. Chem.1989; 264:8443). The isozymes of PKA were found to have two types of R subunits (RI and RII), each of which has both the alpha and beta subtypes (Scott, pharmacol. Ther.1991; 50:123). Thus, four subtypes of PKA regulatory subunits are RIα, RIβ, RIIα and RIIIβ, each of which includes the amino acid sequence of the DDD moiety. The RII alpha subunit was isolated only as a stable dimer, and the dimerization domain has been shown to consist of the first 44 amino-terminal residues (Newlon et al., nat. Struct. Biol.1999; 6:222). Similar portions of the amino acid sequences of other regulatory subunits, each located near the N-terminus of the regulatory subunit, participate in dimerization and docking, as described below. Binding of cAMP to R subunits results in release of active catalytic subunits with broad spectrum serine/threonine kinase activity, which are targeted to a selected substrate by PKA compartmentalization with AKAP (Scott et al, J.biol. Chem.1990;265; 21561).

Since the first AKAP (microtubule-associated protein-2) was characterized in 1984 (Lohmann et al, proc. Natl. Acad. Sci USA 1984; 81:6723), more than 50 AKAPs have been identified with different structures in species ranging from yeast to humans, which are localized to different subcellular sites including plasma membrane, actin cytoskeleton, nucleus, mitochondria and endoplasmic reticulum (Wong and Scott, nat. Rev. Mol. Cell biol.2004; 5:959). The AD of AKAP for PKA is an amphipathic helix of 14-18 residues (Carr et al, J.biol. Chem.1991; 266:14188). The amino acid sequence of AD varies from AKAP to AKAP, with the binding affinity of the RII dimer reported to range from 2 to 90nM (Alto et al, proc. Natl. Acad. Sci. USA 2003; 100:4445). AKAP binds only to the dimeric R subunit. For human RIIα, AD binds to a hydrophobic surface formed by 23 amino-terminal residues (Colledge and Scott, TRENDS CELL biol.1999; 6:216). Thus, both the dimerization domain and AKAP binding domain of human riiα are located within the same N-terminal 44 amino acid sequence (Newlon et al., nat. Struct. Biol.1999;6:222;Newlon et al., EMBO j.2001; 20:1651), which is referred to herein as DDD.

As described herein, DDD of human PKA regulatory subunit and AD of AKAP serve as a binding pair of linker modules for docking any two entities into a non-covalent complex that can be further locked by introducing cysteine residues at critical positions of DDD and AD to promote disulfide bond formation.

FIGS. 10A-10D are schematic illustrations of four exemplary forms or models of protein complexes described herein. As shown in fig. 10A and 10B, two light chains of one antibody (e.g., an anti-TAA antibody) are linked to two DDD sequences, while another protein (e.g., a single chain anti-CD 3 antibody) is linked to an AD sequence. Because two DDD sequences affect the spontaneous formation of dimers, two DDD-containing chains dimerize via a DDD sequence. The DDD dimer motif contained in the light chain creates a docking site for binding to AD sequences contained in other proteins, thereby facilitating rapid binding of the two light chains to other proteins (in this particular example, anti-CD 3 antibodies) forming a binary trimeric complex with two antigenic sites for one antigen (e.g., TAA) and one antigenic site for the other antigen (e.g., CD 3), i.e., the "2+1" form.

Similarly, as shown in fig. 10C and 10D, two heavy chains of one antibody (e.g., an anti-TAA antibody) are linked to (fig. 10D) or two DDD sequences are inserted (fig. 10C), while the other protein (e.g., a single chain anti-CD 3 antibody) is linked to an AD sequence. The DDD dimer motif contained in the heavy chain creates a docking site for binding to AD sequences contained in other proteins, thereby facilitating rapid binding of the two heavy chains to other proteins (in this particular example, anti-CD 3 antibodies), thereby also forming a binary trimeric complex in the form of "2+1".

This binding event can be stabilised by a subsequent reaction to covalently fix two or three entities of the trimeric complex by disulfide bonds, which can happen very effectively based on the principle of effective local concentration, since the initial binding interaction should bring the reactive thiol groups in the vicinity of DDD and AD (Chmura et al., proc. Natl. Acad. Sci. USA 2001; 98:8480) for site-specific ligation. For example, as shown in FIG. 10B, the two linkers connecting each pair of light chain and DDD sequences are stabilized by disulfide bonds. Such one or more disulfide bonds may also be strategically placed between any two or more chains in the complex. Using various combinations of linkers, linker modules, and precursors, a variety of constructs of different stoichiometries can be generated and used (see, e.g., U.S. patent nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787, and 7,666,400).

Unexpectedly, all four forms in the present disclosure can form a functional binary trimeric complex, although multiple protein chains are bound together by multiple dimerization and trimerization domains. Although two free DDD fusion molecules (e.g., two Fab-DDD molecules or two interferon-DDD molecules) can dimerize and assemble with an AD fusion molecule (e.g., scFv-AD2 molecules) via disulfide covalent linkages to form a stable structure, it is unpredictable whether the two DDD moieties can dimerize correctly (intramolecular dimerization, intermolecular crosslinking, or both) when fused to two light or heavy chains of one immunoglobulin molecule. As disclosed herein, unexpectedly, both IgG monomers and covalently linked IgG dimers or multimers are formed in forms a and D as shown in fig. 11A and 11D without the addition of AD-linked scFv. Furthermore, as detected by HPLC (data not shown), some IgG monomers and most non-covalently linked dimeric IgG aggregate in forms B and C without addition of AD-linked scFv. On the other hand, without the addition of DDD-linked IgG, when AD-linked scFv was produced alone, it formed monomers and covalently linked dimers (fig. 11A-11D). Indeed, it was surprising that when AD-linked scFv and DDD-linked IgG were co-produced in one cell, most of them assembled to form stable IgG-scFv monomers with only a minimal amount of dimers (fig. 11A-11D) or a small amount of aggregates.

For forms a and B, it was surprisingly found that the attachment of a third protein (e.g. a single chain anti-CD 3 antibody) to the light chain by DDD/AD trimer does not interfere with the assembly and function of the two heavy chain-light chain pairs. For form C, it was surprisingly found that insertion of sequences (e.g., DDD sequences) in the hinge or flanking regions does not prevent the formation and function of the two heavy chain-light chain pairs. It was also unexpected that, despite the limited space, DDD inserted into both heavy chains could dimerize and dock with AD sequences to form trimers. As for form D, unexpectedly, the formation of DDD/AD trimer at the C-terminus of the heavy chain does not interfere with the function of the Fc region. Of the four forms of the complex, form C is the most unpredictable design because of potential steric constraints and unpredictable effects on DDD dimerization, DDD-AD interactions, and structure/function of IgG molecules when two DDD moieties are inserted into the flexible hinge region. Unexpectedly, however, this form yields the best quality, purity, yield and bioactivity of the product.

Furthermore, by linking DDD and AD to functional groups remote from the first and second moieties, such site-specific linking is expected to preserve the original activity of both moieties. This approach is modular in nature and can be applied to site-specific and covalent attachment of a variety of substances, including peptides, proteins, antibodies, antibody fragments, nucleic acids, chemicals and other effectors or targeting moieties having a wide range of activities. Virtually any protein or peptide may be incorporated using the fusion protein method described in the examples below to construct an effector or targeting moiety for AD and DDD binding. However, the technique is not limiting and other bonding methods may be utilized.

A variety of methods are known for preparing fusion proteins, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest. Such double stranded nucleic acids can be inserted into expression vectors by standard molecular biology techniques to produce fusion proteins. For further guidance, those skilled in the art can refer to Frederick M.Ausubel et al, current Protocols in Molecular Biology, john Wiley & Sons,2003, and Sambrook et al.,Molecular Cloning,A Laboratory Manual,"Cold Spring Harbor Press,Cold Spring Harbor,NY,2001).

In preferred embodiments, the AD and/or DDD moiety may be linked to the N-terminus or C-terminus or middle of an effector or targeting protein or peptide. However, one skilled in the art will recognize that the attachment site of the AD or DDD moiety to the effector moiety may vary, depending on the chemical nature of the effector moiety and the moiety involved in the physiological activity in the effector moiety or targeting moiety. Site-specific ligation of various effectors or targeting moieties may be performed using techniques known in the art, for example, using divalent crosslinking reagents and/or other chemical conjugation techniques.

A variety of different AD or DDD sequences may be used. Exemplary DDD and AD sequences include the sequences described in US9315567 and functional variants thereof.

The structure-function relationship of the AD and DDD domains has been the subject of research. See, for example, ,Burns-Hamuro et al.,2005,Protein Sci 14:2982-92;Carr et al.,2001,J Biol Chem 276:17332-38;Alto et al.,2003,Proc Natl Acad Sci USA 100:4445-50;Hundsrucker et al.,2006,Biochem J 396:297-306;Stokka et al.,2006,Biochem J 400:493-99;Gold et al.,2006,Mol Cell 24:383-95;Kinderman et al.,2006,Mol Cell 24:397-408. the contents of these publications are incorporated herein by reference.

KINDERMAN ET al (2006,Mol Cell 24:397-408) examined the crystal structure of AD-DDD binding interactions and concluded that the human DDD sequence contains a number of conserved amino acid residues, important for dimer formation or AKAP binding, which are underlined in the following sequence. (see also KINDERMAN ET al, 2006, fig. 1 which is incorporated herein by reference), one skilled in the art will recognize that in designing sequence variants of DDD sequences, one would wish to avoid altering any underlined residues, while conserved amino acid substitutions may be made for residues that are less important for dimerization and AKAP binding. SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 77)

As discussed in more detail below, conservative amino acid substitutions for each of the 20 common L-amino acids have been characterized. Thus, based on the data of KINDERMAN (2006) and conservative amino acid substitutions, potential alternative DDD sequences can be created based on the above sequences. Those skilled in the art will recognize that many other alternative species within the DDD moiety genus can be constructed by standard techniques, for example using commercial peptide synthesizers or well known site-directed mutagenesis techniques. Some exemplary DDD variants are listed below.

THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:78)

SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:79)

SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:80)

SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:81)

SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:82)

SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:83)

SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:84)

SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:85)

SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:86)

SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFIRLREARA(SEQ ID NO:87)

SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:88)

SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:89)

SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA(SEQ ID NO:90)

SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA(SEQ ID NO:91)

SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA(SEQ ID NO:92)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA(SEQ ID NO:93)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA(SEQ ID NO:94)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA(SEQ ID NO:95)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA(SEQ ID NO:96)

SHIQEPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA(SEQ ID NO:97)

The N-terminal dimer structure of RIIα (1-44) can be subdivided into two functional regions, the first 23 residues, forming the AD binding surface (docking domain), and residues 24-44, which comprise the majority of dimer contacts (dimerization domain) (Newlon et al., nat. Struct. Biol.1999; 6:222). If the N-terminal motif of riiα is inserted into both self-dimerizing heavy chains of an immunoglobulin, the docking domain (DD, at least amino acids 1-23:SHIQIPPGLTELLQGYTVEVLRQ,SEQ ID NO:98) may remain bound to the AD fusion moiety alone without the dimerization domain. Thus, in some embodiments, only the docking motif or functional variant thereof is used to form a complex or fusion protein described herein.

The effect of amino acid substitutions on binding of the AD moiety can also be readily determined by standard binding assays, such as those disclosed in Alto et al (2003,Proc Natl Acad Sci USA 100:4445-50). Specifically, alto et al performed bioinformatic analysis of AD sequences of various AKAP proteins, designed an RII-selective AD sequence called AKAP-IS (SEQ ID NO: 75) with a binding constant to DDD of 0.4nM. AKAP-IS sequences are designed as peptide antagonists of AKAP binding to PKA. Residues in the AKAP-IS sequence are underlined in the following sequence, where substitutions tend to reduce binding to DDD.

QIEYLAKQIVDNAIQQA(SEQ ID NO:75)

QIEYVAKQIVDYAIHQA(SEQ ID NO:99)

One skilled in the art will recognize that in designing sequence variants of the AD sequence, it is desirable to avoid altering any underlined residues, while conservative amino acid substitutions may be made for residues that are less critical for DDD binding. Those skilled in the art will also recognize that based on the data of Alto et al (2003), those skilled in the art can prepare, test and use many other variant species within the genus of possible AD partial sequences. It IS also notable that Gold et al (2006,Mal Cell 24:383-95) developed a SuperAKAP-IS sequence (SEQ ID NO: 99) using crystallography and peptide screening that was five orders of magnitude more selective for the RII subtype of PKA than the RI subtype. It IS contemplated that in certain alternative embodiments, superAKAP-IS sequences may replace the AKAP-IS AD partial sequence to construct any of the four forms of the complex of FIGS. 10A-10D. AKAP-IS and SuperAKAP-IS and variants thereof represent synthetic RII subunit binding peptides, more relevant content being presented in patent US 9315567.

Additional DDD binding sequences are found in a variety of AKAP proteins or are developed as peptide competitors to AKAP binding PKA. The sequences of the various AKAP antagonistic peptides are provided in table 1 of Hundsrucker et al (2006,Biochem J396:297-306). In AKAP, AKAP7 delta (or AKAP18 delta) binds to the riia subunit with high affinity. AKAP7 delta binds to riiα and riiβ subunits with Kd values of 31nM and 20nM, respectively. The truncated AKAP7 delta mutant consisting of amino acid residues 124-353 binds to riiα and riiβ subunits of PKA with higher affinity than the full-length protein (9 nM and 4nM, respectively) (Henn et al, j. Biol. Chem (2004) 279,26654-26665). From the AKAP7 δrii binding domain, hundsrucker et al (2006,Biochem J396:297-306) a 25 amino acid peptide was identified that has a higher binding affinity (kd=0.4 nM) to the RII subunit than the full length protein, N-terminally truncated mutants (as described above) and peptides derived from other AKAPs. The 25 amino acid peptide was designated AKAP7 delta-wt-pep (SEQ ID 100). The introduction of single amino acid substitutions of polar or charged amino acid residues (e.g., the peptides AKAP7 delta-L314E-pep and AKAP7 delta-L304T-pep, SEQ ID NOs 101 and 102) did not significantly alter the affinity of the AKAP7 delta-derived peptide to the human RIIIa subunit. One skilled in the art will recognize that based on Hundsrucker et al data (2006,Biochem J396:297-306), one can prepare, test, and use other variant peptides within the RII binding domain of AKAP7 delta. In clinical applications, the human AKAP7 delta derivative peptide provides a valuable alternative for synthesizing AKAP-IS or SuperAKAP-IS or variant peptides thereof (such as AD 2), and the invention links AKAP7 delta derivative peptide (such as AD 7) with an irrelevant protein for the first time, and successfully forms a stable complex with another DDD fusion protein.

CGPDDAELVRLSKRLVENAVLKAVQQYGC(SEQ ID NO:46)

PEDAELVRLSKRLVENAVLKAVQQY(SEQ ID NO:100)

PEDAELVRTSKRLVENAVLKAVQQY(SEQ ID NO:101)

PEDAELVRLSKRLVENAVEKAVQQY(SEQ ID NO:102)

As with the AD2 sequence shown in SEQ ID NO. 2, the AD moiety may also include additional N-terminal residues cysteine and glycine and C-terminal residues glycine and cysteine.

Joint

The AD or DDD domains described above, as well as other domains disclosed herein, can be linked to another protein by a linker, such as, but not limited to, a chemical modification, a peptide linker, a chemical linker, covalent or non-covalent bonds, or a protein fusion or by any means known to those of skill in the art. The connection may be permanent or reversible. See, for example, U.S. patent nos. 4625014, 5057301, and 5514363, U.S. patent application nos. 20150182596 and 20100063258, and WO2012142515, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, multiple linkers may be included to take advantage of the desired properties of each linker and each protein domain in the complex. For example, it is contemplated to use flexible linkers alone and linkers that increase the solubility of the complex or with other linkers. Peptide linkers can be linked to one or more protein domains in the complex by expression of DNA encoding the linker. The linker may be acid cleavable, photo cleavable and thermosensitive. Bonding methods are well known to those skilled in the art and are included for use in the present invention.

Suitable examples of such linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic substances (e.g., polyethylene glycol). In some embodiments, the linker is a peptide linker. The peptide linker may be about 2-100, 10-50, or 15-30 amino acids in length. In some embodiments, the peptide linker may be at least 10, at least 15, or at least 20 amino acids in length, and no more than 80, no more than 90, or no more than 100 amino acids in length. In some embodiments, the linker is a peptide linker having a single or repeated GGGGS, GGGS, GS, GSGGS, GGSG, GGSGG, GSGSG, GSGGG, GGGSG and/or GSSSG sequence (SEQ ID NOS: 103-112).

In some embodiments, the AD or DDD domain and the other protein domain may be linked by a peptide linker. Peptide linkers can be joined by expressing nucleic acids encoding the two domains and the linker in frame. Optionally, the peptide linker can be attached at one or both of the amino-terminus and the carboxy-terminus of the domain. In some examples, the linker is an immunoglobulin hinge region linker, as disclosed in U.S. patent nos. 6,165,476, 5,856,456, U.S. patent application nos. 20150182596 and 2010/0063258, and international application WO2012/142515, each of which is incorporated herein by reference in its entirety.

Anti-TROP 2 antibodies and anti-HER 2 antibodies

The present disclosure provides novel anti-TROP 2 antibodies or antigen binding fragments thereof. Trop2 is also known as tumor-associated calcium signaling protein 2, trophoblast antigen 2, cell surface glycoprotein Trop-2/Trop2, gastrointestinal tumor-associated antigen GA7331, pancreatic cancer marker protein GA733-1/GA733, chromosome membrane fraction 1 surface marker 1M1S1, epithelial glycoprotein-1 (EGP-1), CAA1, gelatinous water droplet-like corneal dystrophy GDLD, and TTD2. It is encoded by gene Tacstd and the human TACSTD gene.

The gene encodes a cancer-associated antigen that is a member of a family comprising at least two type I membrane proteins. It transduces intracellular calcium signals and acts as a cell surface receptor. Mutation of this gene can lead to diseases including gelatinous water droplet-like corneal dystrophies, an autosomal recessive genetic disease characterized by severe corneal amyloidosis, leading to blindness. The transmembrane glycoprotein Trop2 is highly expressed in many (but not all) cancers and has differential expression in certain normal tissues. About 35kDa. Trop2 spans the cell membrane and has an extracellular domain, a transmembrane domain and an intracellular domain, and a cytoplasmic tail necessary for signal transduction (Shvartsur et al., genes cancer.2015Mar (3-4): 84-105). Trop-2 is upregulated in many cancer types, independent of baseline levels of Trop-2 expression. Trop-2 is an ideal candidate for targeted therapy because it is a transmembrane protein, has an extracellular domain that is overexpressed in a variety of tumors, and is upregulated relative to normal cells. Zaman et al Onco Targets Ther.2019, 12:1781-1790.

The anti-TROP 2 antibodies or antigen binding fragments thereof described herein are useful for treating a variety of cancers and tumor types. Examples include, but are not limited to, breast cancer (e.g., triple negative breast cancer), urothelial cancer (e.g., platinum resistant urothelial cancer), and lung cancer (e.g., small cell lung cancer).

Example 3 and table 2 below show CDR sequences, light chain variable region sequences, and heavy chain variable region sequences of exemplary anti-TROP 2 antibodies L0125 and humanized versions of hL 0125. The antibodies can be used to prophylactically and therapeutically treat and protect a subject against a tumor or cancer.

In some embodiments, the antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) having the heavy chain variable region of the amino acid sequence of SEQ ID NO. 48 (HCDR 1, HCDR2, and HCDR 3), such as SEQ ID Nos. 43-35 shown in Table 2, and three light chain CDRs (LCDRs) having the light chain variable region of the amino acid sequence of SEQ ID NO. 47 (LCDR 1, LCDR2, and LCDR 3), such as SEQ ID Nos. 40-42 shown in Table 2.

VL protein of hL0125 (SEQ ID NO. 47)

DIQLTQSPAIMSASPGERVTMTCRASSSVSSSYLHWYQQRSGQSPKLLIYSTSNLASGVPARFSGSGSGTDYSLTISSLEAEDAATYYCQQYSGSPLTFGSGTKLEIKR

VH protein of hL0125 (SEQ ID No. 48)

QVQLQESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPDKRLEWVAEISSDGFYTYYPDTVTGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARDGNYVDYAMDYWGQGTSVTVSS

In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region having an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SEQ ID NO:47 or 10, or having an amino acid sequence of SEQ ID NO:47 or 10, and a heavy chain variable region having an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SEQ ID NO:48 or 49 or 12, or having an amino acid sequence of SEQ ID NO:48 or 49 or 12.

In some embodiments, an antibody or antigen binding portion/fragment thereof that binds HER2 is used as a targeting moiety or effector moiety in a protein complex described herein. Various anti-HER 2 antibodies are known in the art. Such anti-HER 2 antibodies or antigen binding portions/fragments thereof may be used in protein complexes.

In some embodiments, the anti-HER 2 antibody or antigen binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 of the heavy chain region having the amino acid sequence of SEQ ID NO. 14, e.g., SEQ ID Nos. 24-26 shown in Table 3, and LCDR1, LCDR2, and LCDR3 of the light chain region having the amino acid sequence of SEQ ID NO. 13, e.g., SEQ ID Nos. 21-23 shown in Table 3.

In some embodiments, the anti-HER 2 antibody or antigen binding fragment thereof comprises a light chain variable region having an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SEQ ID NO:13, or having the amino acid sequence of SEQ ID NO:13, and a heavy chain variable region having an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SEQ ID NO:14, or having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the antibody or antigen binding fragment thereof further comprises a variant Fc region (e.g., a variant Fc region comprising E233P/L234V, L235A, G del and S267K substitutions according to EU numbering). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a humanized monoclonal antibody. In some embodiments, the antibody is a single chain antibody, fab, or Fab2 fragment.

In some embodiments, the antibody or antigen binding fragment thereof may be detectably labeled or bound to a toxin, therapeutic agent, polymer (e.g., polyethylene glycol (PEG)), receptor, enzyme, or receptor ligand. For example, the antibodies of the disclosure can be conjugated to a toxin (e.g., tetanus toxin). Such antibodies are useful for treating animals, including humans, suffering from cancer.

In another example, an antibody of the present disclosure may be conjugated to a detectable label. Such antibodies can be used in diagnostic assays to determine whether an animal (e.g., human) has cancer associated with TROP2 expression. Examples of detectable labels include fluorescent proteins (i.e., green fluorescent protein, red fluorescent protein, yellow fluorescent protein), fluorescent labels (i.e., fluorescein isothiocyanate, rhodamine, texas red), radioactive labels (i.e., 3H, 32P, 125I), enzymes (i.e., β -galactosidase, horseradish peroxidase, β -glucuronidase, alkaline phosphatase), or affinity labels (i.e., avidin, biotin, streptavidin). Methods of coupling antibodies to detectable labels are known in the art. Harlow et al, antibodies A Laboratory Manual, page 319 (Cold Spring Harbor Pub.1988).

Fragments

In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, fab '-SH, F (ab') 2, fv, and single chain Fv (scFv) fragments, as well as other fragments described below, such as diabodies, triabodies, tetrabodies, and single domain antibodies. For a review of certain antibody fragments, see Hudson et al, nat.Med.9:129-134 (2003). For reviews of scFv fragments see, e.g., Pluckthun,in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.,(Springer-Verlag,New York),pp.269-315(1994);, see also WO93/16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F (ab') 2 fragments that include salvaged receptor binding epitope residues and have increased in vivo half-life, see U.S. patent No. 5,869,046.

Diabodies are antibody fragments having two antigen binding sites, which may be bivalent or bispecific. See, e.g., EP404,097; WO1993/01161;Hudson et al, nat. Med.9:129-134 (2003), and Hollinger et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Tri-and tetra-antibodies are also described in Hudson et al, nat.Med.9:129-134 (2003).

A single domain antibody is an antibody fragment that includes all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In some embodiments, the single domain antibody is a human single domain antibody (DOMANTI, inc., waltham, mass; see, e.g., U.S. patent No. 6,248,516).

Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies, and by recombinant host cells (e.g., E.coli or phage), as described herein.

Chimeric and humanized antibodies

In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567, and Morrison et al, proc.Natl. Acad.Sci.USA,81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, the chimeric antibody is a "class switch" antibody, wherein the class or subclass has been altered from the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In some embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In general, humanized antibodies include one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are from a non-human antibody and the FRs (or portions thereof) are from a human antibody sequence. The humanized antibody also optionally comprises at least a portion of a constant region of human origin. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or increase antibody specificity or affinity.

For a review of humanized antibodies and Methods of their preparation see, e.g., almagro and Fransson, front. Biosci.13:1619-1633 (2008), and are further described, e.g., in Riechmann et al.,Nature 332:323-329(1988);Queen et al.,Proc.Nat'l Acad.Sci.USA 86:10029-10033(1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; KASHMIRI ET al, methods 36:25-34 (2005) (describing Specific Determinant Region (SDR) grafting), padlan, mol. Immunol.28:489-498 (1991) (describing "surface reconstruction"); dall' Acqua et al, methods 36:43-60 (2005) (describing "FR shuffling"); osbourn et al, methods 36:61-68 (2005) and Klimka et al, br.J. Cancer,83:252-260 (2000) (describing the "guide selection" method of FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., sims et al J.Immunol.151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subsets of the light or heavy chain variable regions (see, e.g., carter et al Proc. Natl. Acad. Sci. USA,89:4285 (1992)), and Presta et al J.Immunol.,151:2623 (1993)), human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., almagro and Fransson, front. Biosci.13:1619-1633 (2008)), framework regions derived from screening FR libraries (see, e.g., baca et al, J.biol. Chem.272:10678-84 (1997) and Rosok et al, J.biol. Chem.271:22611 (1996)).

Human antibodies

In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared using various techniques known in the art or using the techniques described herein. For a general description of human antibodies see van Dijk AND VAN DE WINKEL, curr. Opin. Phacol.5:368-74 (2001) and Lonberg, curr. Opin. Immunol.20:450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody having a variable region of human origin in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, either extrachromosomally or randomly integrated into the animal chromosome. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describe XENOMOUSE technology, U.S. Pat. No. 5,770,429 describes HUMAB technology, U.S. Pat. No. 7,041,870 describes K-M MOUSE technology, and U.S. patent publication No. 2007/0061900 describes VELOCIMOUSE technology. The human variable regions from the whole antibodies produced by such animals may be further modified, for example by binding to different human constant regions.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heterologous myeloma cell lines for the preparation of human monoclonal antibodies have been described (see, e.g., ,Kozbor J.Immunol.,133:3001(1984);Brodeur et al.,Monoclonal Antibody Production Techniques and Applications,pp.51-63(Marcel Dekker,Inc,New York,1987); and Boerner et al, proc.147:86 (1991)). Human antibodies prepared by human B cell hybridoma technology are also described in Li et al, proc.Natl. Acad.Sci.USA,103:3557-3562 (2006). Other methods include, for example, in U.S. Pat. No. 7,189,826 (describing the preparation of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, xiandai Mianyixue,26 (4): 265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, histology and Histopathology,20 (3): 927-937 (2005) Vollmers and Brandlein,Methods and Inventions in Experimental and Clinical Pharmacology,27(3):185-91(2005).

Human antibodies can also be prepared by isolating Fv clones having a variable domain sequence selected from a human phage display library. Such variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from a repertoire of antibodies are described below.

Antibodies of the present disclosure can be isolated by screening a combinatorial library to obtain antibodies having the desired activity. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are reviewed in, for example, Hoogenboom et al.,Methods in Molecular Biology 178:1-37(O'Brien et al.,ed.,Human Press,Totowa,N.J.,2001) and further described in, for example, McCafferty et al.,Nature 348:552-554;Clackson et al.,Nature 352:624-628(1991);Marks et al.,J.Mol.Biol.222:581-597(1992);Marks and Bradbury,Methods in Molecular Biology 248:161-175(Lo,ed.,Human Press,Totowa,N.J.,2003);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004); and Lee et al, J.Immunol. Methods 284 (1-2): 119-132 (2004).

In some phage display methods, VH and VL gene libraries are cloned separately by Polymerase Chain Reaction (PCR) and randomly recombined in phage libraries, and antigen-binding phages can then be screened as described by Winter et al, ann.rev.immunol.,12:433-455 (1994). Phage typically display antibody fragments, either as scFv fragments or Fab fragments. Libraries from immune sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, the initial pool can be cloned (e.g., from humans) as described in GRIFFITHS ET al, EMBO J,12:725-734 (1993) to provide antibodies against a single source of multiple non-self and self antigens without any immunization. Finally, the original library can also be synthesized by cloning unrearranged V gene fragments from stem cells and encoding highly variable CDR3 regions using PCR primers containing random sequences and completing the rearrangement in vitro, as described in Hoogenboom AND WINTER, j.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. patent No. 5,750,373, and U.S. patent publication nos. 2005/0075974, 2005/019455, 2005/0266000, 2007/017126, 2007/0160598, 2007/027764, 2007/0292936, and 2009/0002360. Antibodies or antibody fragments isolated from a human antibody library are herein considered human antibodies or human antibody fragments.

Variants

In some embodiments, amino acid sequence variants of the antibodies and any component of the protein complexes described above are contemplated. These components may be any immunoglobulin chain, targeting moiety/agent, effector moiety/agent, DDD moiety and AD moiety. Thus, the protein complexes and fusion proteins disclosed herein include those having one or more component variants.

For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to obtain the final construct, provided that the final construct has the desired properties, such as antigen binding.

Substitution, insertion and deletion variants

In some embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVRs and FR. Conservative substitutions are defined herein. Amino acid substitutions may be introduced into the antibody of interest and the products screened to obtain the desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Thus, an antibody of the disclosure may include one or more conservative modifications of the CDRs, heavy chain variable regions, or light chain variable regions described herein. Conservative modifications or functional equivalents of a peptide, polypeptide or protein disclosed in the present disclosure refer to polypeptide derivatives of the peptide, polypeptide or protein, such as proteins having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. It substantially retains the activity of the parent peptide, polypeptide or protein (e.g., those disclosed in this disclosure). In general, a conservative modification or functional equivalent is one that has at least 60% (e.g., any number between 60% and 100%) identity with the parent, including the endpoints, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%). Thus, antibodies having heavy chain variable or light variable regions of one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof, and having variant regions are also within the scope of the present disclosure.

As used herein, the percent homology between two amino acid sequences corresponds to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,%homology = number of identical positions/total number of positions x 100), which gaps need to be introduced to achieve optimal alignment of the two sequences, taking into account the number of gaps and the length of each gap. Comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the algorithm of e.meyers and w.miller (comp.appl.biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap penalty of 12, and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences may be calculated using the Needleman and Wunsch (j.mol. Biol.48:444-453 (1970)) algorithm, which has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum62 matrix or PAM250 matrix, with a GAP weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

Additionally or alternatively, the protein sequences of the present disclosure may also be used as "query sequences" to search against a public database, such as to identify related sequences. Such searches may be performed using the XBLAST program of Altschul et al (version 2.0). Altschul, et al (1990) J.mol.biol.215:403-10. BLAST protein searches can be performed using the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain a gap alignment for comparison purposes, the Gapped BLAST program can be used, as described in Altschul et al, (1997) Nucleic Acids Res.25 (17): 3389-3402. When using BLAST and Gapped BLAST programs, default parameters (e.g., XBLAST and NBLAST) for the respective programs can be used. (see www.ncbi.nlm.nih.gov).

Exemplary substitution variants are affinity matured antibodies, which may, for example, use phage display-based affinity maturation techniques, such as described in Hoogenboom et al.,Methods in Molecular Biology 178:1-37(O'Brien et al.,ed.,Human Press,Totowa,N.J.,2001). Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions, ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusion of the N-terminus or C-terminus of an antibody with an enzyme (e.g., ADEPT) or polypeptide that increases the serum half-life of the antibody.

In some embodiments, the disclosed methods and compositions may relate to the preparation and use of proteins or peptides having one or more substituted amino acid residues. For example, DDD and/or AD sequence variants have been discussed above.

One skilled in the art will appreciate that in general, amino acid substitutions generally involve the substitution of one amino acid with another having relatively similar properties (i.e., conservative amino acid substitutions). The properties of various amino acids and the effect of amino acid substitutions on protein structure and function have been the subject of extensive research and knowledge in the art.

For example, the hydropathic index of amino acids may be considered (Kyte & Doolittle,1982, J.mol. Biol., 157:105-132). The relatively hydrophilic nature of the amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules. Each amino acid is assigned a hydropathic index (Kyte & Doolittle, 1982) based on its hydrophobicity and charge characteristics, which are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5). In performing conservative substitutions, amino acids with a hydropathic index within ±2 are preferably used, more preferably within ±1, and even more preferably within ±0.5.

Amino acid substitutions may also take into account the hydrophilicity of the amino acid residues (e.g., U.S. Pat. No.4,554,101). The hydrophilicity values of the amino acid residues were arginine (+3.0), lysine (+3.0), aspartic acid (+3.0), glutamic acid (+3.0), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5.+ -. 0.1), alanine (-0.5), histidine (-0.5), cysteine (-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), tryptophan (-3.4). Amino acid substitutions with similar hydrophilicity are preferred.

Other considerations include the size of the amino acid side chains. For example, it is generally not preferred to replace an amino acid with a compact side chain (e.g., glycine or serine) with an amino acid with a bulky side chain (e.g., tryptophan or tyrosine). The effect of various amino acid residues on the secondary structure of proteins is also a consideration. Through empirical studies, the effect of different amino acid residues on the tendency of a protein domain to adopt an α -helix, β -sheet or reverse secondary structure has been determined and is known in the art (see, e.g. ,Chou&Fasman,1974,Biochemistry,13:222-245;1978,Ann.Rev.Biochem.,47:251-276;1979,Biophys.J.,26:367-384).

Based on such considerations and extensive empirical studies, conservative amino acid substitution tables have been constructed and are known in the art. Such as arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, and valine, leucine and isoleucine. Or (A) leucine, isoleucine, valine, arginine (R) glutamine, asparagine, lysine, asparagine (N) histidine, aspartic acid, lysine, arginine, glutamine, aspartic acid (D) asparagine, glutamic acid, cysteine (C) alanine, serine, glutamine (q) glutamic acid, asparagine, glutamic acid (E) glutamine, aspartic acid, glycine (G) alanine, histidine (H) asparagine, glutamine, lysine, arginine, isoleucine (I) valine, methionine, propylamino acid, phenylalanine, leucine (L) valine, methionine, propylamino acid, phenylalanine, isoleucine, lysine (K) glutamine, asparagine, arginine, methionine (M) phenylalanine, isoleucine, leucine, phenylalanine (F) leucine, valine, isoleucine, propylamino acid, tyrosine, proline (P) alanine, serine (S), threonine (T) serine, tryptophan (W) phenylalanine, tyrosine (Y), phenylalanine, tryptophan, serine, leucine (V) isoleucine, phenylalanine, alanine.

Other considerations for amino acid substitutions include whether the residue is located within the protein or exposed to solvents. Conservative substitutions include aspartic acid and asparagine, serine and threonine, serine and propylamine, threonine and alanine, alanine and glycine, isoleucine and valine, valine and leucine, leucine and isoleucine, leucine and methionine, phenylalanine and tyrosine, tyrosine and tryptophan. Conservative substitutions (see, e.g., PROWL website winder.edu) for residues exposed to solvents include aspartic acid and asparagine, aspartic acid and glutamic acid, glutamic acid and glutamine, glutamic acid and alanine, glycine and aspartic acid, alanine and alanine, alanine and glycine, alanine and serine, alanine and lysine, serine and threonine, lysine and arginine, valine and leucine, leucine and isoleucine, isoleucine and valine, phenylalanine and tyrosine. Various matrices have been constructed (supra) to aid in the selection of amino acid substitutions, such as PAM250 scoring matrix, dayhoff matrix, grantham matrix, MCLACHLAN matrix, doolittle matrix, henikoff matrix, miyata matrix, fitch matrix, jones matrix, rao matrix, levin matrix, and Risler matrix (supra).

In determining amino acid substitutions, the presence of intermolecular or intramolecular bonds, such as ionic bonds (salt bridges) formed between positively charged residues (e.g., histidine, arginine, lysine) and negatively charged residues (e.g., aspartic acid, glutamic acid) or disulfide bonds formed between nearby cysteine residues, may also be considered.

Any amino acid is known to those skilled in the art and can be substituted for any other amino acid in the encoded protein sequence by routine experimentation, for example by site-directed mutagenesis techniques or by synthesis and assembly of oligonucleotides encoding the amino acid substitutions and splicing them into expression vector constructs.

Glycosylation variants

In some embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. The addition or deletion of antibody glycosylation sites can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

For example, non-glycosylated antibodies (i.e., antibodies lacking glycosylation) may be prepared. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modification may be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions may be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such deglycosylation may increase the affinity of the antibody for the antigen. Such a process is described in more detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co et al.

Glycosylation of the constant region at N297 may be prevented by mutating the N297 residue to another residue (e.g., N297A) and/or by mutating adjacent amino acids (e.g., 298) to reduce glycosylation at N297.

Additionally or alternatively, antibodies with altered glycosylation types, such as low fucosylation antibodies with reduced fucose residues or antibodies with increased bisecting GlcNac structure, can be prepared. This pattern of glycosylation alterations has been demonstrated to enhance the ADCC capacity of antibodies. Such carbohydrate modification may be achieved, for example, by expressing the antibody in a host cell having a mechanism for glycosylation modification. Cells having a mechanism of glycosylation alteration have been described in the art and can be used as host cells in which the recombinant antibodies described herein are expressed, thereby producing antibodies having glycosylation alterations. For example, EP1,176,195 to Hanai et al describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such a cell line exhibit low fucosylation. PCT publication WO03/035835 to Presta describes a variant Chinese hamster ovary cell line Led3 cell with reduced ability to attach fucose to asparagine (297) -linked carbohydrates, also resulting in low degrees of fucosylation of antibodies expressed in the host cell (see also Shields, R.L.et al, (2002) J.biol. Chem. 277:26733-26740). PCT publication WO99/54342 to Umana et al describes cell lines engineered to express glycoprotein-modified glycosyltransferases (e.g., beta (1, 4) -N-acetylglucosaminyl transferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisected GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al, (1999) Nat. Biotech.17:176-180).

Fc region variants

The variable regions of the antibodies described herein can be linked (e.g., covalently linked or fused) to an Fc, such as an IgG1, igG2, igG3, or IgG4 Fc, which can be any allotype (allotype) or allotype (isoallotype), such as for IgG1: glm, glm1 (a), glm2 (x), glm3 (f), glm (z), for IgG2: G2m, G2m23 (n), for IgG3：G3m、G3m21(gl)、G3m28(g5)、G3ml1(b0)、G3m5(bl)、G3ml3(b3)、G3ml4(b4)、G3ml0(b5)、G3ml5(s)、G3ml6(t)、G3m6(c3)、G3m24(c5)、G3m26(u)、G3m27(v);, for K: km, kml, km2, km3 (see, e.g., jefferies et al., (2009) mAbs 1: 1). In some embodiments, the antibody variable regions described herein are linked to an Fc that binds to one or more activated Fc receptors (fcγ I, fc γiia or fcγiiia), thereby stimulating ADCC and possibly causing T cell depletion. In some embodiments, the antibody variable regions described herein are linked to Fc that results in depletion.

In some embodiments, the antibody variable regions described herein may be linked to an Fc that includes one or more modifications, typically in order to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, fc receptor binding, and/or antigen-dependent cytotoxicity. Furthermore, the antibodies described herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, thereby altering one or more functional properties of the antibody. The numbering of the residues in the Fc region is that of the EU index of Kabat.

The Fc region includes domains derived from immunoglobulin (preferably human immunoglobulin) constant regions, including fragments, analogs, variants, mutants or derivatives of the constant regions. Suitable immunoglobulins include IgG1, igG2, igG3, igG4 and other classes such as IgA, igD, igE and IgM. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide homologous to the C-terminal region of the immunoglobulin and may include a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, respectively, alone or in combination. The antibodies can have Fc regions from IgG (e.g., igG1, igG2, igG3, and IgG 4) or other classes such as IgA, igD, igE and IgM.

The constant region of the immunoglobulin is responsible for many important antibody functions, including Fc receptor (FcR) binding and complement binding. The heavy chain constant regions are of five main types, igA, igG, igD, igE, igM, each with characteristic effector functions specified by the isotype. For example, igG is divided into four subclasses, called IgG1, igG2, igG3, and IgG4.

Immunoglobulin molecules interact with a variety of cellular receptors. For example, igG molecules interact with three classes of fcγ receptors (fcγr) specific for IgG class antibodies, namely fcγri, fcγrii, and fcγ RIIL. Important sequences for IgG binding to fcγr receptors are reported to be located in the CH2 and CH3 domains. The serum half-life of an antibody is affected by the ability of the antibody to bind FcRn.

In some embodiments, the Fc region is a variant Fc region, e.g., an Fc sequence (e.g., an unmodified Fc polypeptide, which is subsequently modified to produce a variant) that has been modified (e.g., by amino acid substitutions, deletions, and/or insertions) relative to a parent Fc sequence, to provide the desired structural features and/or biological activity. For example, modifications can be made in the Fc region to produce Fc variants that (a) have increased or decreased ADCC, (b) have increased or decreased CDC, (C) have increased or decreased affinity for C1q and/or (d) have increased or decreased affinity for Fc receptors relative to the parent Fc. Such Fc region variants typically include at least one amino acid modification in the Fc region. Combined amino acid modifications are considered particularly desirable. For example, the variant Fc region may include two, three, four, five, etc. substitutions therein, e.g., substitutions at the positions of the particular Fc region identified herein.

Variant Fc regions may also include sequence alterations in which amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cells used to produce the antibodies described herein. Even with cysteine residues removed, the single chain Fc domains can still form dimeric Fc domains that are non-covalently bound together. In other embodiments, the Fc region may be modified to render it more compatible with the host cell of choice. For example, PA sequences near the N-terminus of a typical native Fc region can be removed, which sequences can be recognized by digestive enzymes in e.coli (e.g., proline iminopeptidase). In other embodiments, one or more glycosylation sites within the Fc domain can be removed. Residues that are normally glycosylated (e.g., asparagine) may produce a cytolytic reaction. Such residues may be deleted or replaced with residues that are not glycosylated (e.g., alanine). In other embodiments, a site involved in interaction with complement, such as a C1q binding site, may be removed from the Fc region. For example, the EKK sequence of human IgG1 may be deleted or replaced. In some embodiments, sites that affect binding to Fc receptors may be removed, preferably sites other than rescue receptor binding sites. In other embodiments, the Fc region may be modified to remove ADCC sites. ADCC sites are known in the art, see, for example, molecular immunol 29 (5): 633-9 (1992) for ADCC sites in IgG 1. Specific examples of variant Fc domains are disclosed, for example, in WO97/34631 and WO 96/32478.

In one embodiment, the hinge region of the Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. Such a process is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine residues in the Fc hinge region is altered, for example, to promote assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment, such that the antibody binds to staphylococcal protein a (SpA) more poorly than the native Fc hinge domain. This method is described in further detail in U.S. Pat. No. 6,165,745 to Ward et al.

In other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 may be substituted with a different amino acid residue such that the affinity of the antibody for the effector ligand is altered but the antigen binding capacity of the parent antibody is retained. The affinity-altering effector ligand may be, for example, an Fc receptor or a CI component of complement. This method is described in more detail in Winter et al, U.S. Pat. Nos. 5,624,821 and 5,648,260.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 may be substituted with a different amino acid residue such that the antibody alters C1q binding and/or reduces or eliminates CDC. This process is described in more detail in U.S. Pat. No. 6,194,551 to Idusogie et al.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered, thereby altering the ability of the antibody to bind complement. This method is further described in PCT publication WO94/29351 to Bodmer et al.

In yet another example, the Fc region may be modified by modifying one or more amino acids at the following positions to increase ADCC and/or increase affinity ：234、235、236、238、239、240、241、243、244、245247、248、249、252、254、255、256、258、262、263、264、265、267、268、269、270、272、276、278、280、283、285、286、289、290、292、293、294、295、296、298、299、301、303、305、307、309、312、313、315、320、322、324、325、326、327、329、330、331、332、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、433、434、435、436、437、438 or 439 for fcγ receptors. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T and 267E/268F7324T. Other modifications for enhancing fcγr and complement interactions include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl,2009,Current Opinion in Biotechnology 20:685-691.

Fc modifications that increase binding to fcγ receptors include an Fc region with amino acid modifications ：238、239、248、249、252、254、255、256、258、265、267、268、269、270、272、279、280、283、285、298、289、290、292、293、294、295、296、298、301、303、305、307、312、315、324、327、329、330、335、337、338、340、360、373、376、379、382、388、389、398、414、416、419、430、434、435、437、438 or 439 at any one or more of the following amino acid positions, wherein the numbering of the residues in the Fc region is that of the EU index as in abat (WO 00/42072).

Other Fc modifications that may be made by Fc are those that are used to reduce or eliminate binding to fcγr and/or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, antibody Dependent Cell Phagocytosis (ADCP), and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, where the numbering is according to the EU index. Exemplary permutations include, but are not limited to, 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein the numbering is according to the EU index. The Fc variant may comprise 236R/328R. Other modifications for reducing fcγr and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P and 234V, and glycosylation at position 297 is removed by mutation or enzymatic means or by preparation in organisms (e.g. bacteria) that do not glycosylate the protein. These and other modifications are reviewed in Strohl,2009,Current Opinion in Biotechnology 20:685-691.

Optionally, the Fc region may include non-naturally occurring amino acid residues at addition and/or substitution positions known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; ;WO00/42072;WO01/58957;WO02/06919;WO04/016750;WO04/029207;WO04/035752;WO04/074455;WO04/099249;WO04/063351;WO05/070963;WO05/040217;WO05/092925 and WO 06/020114).

Fc variants that enhance affinity for the inhibitory receptor fcγriib can also be used. Such variants can provide Fc fusion proteins having immunomodulatory activity associated with fcyriib cells (including, for example, B cells and monocytes). In one embodiment, the Fc variant provides a selectively enhanced affinity for fcyriib relative to one or more activating receptors. Modifications for altering binding to fcyriib according to the EU index include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332. Exemplary substitutions for enhancing the affinity of feγriib include, but are not limited to 234D、234E、234F、234W、235D、235F、235R、235Y、236D、236N、237D、237N、239D、239E、266M、267D、267E、268D、268E、327D、327E、328F、328W、328Y and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants useful for enhancing binding to FcgammaRIIB include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E and 267E/328F.

The affinity and binding characteristics of an Fc region for its ligand can be determined by a variety of in vitro assay methods known in the art (biochemical or immunological based assays), including but not limited to equilibration methods (e.g., ELISA or radioimmunoassays) or kinetics (e.g., BIACORE analysis), as well as other methods such as indirect binding assays, competitive inhibition assays, fluorescence Resonance Energy Transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labels on one or more components being examined and/or employ a variety of detection methods including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W.E., ed., fundamental Immunology,4th Ed, lippincott-Raven, philadelphia (1999), which focuses on antibody-immunogen interactions.

In some embodiments, the antibody is modified to increase its biological half-life. Various methods are possible. For example, this can be achieved by increasing the binding affinity of the Fc region for FcRn. For example, one or more of the following residues 252, 254, 256, 433, 435, 436 may be mutated as described in U.S. Pat. No. 6,277,375. Specific exemplary permutations include one or more of T252L, T254S and/or T256F. Alternatively, as described in Presta et al, U.S. Pat. Nos. 5,869,046 and 6,121,022, to increase biological half-life, antibodies can be altered within the CH1 or CL region to include rescue receptor binding epitopes taken from both loops of the CH2 domain of the IgG Fc region. Other exemplary variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include ：250E、250Q、428L、428F、250Q/428L(Hinton et al.,2004,J.Biol.Chem.279(8)：6213-6216;Hinton et al.,2006Journal of Immunology 176:346-356)、256A、272A、286A、305A、307A、307Q、311A、312A、376A、378Q、380A、382A、434A(Shields et al.,Journal of Biological Chemistry,2001,276(9):6591-6604)、252F、252T、252Y、252W、254T、256S、256R、256Q、256E、256D、256T、309P、311S、433R、433S、433I、433P、433Q、434H、434F、434Y、252Y/254T/256E、433K/434F/436H、308T/309P/311S(Dall Acqua et al.,Journal of Immunology,2002,169:5171-5180;Dall'Acqua et al.,2006,Journal of Biological Chemistry 281:23514-23524). other modifications for modulating FcRn binding are described in Yeung et al, 2010, jimmunol,182:7663-7671. In some embodiments, hybrid IgG isotypes with specific biological characteristics may be used. For example, an IgG1/IgG3 hybrid variant can be constructed by substituting the IgG1 position in the CH2 and/or CH3 region with an amino acid from IgG3 at a different position of the two isotypes. Thus, hybrid variant IgG antibodies can be constructed that include one or more substitutions, such as 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments described herein, igG1/IgG2 hybrid variants can be constructed by substituting amino acids from IgG1 at different positions in the CH2 and/or CH3 region for the IgG2 position. Thus, hybrid variant IgG antibodies can be constructed that include one or more substitutions, such as one or more of the following amino acid substitutions 233E, 234L, 235L, 236G (meaning insertion of glycine at position 236) and 321H.

Furthermore, binding sites for FcgammaRI, fcgammaRII, fcgammaRIII and FcRn on human IgG1 have been mapped and variants with improved binding have been described (see Shields, R.L.et al, (2001) J.biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 were shown to improve binding to fcyriii. In addition, the combination mutants, which have been shown to exhibit enhanced FcgammaRIIIa binding and ADCC activity, demonstrate improved FcgammaRIIIa binding to T256A/S298A, S A/E333A, S A/K224A and S298A/E333A/K334A (SHIELDS ET al., 2001). Other IgG1 variants have been identified that have strongly enhanced binding to fcyriiia, including variants with S239D/I332E and S239D/I332E/a330L mutations, which exhibit maximal increase in affinity to fcyriiia, reduced fcyriib binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al, 2006). Triple mutations were introduced into antibodies such as alemtuzumab (CD 52-specific), trastuzumab (HER 2/neu-specific), rituximab (rituximab) (CD 20-specific), and cetuximab (cetuximab) (EGFR-specific), which greatly enhanced ADCC activity in vitro, and the S239D/I332E variant showed enhanced ability to deplete monkey B cells (Lazar et al, 2006). Furthermore, igG1 mutants containing the L235V, F243L, R292P, Y L and P396L mutations have been identified, which mutants show enhanced binding to fcyriiia and at the same time enhanced ADCC activity in transgenic mice expressing human fcyriiia in B cell malignancy and breast cancer models (STAVENHAGEN ET al.,2007;Nordstrom et al, 2011). Other Fc mutants that may be used include S298A/E333A/L334A, S D/I332E, S239D/I332E/A330L, L V/F243L/R292P/Y300L/P396L and M428L/N434S.

In some embodiments, fc is selected that has reduced binding to fcγr. Exemplary fcγr binding reduced fcs, e.g., igG1Fc, include the following three amino acid substitutions L234A, L235E and G237A.

In some embodiments, fc with reduced complement fixation is selected. An exemplary Fc with reduced complement fixation, such as an IgG1 Fc, has the following two amino acid substitutions A330S and P331S.

In some embodiments, an Fc that has substantially no effector function is selected, i.e., its binding to fcγr is reduced and complement fixation is reduced. Exemplary fcs without effectors, such as IgG1 Fc, include the five mutations L234A, L235E, G237A, A S and P331S.

When using an IgG4 constant domain, it is generally preferred to include the substitution S228P, which mimics the hinge sequence in IgG1, thereby stabilizing the IgG4 molecule.

Multivalent antibodies

In one embodiment, the antibodies of the disclosure may be monovalent or multivalent (e.g., bivalent, trivalent, etc.). As used herein, the term "titer" refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds to a target molecule or a specific location or site on a target molecule. When the antibody is monovalent, each binding site of the molecule will specifically bind to a single antigen position or epitope. When an antibody includes more than one target binding site (multivalent), each target binding site can specifically bind to the same or different molecules (e.g., can bind to different ligands or different antigens, or different epitopes or locations on the same antigen). See, for example, U.S. patent publication No. 2009/0129125.

In one embodiment, the antibody is a bispecific antibody, wherein the two chains have different specificities. Other embodiments include antibodies with additional specificity, such as trispecific antibodies. Other more complex compatible multispecific constructs and methods for their preparation are described in U.S. patent No. 2009/0155255, and WO94/04690;Suresh et al, 1986,Methods in Enzymology,121:210, and WO96/27011.

As described above, multivalent antibodies can immunospecifically bind to different epitopes of a desired target molecule, or can immunospecifically bind to more than one target molecule as well as to a heterologous epitope, such as a heterologous polypeptide or solid support material. In some embodiments, the multivalent antibody may comprise a bispecific antibody or a trispecific antibody. Bispecific antibodies also include cross-linked or "heteroconjugate" antibodies. For example, one of the heteroconjugate antibodies may be conjugated to avidin and the other to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO92/200373 and EP 03089). The heteroconjugate antibodies may be prepared using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, as well as some crosslinking techniques.

In some embodiments, the antibody variable domain having the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant domain sequence, such as an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, CH2 and/or CH3 region, using methods well known to those of ordinary skill in the art.

Antibody derivatives

The antibodies provided herein can be further modified to include additional non-protein moieties known and readily available in the art. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers.

Non-limiting examples of water soluble polymers include, but are not limited to, PEG, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly 1, 3-dioxolane, poly 1,3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers) and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in preparation due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular nature or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, and the like.

In another embodiment, conjugates of antibodies and non-protein moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al, proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells but heat the non-protein fraction to a temperature that kills cells near the antibody-non-protein fraction.

Another modification of the antibodies described herein is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. For pegylation of antibodies, the antibody or fragment thereof is typically reacted with PEG (e.g., a reactive ester or aldehyde derivative of PEG) under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, the pegylation is performed by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (CI-CIO) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP0154316 to Nishimura et al, and EP0401384 to Ishikawa et al.

The present disclosure also encompasses human monoclonal antibodies described herein conjugated to a therapeutic agent, polymer, detectable label, or enzyme. In one embodiment, the therapeutic agent is a cytotoxic agent. In one embodiment, the polymer is PEG.

Nucleic acids, expression cassettes and vectors

The present disclosure provides isolated nucleic acid fragments encoding the polypeptides, peptide fragments, conjugated proteins, antibodies, and protein complexes of the present disclosure. Nucleic acid fragments of the present disclosure also include fragments encoding the same amino acid due to the degeneracy of the genetic code. For example, the amino acid threonine is encoded by ACU, ACC, ACA and ACG and is therefore degenerate. The present disclosure is intended to include all variants of a polynucleotide fragment encoding the same amino acid. Such mutations are known in the art (Watson et al Molecular Biology of the Gene, benjamin Cummings 1987). Mutations also include alterations to nucleic acid fragments to encode conservative amino acid substitutions, such as substitution of isoleucine with leucine, and the like. Such mutations are also known in the art. Thus, the genes and nucleotide sequences of the present disclosure include naturally occurring sequences and mutated forms.

The nucleic acid fragments of the present disclosure may be contained within a vector. Vectors may include, but are not limited to, any plasmid, phagemid, factor F, virus, cosmid, or phage, in double-stranded or single-stranded linear or circular form, which may or may not be self-delivering or mobile. The vector may also transform a prokaryotic or eukaryotic host by integration into the cell genome or by presence extrachromosomal (e.g., an autonomously replicating plasmid with an origin of replication).

Preferably, the nucleic acid fragment in the vector is under the control of and operably linked to a suitable promoter or other regulatory element when transcribed in vitro or in a host cell (e.g., eukaryotic cell) or microorganism (e.g., bacteria). The vector may be a shuttle vector that functions in multiple hosts. The vector may also be a cloning vector, which typically contains one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences may be inserted in a determinable fashion. Such insertion may occur without losing the basic biological function of the cloning vector. The cloning vector may also contain a marker gene suitable for identifying and screening cells transformed with the cloning vector. Examples of marker genes are tetracycline resistance or ampicillin resistance. Many cloning vectors are commercially available (Stratagene, NEW ENGLAND Biolabs, clonetech).

The nucleic acid fragments of the invention may also be inserted into expression vectors. Typically, expression vectors contain prokaryotic DNA elements encoding bacterial origins of replication and antibiotic resistance genes to provide for amplification and selection of the expression vector in a bacterial host, regulatory elements such as promoters to control transcription initiation, and DNA elements such as introns or transcription termination/polyadenylation sequences to control transcription product processing.

Methods of introducing nucleic acid fragments into vectors are those available in the art and, briefly, (Sambrook et al.,Molecular Cloning:A Laboratory Manual,3rd edition,Cold Spring Harbor Press,Cold Spring Harbor,N.Y.(2001))., the vector into which the nucleic acid fragment is to be inserted is treated with one or more restriction enzymes (restriction endonucleases) to produce linearized vectors having blunt ends, "sticky" ends with 5 'or 3' overhangs, or any combination of the above. The vector may also be treated with a restriction enzyme, followed by another modification enzyme, such as a polymerase, exonuclease, phosphatase or kinase, to produce a linearized vector having properties useful for ligating nucleic acid fragments into the vector. The nucleic acid fragment to be inserted into the vector is treated with one or more restriction enzymes to produce a linearized fragment having blunt ends, a "sticky" end with 5 'or 3' overhangs, or any combination thereof. The nucleic acid fragment may also be treated with a restriction enzyme, followed by another DNA modification enzyme. Such DNA modifying enzymes include, but are not limited to, polymerases, exonucleases, phosphatases, or kinases to produce nucleic acid fragments having properties useful in ligating the nucleic acid fragments into vectors.

The treated vector and nucleic acid fragments are then ligated together according to methods available in the art to form a nucleic acid fragment-containing construct (Sambrook et al.,Molecular Cloning:A Laboratory Manual,3rd edition,Cold Spring Harbor Press,Cold Spring Harbor,N.Y.(2001)). briefly, and the treated nucleic acid fragments and treated vector are combined in the presence of a suitable buffer and ligase. The mixture is then incubated under appropriate conditions to allow the ligase to ligate the nucleic acid fragments into the vector.

The disclosure also provides expression cassettes containing nucleic acid sequences capable of directing expression of the specific nucleic acid fragments of the disclosure in vitro or in a host cell. Furthermore, the nucleic acid fragments of the present disclosure may be inserted into an expression cassette, thereby generating antisense information. The expression cassette is a separable unit such that the expression cassette can be in a linear form and functional for in vitro transcription and translation assays. Materials and procedures for performing these assays are available from Promega corp (Madison, WI). For example, in vitro transcripts may be produced by placing a nucleic acid sequence under the control of a T7 promoter and then using T7 RNA polymerase to produce in vitro transcripts. The transcript can then be translated in vitro by using rabbit reticulocyte lysate. Alternatively, the expression cassette may be integrated into a vector, allowing replication and amplification of the expression cassette within a host cell or in vitro transcription and translation of a nucleic acid fragment.

Such an expression cassette may contain one or more restriction sites that allow for the nucleic acid fragments to be placed under the control of regulatory sequences. The expression cassette may also contain a termination signal operably linked to the nucleic acid fragment and regulatory sequences required for proper translation of the nucleic acid fragment. The expression cassette containing the nucleic acid fragment may be chimeric, meaning that at least one component thereof is heterologous with respect to at least one other component thereof. The expression cassette may also be naturally occurring, but has been obtained in recombinant form that can be used for heterologous expression. Expression of the nucleic acid fragment in the expression cassette may be under the control of a constitutive or inducible promoter, which initiates transcription only when the host cell is exposed to certain specific external stimuli.

The expression cassette may include transcription and translation initiation regions, nucleic acid fragments, and transcription and translation termination regions that function in vivo and/or in vitro in the 5'-3' direction of transcription. The termination region may be homologous to the transcription initiation region, may be homologous to the nucleic acid fragment, or may be derived from another source.

The regulatory sequence may be a polynucleotide sequence located upstream (5 'non-coding sequence), internal or downstream (3' non-coding sequence) of the coding sequence and which affects transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, enhancers, promoters, repressor binding sites, translation leader sequences, introns, and polyadenylation signal sequences. They may include natural sequences and synthetic sequences and may be a combination of synthetic and natural sequences. Although regulatory sequences are not limited to promoters, some useful regulatory sequences include constitutive promoters, inducible promoters, regulatable promoters, tissue-specific promoters, viral promoters, and synthetic promoters.

A promoter is a nucleotide sequence that controls the expression of a coding sequence by providing for the recognition of RNA polymerase and other factors required for proper transcription. Promoters include minimal promoters which consist only of all basic elements required for transcription initiation, such as TATA-boxes and/or initiators, which are short DNA sequences consisting of TATA-boxes and other sequences for specifying transcription initiation sites, to which regulatory elements are added to control expression. Promoters may be derived entirely from a natural gene, or consist of different elements derived from different promoters found in nature, or even of synthetic DNA fragments. Promoters may contain DNA sequences involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

The disclosure also provides constructs comprising the vectors and expression cassettes. The carrier may be selected from, but is not limited to, any of the carriers previously described. Insertion (Sambrook et al.,Molecular Cloning:A Laboratory Manual,3rd edition,Cold Spring Harbor Press,Cold Spring Harbor,N.Y.(2001)). of an expression cassette into the vector by methods known in the art and previously described in one embodiment, the regulatory sequences of the expression cassette shown may be derived from sources other than the vector into which the expression cassette is inserted. In another embodiment, a construct comprising a vector and an expression cassette is formed upon insertion of a nucleic acid fragment of the disclosure into a vector that itself comprises a regulatory sequence. Thus, an expression cassette is formed after insertion of the nucleic acid fragment into the vector. Vectors containing regulatory sequences are commercially available and methods of their use are known in the art (Clonetech, promega, stratagene).

In another aspect, the disclosure also provides (i) one or more nucleic acid molecules encoding the polypeptide chains of an antibody or antigen binding fragment or protein complex thereof described herein, (ii) a vector comprising the one or more nucleic acid molecules, and (iii) a cultured host cell comprising the vector. Also provided is a method of producing a polypeptide comprising (a) obtaining the cultured host cell, (b) culturing the cultured host cell in a medium under conditions allowing expression of the polypeptide encoded by the vector and assembly of the antibody or fragment or protein complex thereof, and (c) purifying the antibody or fragment or protein complex thereof from the cultured cell or cell culture medium.

Preparation method

Antibodies or antibody fragments or protein complexes can be prepared using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acids may encode amino acid sequences comprising the VL of an antibody and/or amino acid sequences comprising the VH of an antibody (e.g., the light chain and/or heavy chain of an antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed) a (1) vector comprising nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising nucleic acid encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising nucleic acid encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, sp20 cell). In one embodiment, a method of producing an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant methods of making antibodies, nucleic acids encoding the antibodies (e.g., as described herein) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton,Methods in Molecular Biology,Vol.248(B.K.C.Lo,ed.,Humana Press,Totowa,N.J.,2003,pp.245-254, for expression of antibody fragments in E.coli), the antibodies can be isolated as soluble fractions from the bacterial cell paste and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22:1409-1414 (2004) and Li et al, nat. Biotech.24:210-215 (2006).

Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda cells.

Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing plant antibody (PLANTBODIES) techniques for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell lines transformed with SV40 (COS-7), human embryonic kidney cell lines (293 or 293 cells as described, for example, in Graham et al, J.Gen. Virol.36:59 (1977)), hamster kidney cells (BHK), mouse support cells (TM 4 cells as described in Mather, biol. Reprod.23:243-251 (1980)), monkey kidney cells (CV 1), african green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumors (MMT 060562), cells as described in Mather et al, annals N.Y. Acad. Sci.383:44-68 (1982), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include CHO cells, including DHFR-CHO cells (Urlaub et al, proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines, e.g., Y0, NS0, and Sp2/0. For reviews of certain mammalian host cell lines suitable for the production of antibodies, see, for example Yazaki and Wu,Methods in Molecular Biology,Vol.248(B.K.C.Lo,ed.,Humana Press,Totowa,N.J.),pp.255-268(2003).

Composition and formulation

The antibodies or protein complexes of the present disclosure represent excellent pathways for development of therapeutic therapies for treating various disorders, alone or in combination with other therapeutic agents.

In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or protein complex of the present disclosure described herein formulated with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or therapeutic agent.

The pharmaceutical compositions may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, etc. In some embodiments, the compositions comprise an antibody or protein complex of the present disclosure at a concentration of at least 1mg/ml, 5mg/ml, 10mg/ml, 50mg/ml, 100mg/ml, 150mg/ml, 200mg/ml, 1-300mg/ml, or 100-300mg/ml.

In some embodiments, the second therapeutic agent comprises an anti-inflammatory or antiviral compound. In some embodiments, the antiviral compound comprises a nucleoside analog, a peptoid, an oligopeptide, a polypeptide, a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an RNA-dependent RNA polymerase inhibitor. In some embodiments, the antiviral compound may include acyclovir, ganciclovir, vidarabine, foscarnet, cidofovir, amantadine, ribavirin, trifluoretortin, zidovudine, didanosine, zalcitabine, or interferon. In some embodiments, the interferon is interferon- α, interferon- β, interferon- γ, or interferon- λ.

The use of the pharmaceutical compositions shown in the manufacture of a medicament for the diagnosis, prevention, treatment or combination thereof of a disease condition (e.g. cancer or pathogen infection) is also within the scope of the present disclosure.

The pharmaceutical composition may include any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, colorants, flavorants, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents and combinations of the above. The selection and use of suitable excipients is taught at Gennaro,ed.,Remington:The Science and Practice of Pharmacy,20th Ed.(Lippincott Williams&Wilkins2003),, the disclosure of which is incorporated herein by reference.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect it from acids and other natural conditions which may inactivate it. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibodies described herein may be administered by a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration.

The pharmaceutical compositions of the present disclosure may be prepared in a variety of forms including tablets, hard or soft gelatin capsules, aqueous solutions, suspensions and liposomes and other sustained release formulations, such as shaped polymer gels. Oral dosage forms may be formulated so that the antibody is released into the intestine after passing through the stomach. Such formulations are described in U.S. patent No. 6,306,434 and the references contained therein.

Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.

The antibody or protein complex may be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and may be presented in unit dosage form in preservative-added ampoules, pre-filled syringes, small volume infusion containers or multi-dose containers. The pharmaceutical composition may take the form of a suspension, solution or emulsion, such as in an oily or aqueous vehicle, and may contain formulatory agents, such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions suitable for rectal administration may be prepared as unit dose suppositories. Suitable carriers include saline solutions and other materials commonly used in the art.

For administration by inhalation, the antibody or protein complex may be conveniently delivered from an insufflator, nebulizer or pressurized pack or other convenient means of delivering an aerosol spray. The pressurized pack may include a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of pressurized aerosols, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively for administration by inhalation or insufflation, the antibody or protein complex may take the form of a dry powder composition such as a powder mix of the modulator and a suitable powder base such as lactose or starch. The powder composition may be present in unit dosage form, for example in a capsule or cartridge or in a package, for example gelatin or blisters, from which it may be administered by means of an inhaler or insufflator. For intranasal administration, the antibodies may be administered by liquid spray, for example by a plastic bottle nebulizer.

The pharmaceutical composition may also contain other ingredients, such as flavouring agents, colouring agents, antimicrobial agents or preservatives. It will be appreciated that the amount of antibody required for use in therapy will vary not only with the particular carrier selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient. Ultimately, the caregiver can determine the appropriate dosage. In addition, the pharmaceutical composition may be formulated as a single unit dosage form.

The pharmaceutical compositions of the present disclosure may be in the form of a sterile aqueous solution or dispersion. It may also be formulated as a microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

The antibodies or protein complexes described in the present disclosure may be administered as a slow release formulation, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the antibody or protein complex in the patient. Generally, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a longer period of time. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the patient's symptoms of the disease are partially or fully ameliorated. Thereafter, the patient may be subjected to prophylactic treatment.

The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration, and is generally the amount of the composition that produces a therapeutic effect. Generally, the active ingredient is admixed with a pharmaceutically acceptable carrier in an amount ranging from about 0.01% to about 99%, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% by weight percent.

The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, the administration may be a single bolus, may be multiple administrations over time, or the dosage may be proportionally reduced or increased depending on the urgency of the treatment situation. For ease of administration and dose consistency, it is particularly advantageous to formulate parenteral compositions in unit dosage form. The unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Or the antibody or protein complex may be administered as a slow release formulation, in which case a lower frequency of administration is required. For administration of the antibody or protein complex, the dosage range is about 0.0001 to 800mg/kg, more typically 0.01 to 5mg/kg of host body weight. For example, the dosage may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, or 10mg/kg body weight or in the range of 1-10mg/kg body weight. Exemplary treatment regimens require administration weekly, biweekly, tricyclically, quarterly, monthly, tricyclically, or three to six months. Preferred dosage regimens for antibodies of the disclosure include administration of the antibody by intravenous administration of 1mg/kg body weight or 3mg/kg body weight, using one of (i) six times a week and then three months, (ii) once every three weeks, and (iii) once every 3mg/kg body weight, followed by once every three weeks of 1mg/kg body weight. In some methods, the dosage is adjusted to achieve a plasma antibody or protein complex concentration of about 1-1000 μg/ml, and in some methods about 25-300 μg/ml. A "therapeutically effective dose" of an antibody of the present disclosure preferably results in a decrease in severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of injury or disability due to the disease.

The pharmaceutical compositions may be controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. See, for example ,Sustained and Controlled Release Drug Delivery Systems,J.R.Robinson,ed.,Marcel Dekker,Inc.,New York,1978.

The therapeutic composition may be administered by medical devices such as (1) needleless subcutaneous injection devices (e.g., U.S. Pat. No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) mini-infusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224), and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

In some embodiments, the antibodies or protein complexes described in the present disclosure may be formulated to ensure proper distribution in vivo. For example, to ensure that therapeutic compounds of the present disclosure cross the blood brain barrier, they may be formulated in liposomes, which may additionally contain targeting moieties to enhance selective transport to specific cells or organs. See, for example, U.S. Pat. Nos. 4,522,811, 5,374,548, 5,416,016, and 5,399,331, ;V.V.Ranade(1989)Clin.Pharmacol.29:685;Umezawa et al.,(1988)Biochem.Biophys.Res.Commun.153:1038;Bloeman et al.(1995)FEBS Lett.357:140;M.Owais et al.(1995)Antimicrob.Agents Chemother.39:180;Briscoe et al.(1995)Am.Physiol.1233:134;Schreier et al.(1994).Biol.Chem.269:9090;Keinanen and Laukkanen(1994)FEBS Lett.346:123; and Killion AND FIDLER (1994) Immunomethods 4:273.

In some embodiments, the initial dose may be followed by a second or more subsequent doses of antibody or antigen-binding fragment thereof or protein complex in an amount that may be about the same or less than the initial dose, wherein the subsequent doses are spaced at least 1 to 3 days apart, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks apart.

A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, for example, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (Wu et al (1987) j. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other bioactive agents. Administration may be systemic or local. The pharmaceutical compositions may also be delivered in vesicles, particularly liposomes (see, e.g., langer (1990) Science 249:1527-1533).

The use of nanoparticles to deliver the antibodies or protein complexes of the present disclosure is also contemplated herein. Antibody-conjugated nanoparticles are useful in therapeutic and diagnostic applications. Arruebo, m. et al, incorporated herein by reference, describe in detail antibody-conjugated nanoparticles and methods of making and using ("Antibody-conjugated nanoparticles for biomedical applications"in J.Nanomat.Volume 2009,Article ID 439389),. Nanoparticles can be developed and conjugated to antibodies or protein complexes contained in the pharmaceutical composition to target cells. Nanoparticles for drug delivery are also described in, for example, US8257740 or US8246995, each of which is incorporated herein in its entirety.

In certain instances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed near the target site of the composition, thus requiring only a small portion of the systemic dose.

The injection preparation may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intracranial injection, intraperitoneal injection, intramuscular injection, instillation, and the like. These injectable preparations can be prepared by known methods. For example, injectable formulations can be prepared, for example, by dissolving, suspending or emulsifying the antibodies or salts thereof described herein in a sterile aqueous or oily medium conventionally used for injection. As the aqueous medium for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as alcohol (e.g., ethanol), polyol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As the oily medium, for example, sesame oil, soybean oil, etc. may be used, which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in a suitable ampoule.

The pharmaceutical compositions of the present disclosure may be delivered subcutaneously or intravenously with standard needles and syringes. In addition, with respect to subcutaneous delivery, pen delivery devices are readily applicable to delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device may then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with a pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

Many reusable pen and auto-injector delivery devices are applicable to subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include, but are certainly not limited to AUTOPEN ^TM (European Fund, wood Stokes, UK), DISETRONIC ^TM pen (Disetronic MEDICAL SYSTEMS, bungedorf, switzerland), HUMALOG MIX 75/25 ^TM pen, HUMALOG ^TM, HUMALIN/30 ^TM (Gift Corp., indianapolis, indianana), NOVOPEN ^TM I, II and III (Norand Norde, copenhagen, denmark), NOVOPEN JUNIOR ^TM (norand nod, copenhagen, denmark), BD ^TM pen (Becton Dickinson, franklin lake, new jersey), OPTIPEN ^TM、OPTIPEN PRO^TM、OPTIPEN STARLET^TM and OPTICLIK ^TM (cinofiran vant, germany frankfurt), to name a few. Examples of disposable pen delivery devices that are useful for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are certainly not limited to SOLOSTAR ^TM pens (cinofirant), FLEXPEN ^TM (norand nod) and KWIKPEN ^TM (gill corporation), SURECLICK ^TM auto-injectors (Anin, kiku, calif.), PENLET ^TM (HASELMEIER, stuttgart, germany), EPIPEN (Dey, L.P.), and HUMIRA ^TM pens (Abbott Labs, atbang park, illinois), to name a few.

Advantageously, the pharmaceutical compositions described herein for oral or parenteral use are prepared in unit dosage forms suitable for dosages of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of antibody contained in each dosage form in a unit dose is typically from about 5 to about 500mg, preferably the amount of antibody contained in each dosage form is typically from about 5mg to about 500mg. In particular, the injectable form preferably contains about 5 to about 300mg of antibody, and for other dosage forms preferably contains about 10 to about 300mg of antibody.

Other therapeutic Agents

Various other therapeutic agents may be included in the above-described pharmaceutical compositions or co-administered simultaneously, before or after the compositions. Such therapeutic agents may also be conjugated to antibodies or incorporated into protein complexes as effectors described herein. Examples of such therapeutic agents include, but are not limited to, cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, cytokines, complement agents, checkpoint inhibitors, immune co-stimulators/agonists, immune co-inhibitors/antagonists and enzymes. The drug used may have a pharmaceutical property selected from the group consisting of antimitotic agents, anti-kinase agents, alkylating agents, antimetabolites, antibiotics, alkaloids, antiangiogenic agents, pro-apoptotic agents, and combinations thereof.

Exemplary drugs for use may include, but are not limited to, 5-fluorouracil, afatinib, april (aplidin), azaribine (azaribine), anastrozole (anastrozole), anthracyclines, axitinib (axitinib), AVL-101, AVL-291, bendamustine (bendamustine), bleomycin, bortezomib (bortezomib), bosutinib (bosutinib), bryostatin-1, busulfan (busulfan), busulfan (Amersham), Calicheamicin (CALICHEAMYCIN), camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine (carmustine), celecoxib (celebrex), chlorambucil, cisplatin (CDDP), cox-2 inhibitor, irinotecan (CPT-11), SN-38, carboplatin, cladribine (cladribine), camptothecin (camptothecans), crizotinib (crizotinib), cyclophosphamide, cytarabine, dacarbazine (dacarbazine), dasatinib (dasatinib), and pharmaceutical compositions containing the same, Denafil (dinaciclib), docetaxel, dactinomycin (dactinomycin), daunorubicin, doxorubicin, 2-pyrrolinodoxorubicin (2P-DOX), cyanomorpholindoxorubicin, doxorubicin glucuronide, epirubicin glucuronide (epirubicin glucuronide), erlotinib, estramustine (estramustine), epirubicin (epidophyllotin), erlotinib, entinostat (entinostat), and pharmaceutical compositions, Estrogen receptor binding agents, etoposide (VP 16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod (fingolimod), fluorouridine (FUdR), 3',5' -O-dioleoyl-FudR (FUdR-dO), fludarabine (fludarabine), flutamide (flutamide), farnesyl protein transferase inhibitors, huang Bi alcohol (flavopiridol), fumagtinib (fostamatinib), ganetetrazine (ganetespib), GDC-0834, GS-1101, gefitinib (gefitinib), gemcitabine, hydroxyurea, ibutinib (ibrutinib), idarubicin (idarubicin), idarubicin (idelalisib), ifosfamide, imatinib (imatinib), L-asparaginase, lapatinib (lapatinib), lenalidomide (lenolidamide), folinic acid, LFM-A13, lomustine (lomustine), nitrogen mustard, melphalan, mercaptopurine, 6-mercaptopurine, Methotrexate, mitoxantrone (mitoxantrone), mithramycin (mithramycin), mitomycin, mitotane, vinorelbine (navlbine), nelatinib (neratinib), nilotinib (nilotinib), nitrourea (nitrosurea), olaparib (olaparib), plicomycin (plicomycin), procarbazine (procarbazine), paclitaxel, PCI-32765, penostatin (pentastatin), PSI-341, raloxifene (raloxifene), semustine (semustine), sorafenib (sorafenib), streptozotocin, SU11248, sunitinib (sunitinib), tamoxifen, temustine Maan (aqueous form of DTIC), transplatin (transplatinum), thalidomide, thioguanine (thioguanine), thiotepa (thiotepa), teniposide (teniposide), topotecan (topotecan), uracil mustard (uracil mustard), Vatalanib, vinorelbine, vincristine, vinblastine, vinca alkaloids (vinca alkaloids) and ZD1839.

Toxins used may include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), such as onconase (onconase), dnase I, staphylococcal enterotoxin-a, pokeweed antiviral protein, gelonin (gelonin), diphtheria toxin, pseudomonas exotoxin, and pseudomonas endotoxin.

Chemokines used may include RANTES, MCAF, MIP-alpha, MIP1-Beta, and IP-10.

In some embodiments of the present invention, in some embodiments, anti-angiogenic agents such as angiostatin, baculostatin (baculostatin), compstatin (canstatin), maspin, anti-VEGF antibodies, anti-PlGF peptides and antibodies, anti-angiogenic factor antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF (macrophage migration inhibitory factor) antibodies, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin-12, gro-beta, thrombospondin, 2-methoxyestradiol, proliferative-related protein, carboxyamidotriazole (carboxiamidotriazole), CM101, marsinta (MARIMASTAT), pentosan polysulfate (pentosan polysulphate), angiopoietin-2, interferon-alpha, herbicidal mycin A (herbimycin A), PNU145156E, 16K prolactin fragment, li Nuomi t (Luo Kuimei) (Linomide (roquinimex)), thalidomide, pentoxifylline, aureomide, aureoxin, plasmin, plasminogen 470, vascular alcohol, AGstatin, cixin, vincristine, vinpocetin (1470), minocycline, or other factors.

The immunomodulator used may be selected from the group consisting of cytokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony Stimulating Factors (CSF), interferons (IFN), erythropoietin, thrombopoietin and combinations thereof. Particularly useful are lymphotoxins, such as Tumor Necrosis Factor (TNF), hematopoietic factors, such as Interleukins (IL), colony stimulating factors, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte-macrophage-colony stimulating factor (GM-CSF), interferons, such as interferon- α, - β, - γ or- λ, and stem cell growth factors, such as the designated "S1 factor". Cytokines include growth hormone such as human growth hormone, N-methylmercapto human growth hormone and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, progastrin, glycoprotein hormones such as Follicle Stimulating Hormone (FSH), thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH), liver growth factors, prostaglandins, fibroblast growth factors, prolactin, placental prolactin, OB protein, tumor necrosis factor-alpha and-beta, miao-tube inhibiting substance (mullerian-inhibiting substance), mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factors such as NGF-beta, platelet growth factors, transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta, insulin-like growth factors-I and-II, erythropoietin (EPO), osteoinductive factors, interferons such as interferon-alpha, -beta and-gamma, colony Stimulating Factors (CSF), such as CSF-protein, tumor necrosis factor-alpha and-beta, vascular Endothelial Growth Factor (VEGFR) such as IL-F-beta, IL-F-CSF, IL-gamma, or IL-53.

The radionuclides used include, but are not limited to ¹¹¹In、¹⁷¹Lu、²¹²Bi、²¹³Bi、²¹¹At、⁶²Cu、⁶⁷Cu、⁹⁰Y、¹²⁵I、¹³¹I、³²P、³³P、⁴⁷Sc、¹¹¹Ag、⁶⁷Ga、¹⁴²Pr、¹⁵³Sm、¹⁶¹Tb、¹⁶⁶Dy、¹⁶⁶Ho、¹⁸⁶Re、¹⁸⁸Re、¹⁸⁹Re、²¹²Pb、²²³Ra、²²⁵Ac、⁵⁹Fe、⁷⁵Se、⁷⁷As、⁸⁹Sr、⁹⁹Mo、¹⁰⁵Rh、¹⁰⁹Pd、¹⁴³Pr、¹⁴⁹Pm、¹⁶⁹Er、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹Pb、²²⁷Th. therapeutic radionuclides preferably having a decay energy in the range of 20 to 6,000keV, preferably in the range of 60 to 200keV for auger emitters, preferably in the range of 100-2,500keV for beta emitters, and preferably in the range of 4,000-6,000keV for alpha emitters. The maximum decay energy of useful beta particle emitting nuclides is preferably 20-5,000keV, more preferably 100-4,000keV, most preferably 500-2,500keV. Also preferred is a radionuclide that significantly decays by auger emission of the particle. For example, co-58, ga-67, br-80m, tc-99m, rh-103m, pt-109, in-111, sb-119, 1-125, ho-161, os-189m and Ir-192. The decay energy of useful beta particle emitting nuclides is preferably <1,000keV, more preferably <100keV, and most preferably <70keV. Also preferred are radionuclides that decay significantly with the production of alpha-particles. Such radionuclides include, but are not limited to Dy-152, at-211, bi-212, ra-223, rn-219, po-215, bi-211, ac-225, fr-221, at-217, bi-213, th-227, and Fm-255. Useful alpha particle emitting radionuclides preferably have decay energies of 2,000 to 10,000keV, more preferably 3,000 to 8,000keV, and most preferably 4,000 to 7,000keV. Other useful radioisotopes include ¹¹C、¹³N、¹⁵O、⁷⁵Br、¹⁹⁸Au、²²⁴Ac、¹²⁶I、¹³³I、⁷⁷Br、^113mIn、⁹⁵Ru、⁹⁷Ru、¹⁰³Ru、¹⁰⁵Ru、¹⁰⁷Hg、²⁰³Hg、12^1mTe、^122mTe、^125mTe、¹⁶⁵Tm、¹⁶⁷Tm、¹⁶⁸Tm、¹⁹⁷Pt、¹⁰⁹Pd、¹⁰⁵Rh、¹⁴²Pr、¹⁴³Pr、¹⁶¹Tb、¹⁶⁶Ho、¹⁹⁹Au、⁵⁷Co、⁵⁸Co、⁵¹Cr、⁵⁹Fe、⁷⁵Se、²⁰¹Tl、²²⁵Ac、⁷⁶Br、¹⁶⁹Yb and the like. Some useful diagnostic nuclides may include ¹⁸F、⁵²Fe、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁸⁹Zr、⁹⁴Tc、^94mTc、^99mTc or ¹¹¹ In.

The therapeutic agent may include a photoactive agent or a dye. Fluorescent compositions, such as fluorescent dyes and other chromogens or dyes, such as porphyrins that are sensitive to visible light, have been used to detect and treat lesions by directing appropriate light to the lesion. In therapy, this is called light radiation, phototherapy or photodynamic therapy. See Joni et al.(eds.),PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES(Libreria Progetto 1985);van den Bergh,Chem.Britain(1986),22:430. in addition, monoclonal antibodies have been conjugated to photoactivated dyes to effect phototherapy. See Mew et al.,J.Immunol.(1983),130:1473;Mew et al.,Cancer Res.(1985),45:4380;Oseroff et al.,Proc.Natl.Acad.Sci.USA(1986),83:8744;Oseroff et al.,Photochem.Photobiol.(1987),46:83;Hasan et al.,Prog.Clin.Biol.Res.(1989),288:471;Tatsuta et al.,Lasers Surg.Med.(1989),9:422;Pelegrin et al.,Cancer(1991),67:2529.

Other useful therapeutic agents may comprise oligonucleotides, particularly antisense oligonucleotides directed against oncogenes and oncogene products such as bcl-2 or p53 are preferred. A preferred form of therapeutic oligonucleotide is siRNA. One of skill in the art will recognize that any siRNA or interfering RNA species may be attached to an antibody or fragment thereof for delivery to a target tissue. Many siRNA species against a variety of targets are known in the art, and any such known siRNA can be used in the claimed methods and compositions.

Known potentially useful siRNA classes include those that are specific for IKK-gamma (U.S. Pat. No. 7,022,828), VEGF, flt-1 and Flk-1/KDR (U.S. Pat. No. 7,148,342), bcl2 and EGFR (U.S. Pat. No. 7,541,453), CDC20 (U.S. Pat. No. 7,550,572), transducin (β) like 3 (U.S. Pat. No. 7,576,196), KRAS (U.S. Pat. No. 7,576,197), carbonic anhydrase II (U.S. Pat. No. 7,579,457), complement component 3 (U.S. Pat. No. 7,582,746), interleukin-1 receptor associated kinase 4 (IRAK 4) (U.S. Pat. No. 7,592,443), survivin (U.S. Pat. No. 7,608,7070), superoxide dismutase 1 (U.S. No. 7,632,938), MET protooncogene (U.S. Pat. No. 7,632,939), amyloid β precursor protein (APP) (U.S. Pat. No. 7,635,771), IGF-1R (U.S. 7,638,621), factor B (U.S. Pat. No. 7,642,349), and complement factor B (U.S. No. 7,696,344), and lipocalin (37B) are each incorporated herein by reference.

Other siRNA species are available from known commercial sources, such as Sigma-Aldrich (St.Louis, misu Rich), invitrogen (Calif. ), santa Cruz Biotechnology (St.Jous, calif.), ambion (Ostine, tex.), dharmacon (Lafeet, colorado, wisconsin), promega (Madison, wis.), mirus Bio (Madison, wis.), qiagen (Basil, calif.), and the like. Other published sources of siRNA species include siRNAdb database from the Stockholm bioinformatics center, MIT/ICBP siRNA database, RNAi Consortium shRNA database from the Broad institute, and Probe database from NCBI. For example, NCBIProbe databases have 30,852 siRNA species. Those skilled in the art will recognize that for any gene of interest, either an siRNA species has been designed or one can be readily designed using publicly available software tools. Any such siRNA species may be delivered using the subject complexes described herein.

Method and use

The protein complexes, antibodies and methods disclosed herein have a wide range of uses. Therefore, they have wide application in research, diagnosis, and therapy.

Therapeutic method

The protein complexes, antibodies, compositions, and formulations described herein can be used to treat a variety of diseases or disorders, including cancer (e.g., breast cancer, lung cancer, gastric cancer, colorectal cancer, bladder cancer, liver cancer, prostate cancer, pancreatic cancer, melanoma, leukemia, lymphoma, multiple myeloma), immune diseases (e.g., autoimmune diseases), and pathogen infection (e.g., viruses, bacteria, fungi, or parasites). Accordingly, various embodiments relate to methods of treating such diseases or disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein complex, antibody, composition or formulation described herein.

In one embodiment, the immune disorder that may be treated with the protein complex or antibody may include, for example, autoimmune disorders such as Systemic Lupus Erythematosus (SLE), joint disorders such as ankylosing spondylitis, juvenile rheumatoid arthritis, neurological disorders such as multiple sclerosis and myasthenia gravis, pancreatic disorders such as diabetes, especially juvenile onset diabetes, gastrointestinal disorders such as chronic active hepatitis, celiac disease, ulcerative colitis, crohn's disease, pernicious anaemia, skin disorders such as psoriasis or scleroderma, allergic disorders such as asthma, and transplant-related disorders such as graft-versus-host disease and allograft rejection.

The administration of the protein complex or antibody may be supplemented by simultaneous or sequential administration of a therapeutically effective amount of another antibody that binds to or reacts with another antigen on the surface of the target cell. Preferred additional monoclonal antibodies comprise at least one humanized, chimeric or human monoclonal antibody, it is selected from the group consisting of a protein selected from the group consisting of CD4、CD5、CD8、CD14、CD15、CD16、CD19、IGF-1R、CD20、CD21、CD22、CD23、CD25、CD27、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD45、CD46、CD47、CD52、CD54、CD66、CD70、CD74、CD79a、CD79b、CD80、CD95、CD126、CD133、CD138、CD154、CD160、CD166、CD229、CEACAM5、CEACAM6、B7、AFP、PSMA、EGP-1、EGP-2、GPRC5、FcRH5、ROR1、BCMA、IGF-1R、EGFR、HER2、HER3、TNF-α、ICOS、ICOSL、 Hypoxia Inducible Factor (HIF), folate receptor, TDGF1, tfR, mesothelin, PSMA, B7, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda, C1R, C1s, C2, C3, C5a, C5aR1, C6, MASP, MSAP2, MASP3, FB, FD, properdin, lag-3, CTLA-4, PD-1, PD-L1, TITIGIT, OX40L, 4-1BB, BTLA, GITR, GITRL, TCR, nectin-4, C-Met, LIV1, mesothelin DLL3, DLL4, tissue factor, TGF-beta receptor, CLDN18.2, carbonic anhydrase IX, PAM4 antigen, MUC1, MUC2, MUC3, MUC4, MUC5, MUC16, ia, MIF, HM.24, HLA-DR, tenascin, flt-3, VEGFR, plGF, ILGF, IL-1β, IL2, IL-6R, IL-15, IL-15R, IL-17, IL-17R, IL-12, IL-25, tenascin, TRAIL-R1, TRAIL-R2, complement factor C5, oncogene products, or combinations thereof. The various antibodies used, such as anti-CD 19, anti-CD 20 and anti-CD 22 antibodies, are known to those skilled in the art. See, for example, ,Ghetie et al.,Cancer Res.48:2610(1988);Heiman et al.,Cancer Immunol.Immunother.32:364(1991);Longo,Curr.Opin.Oncol.8:353(1996),, U.S. patent No. 5,798,554, 6,187,287, 6,306,393, 6,676,924, 7,109,304, 7,151,164, 7,230,084, 7,230,085, 7,238,785, 7,238,786, 7,282,567, 7,300,655, 7,312,318, 7,501,498, 7,612,180, 7,670,804, and U.S. patent publication No. 20080131363, U.S. patent publication No. 20070172920, U.S. patent publication No. 20060193865, and U.S. patent publication No. 20080138333, the examples of each of which are incorporated herein by reference.

The treatment may be further supplemented by the simultaneous or sequential administration of at least one therapeutic agent. For example, "CVB" (1.5 g/m ² cyclophosphamide, 200-400mg/m ² etoposide, and 150-200mg/m ² carmustine) is a treatment regimen for the treatment of non-Hodgkin's lymphoma. Patti et al, eur.J.Haemato.51:18 (1993). Other suitable combination chemotherapy regimens are well known to those skilled in the art. See, e.g., ,Freedman et al.,"Non-Hodgkin's Lymphomas,"in CANCER MEDICINE,VOLUME 2,3rd Edition,Holland et al.(eds.),pages 2028-2068(Lea&Febiger1993). for first generation chemotherapy regimens for the treatment of intermediate non-hodgkin lymphoma (NHL) including C-MOPP (cyclophosphamide, vincristine, procarbazine, and prednisone) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Useful second generation chemotherapy regimens are m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, and folinic acid), while suitable third generation regimens are MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, and folinic acid). Other useful drugs include phenyl butyrate, bendamustine, and bryostatin-1.

The subject protein complexes or antibodies can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby bsAb is combined in a mixture with pharmaceutically suitable excipients. Sterile phosphate buffered saline is one example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art. See, e.g., ,Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS,5th Edition(Lea&Febiger 1990), and Gennaro (ed.), REMINGTON' S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revisions thereof.

The subject protein complexes or antibodies may be formulated for intravenous administration by, for example, bolus injection or continuous infusion. Preferably, the protein complex or antibody is infused in less than about 4 hours, and more preferably, in less than about 3 hours. For example, the first bolus may be infused within 30 minutes, preferably even within 15 minutes, and the remainder infused within the next 2-3 hours. The injectable preparation may be presented in unit dosage form, for example, in ampules or multi-dose containers, with the addition of preservatives. The composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

Other pharmaceutical methods may be employed to control the duration of action of the protein complex or antibody. Controlled release formulations may be prepared by complexing or adsorbing protein complexes or antibodies using polymers. For example, biocompatible polymers include poly (ethylene-co-vinyl acetate) matrices and polyanhydride copolymer matrices of stearic acid dimer and sebacic acid. Shermwood et al, bio/Technology 10:1446 (1992). The release rate from such a matrix depends on the molecular weight of the protein complex or antibody, the amount of protein complex or antibody within the matrix, and the size of the dispersed particles. Saltzman et al, biophys.J.55:163 (1989); shermood et al, supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS,5th Edition(Lea&Febiger 1990), and Gennaro (ed.), REMINGTON' S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revisions thereof.

The protein complex or antibody may also be administered to the mammal subcutaneously or even by other parenteral routes (e.g., intravenous, intramuscular, intraperitoneal, or intravascular). Furthermore, administration may be by continuous infusion or by single or multiple bolus injections. Preferably, the protein complex or antibody is infused in less than about 4 hours, and more preferably, in less than about 3 hours.

More generally, the dosage of protein complex or antibody administered to a human will vary depending on factors such as the age, weight, height, sex, general medical condition, and past medical history of the patient. It may be desirable to provide the protein complex or antibody to the recipient in a single intravenous infusion at a dose of about 1mg/kg to 25mg/kg, although lower or higher doses may also be administered. And (5) determining the situation. For example, a dose of 1-20mg/kg for a 70kg patient is 70-1,400mg, or a dose of 41-824mg/m ² for a 1.7 meter patient. The dosage may be repeated as desired, for example, once a week for 4-10 weeks, once a week for 8 weeks, or once a week for 4 weeks. Depending on the need for maintenance therapy, it may also be administered less frequently, for example once every other week for several months, or once monthly or quarterly for several months.

Alternatively, the protein complex or antibody may be administered in a single dose every 2 or 3 weeks, repeated a total of at least 3 times. Alternatively, the construct may be administered twice weekly for 4-6 weeks. If the dose is reduced to about 200-300mg/m ² (340 mg per dose for a 1.7 m patient, 4.9mg/kg per dose for a 70 kg patient), the administration can be once or even twice a week for 4 to 10 weeks. Or the dose schedule may be reduced, i.e. once every 2-3 weeks for 2-3 months. However, it has been determined that even higher doses, such as 20mg/kg infusions once a week or once every 2-3 weeks, can be administered by slow intravenous injection for repeated dosing cycles. The dosing regimen may optionally be repeated at other intervals, and the doses may be administered by various parenteral routes, with appropriate adjustment of the dose and regimen.

While the protein complex or antibody may be administered as a periodic bolus, in alternative embodiments, the bs protein complex or antibody Ab may be administered by continuous infusion. To increase the Cmax of protein complexes or antibodies in the blood and to prolong PK, continuous infusion may be performed, for example, through an indwelling catheter. Such devices are known in the art, e.g Or (b)Catheters (see, e.g., skolinik et al Ther Drug Monit 32:741-48,2010) and any such known indwelling catheter may be used. A variety of continuous infusion pumps are also known in the art and any such known infusion pump may be used. The dosage range for continuous infusion may be 0.1 to 3.0mg/kg per day. More preferably, the protein complex or antibody may be administered by intravenous infusion over a relatively short period of 2 to 5 hours, more preferably 2-3 hours.

In a preferred embodiment, the protein complex or antibody is useful in the treatment of cancer. Examples of cancers include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More specific examples of such cancers are described below and include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), ewing sarcoma, wilms' cell tumor, astrocytoma, lung cancer (including small cell lung cancer), non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, hepatocellular carcinoma, neuroendocrine tumor, medullary thyroid cancer, differentiated thyroid cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, anal cancer, penile cancer, head and neck cancer, and the like. The term "cancer" includes primary malignant cells or tumors (e.g., those whose cells have not migrated to a site other than the original malignant tumor or tumor site in the subject) and secondary malignant cells or tumors (e.g., cells or tumors that have migrated from metastasis-producing malignant cells or tumor cells to a secondary site different from the original tumor site). Cancers that are amenable to the methods of treatment of the present invention involve cells that express, overexpress, or otherwise abnormally express IGF-1R.

Other examples of cancers or malignancies include, but are not limited to, acute childhood lymphoblastic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphoblastic leukemia, adult acute myelogenous leukemia, adult hodgkin's lymphoma, adult lymphoblastic leukemia, adult non-hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, aids related lymphoma, aids related malignancy, anal carcinoma, astrocytoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, brain stem glioma, Brain tumor, breast cancer, renal pelvis and ureter cancer, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood (primary) hepatocellular carcinoma, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myelogenous leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumor, childhood hodgkin's disease, childhood hodgkin's lymphoma, childhood hypothalamic and vision path glioma, childhood lymphoblastic leukemia, childhood myeloblastoma, childhood non-hodgkin's lymphoma, Pinus koraiensis and supratentoria primitive neuroectodermal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic granulocytic leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine islet cell carcinoma, endometrial carcinoma, ependymoma, epithelial carcinoma, esophageal carcinoma, ewing's sarcoma and related tumors, exocrine pancreatic carcinoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct carcinoma, eye cancer, female breast cancer, gaucher's disease, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal tumors, germ cell tumors, gestational trophoblastoma, Hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal carcinoma, intestinal cancer, intraocular melanoma, islet cell carcinoma, islet cell pancreatic carcinoma, kaposi's sarcoma, renal carcinoma, laryngeal carcinoma, lip and oral cancer, liver cancer, lung cancer, lymphoproliferative diseases, megaloblastic, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic latent primary squamous neck cancer, metastatic squamous neck cancer, multiple myeloma/plasma cell tumor, myelodysplastic syndrome, myelogenous leukemia, Myelogenous leukemia, myeloproliferative diseases, nasal and sinus cancers, nasopharyngeal cancers, neuroblastomas, non-hodgkin lymphomas, non-melanoma skin cancers, non-small cell lung cancers, occult primary metastatic squamous neck cancers, oropharyngeal cancers, osteo/fibrosarcoma, osteosarcoma/malignant fibrous histiocytomas, osteosarcoma/bone malignant fibrous histiocytomas, ovarian epithelial cancers, ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancers, paraproteinemia, polycythemia vera, parathyroid cancer, penile cancers, pheochromocytomas, pituitary tumors, primary central nervous system lymphomas, primary liver cancers, prostate cancer, Rectum cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcomas of the nodes, szeri Syndrome (Sezary syncrome), skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, supratentorial primitive neuroectodermal and pineal tumor, T cell lymphoma, testicular cancer, thymoma, thyroid cancer, renal pelvis and ureter transitional cell carcinoma, transitional renal pelvis and ureter carcinoma, trophoblastic tumor, ureter and renal pelvis cell carcinoma, urethral carcinoma, uterine sarcoma, vaginal carcinoma, visual pathway and hypothalamic glioma, vulval carcinoma, Megaloblastic Fahrenheit (Waldenstrom's Macroglobulinemia), wilms' Tumor, and any other hyperproliferative disease that is located in the organ system above, except neoplasia.

The methods and compositions described and claimed herein are useful for treating malignant or precancerous conditions and preventing progression to a tumor or malignant state, including but not limited to those described above. Such uses are applicable to conditions known or suspected of progressing to neoplasia or cancer, particularly when non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly dysplasia occurs (for reviews of such abnormal growth conditions Robbins AND ANGELL, BASIC PATHOLOGY,2d Ed., w.b. samders co., philiadelphia, pp.68-79 (1976)).

Dysplasia is often a precursor to cancer, and is predominantly found in epithelial cells. It is the most disordered form of non-neoplastic cell growth, involving loss of individual cell uniformity and cell structure orientation. Dysplasia generally occurs where chronic irritation or inflammation is present. Dysplastic conditions that may be treated include, but are not limited to, antiperspirant ectodermal dysplasia, anterior facial dysplasia, asphyxia chest dysplasia, atrial finger dysplasia, bronchopulmonary dysplasia, brain dysplasia, cervical dysplasia, cartilaginous ectodermal dysplasia, collarbone skull dysplasia, congenital ectodermal dysplasia, skull metaphysis, skull metaphyseal dysplasia, dentinal dysplasia, diaphyseal dysplasia, ectodermal dysplasia, enamel dysplasia, brain eye dysplasia, hemipodophyl dysplasia, multiple epiphyseal dysplasia, punctate epiphyseal dysplasia, epithelial dysplasia, facial finger reproductive dysplasia, familial jaw fibrous dysplasia, familial white fold dysplasia, fibromyodysplasia, bone fibrodysplasia, flourescent cementum structural dysplasia, hereditary kidney-retina dysplasia, hyperhidrosis ectodermal dysplasia, hypohidrosis ectodermal dysplasia, lymphocyte thymus dysplasia, mammary gland dysplasia, mandibular face dysplasia, metaphyseal dysplasia, mongolian (Mondini dysplasia), single fibrodysplasia, mucosal epithelium dysplasia, multiple epiphyseal dysplasia, eye-ear vertebral dysplasia, eye-finger dysplasia, eye-vertebra dysplasia, odontogenic dysplasia, eye-mandibular dysplasia, periapical cementum dysplasia, multiple fibrodysplasia, pseudo-cartilage dysplasia, spinal epiphysis dysplasia, retinal dysplasia, optic nerve compartment dysplasia, spinal epiphysis dysplasia and ventral radial dysplasia.

Other preneoplastic conditions that may be treated include, but are not limited to, benign proliferative disorders (e.g., benign tumors, fibrocystic disorders, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia), white spots, keratosis, bao Wenshi diseases (Bowen's disease), chronic actinic dermatitis (norm's Skin), solar cheilitis, and solar keratosis.

In preferred embodiments, the methods of the present disclosure are used to inhibit the growth, progression and/or metastasis of cancers, particularly those cancers listed above.

Other hyperproliferative diseases, disorders and/or conditions include, but are not limited to, the progression and/or metastasis of malignant tumors and related conditions, such as leukemias, including acute leukemias (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia (including myeloblastic leukemia, promyelocytic, granulocytic, monocytic and erythroleukemia) and chronic leukemias (e.g., chronic granulocytic (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., hodgkin's disease and non-hodgkin's disease), multiple myeloma, fahrenheit macroglobulinemia, heavy chain diseases and solid tumors, including, but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphomas, lymphoendotheliosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchi carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyocynoma, ventricular blastoma, pineal tumor, angioblastoma (emangioblastoma), neuroblastoma, oligodendroglioma, meningioma, melanoma neuroblastoma retinoblastoma.

Diagnostic use

The antibodies or protein complexes described herein can be used to detect and/or measure an antigen in a sample, for example for diagnostic purposes. Thus, some embodiments contemplate the use of one or more antibodies or protein complexes described herein in assays for detecting antigens and related diseases or disorders.

Exemplary diagnostic assays can include, for example, contacting a sample obtained from a patient with an antibody or protein complex of the present disclosure, wherein the antibody or protein complex is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate an antigen from the sample. In some embodiments, a detectable label or reporter is integrated into the protein complex as an effector. Alternatively, unlabeled antibodies or protein complexes may be used in diagnostic applications in combination with secondary antibodies that themselves carry a detectable label.

The detectable label or reporter may be a radioisotope, such as H, C, P, S or I, a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine, or an enzyme, such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure targets in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and Fluorescence Activated Cell Sorting (FACS).

In another aspect, the present disclosure further provides a method for detecting the presence of an antigen in a sample comprising the steps of (i) contacting the sample with an antibody or antigen binding fragment or protein complex described herein, (ii) determining the binding of the antibody or antigen binding fragment or protein complex to the antigen, wherein binding of the antibody to the antigen is indicative of the antigen in the sample and the associated disease or disorder.

In some embodiments, the antibody or antigen binding fragment or protein complex is conjugated to a label. In some embodiments, the detecting step comprises contacting the secondary antibody with an antibody or antigen-binding fragment thereof, and wherein the secondary antibody comprises a label. In some embodiments, the label includes a fluorescent label, a chemiluminescent label, a radioactive label, and an enzyme.

In some embodiments, the detecting step comprises detecting fluorescence or chemiluminescence. In some embodiments, the detection step comprises a competitive binding assay or ELISA.

In some embodiments, the methods further comprise binding the sample to a solid support. In some embodiments, the solid support comprises microparticles, microbeads, magnetic beads, and an affinity purification column.

Samples useful in diagnostic assays according to the present disclosure include any tissue or body fluid sample obtainable from a patient that contains a detectable amount of an antigen of interest under normal or pathological conditions. In general, the level of antigen in a particular sample obtained from a healthy patient (e.g., a patient not suffering from an antigen-related disease) will be measured to initially establish a baseline or standard level of antigen. This baseline level may then be compared to the level measured in a sample obtained from an individual suspected of having the antigen and associated disorder or symptoms associated with such disorder.

Kit for detecting a substance in a sample

In another aspect, the present disclosure provides a kit comprising pharmaceutically acceptable dosage units of an antibody or antigen-binding fragment thereof or protein complex or pharmaceutical composition described herein. The scope of the present disclosure also includes kits for diagnosing, prognosing or monitoring the treatment of a disorder in a subject comprising the antibody or antigen binding fragment thereof or protein complex, and at least one detection reagent that specifically binds to the antibody or antigen binding fragment thereof or protein complex thereof.

In some embodiments, the kit further comprises a container comprising the composition and optionally the informational material. The informational material may be descriptive, instructional, marketable, or other material relevant to the methods described herein and/or the therapeutic use of the agent. In one embodiment, the kit further comprises an additional therapeutic agent as described herein. For example, the kit includes a first container containing the composition and a second container containing additional therapeutic agent.

The form of the information material in the kit is not limited. In some embodiments, the informational material may include information about the production, concentration, expiration date, batch or production site information, etc. of the composition. In one embodiment, the informational material relates to a method of administering the composition, such as a method of administering the composition in a suitable dosage, dosage form, or manner of administration (e.g., a dosage, dosage form, or manner of administration described herein) to treat a subject in need thereof. In one embodiment, the informational material provides a dosing regimen, dosing schedule, and/or route of administration of the composition or additional therapeutic agent. The information may be provided in a variety of formats, including printed text, computer readable material, video or audio recordings, or information including links or addresses to substantive material.

The kit may comprise one or more containers containing the composition. In some embodiments, the kit comprises separate containers, compartments or compartments for the composition and the informational material. For example, the composition may be contained in a bottle or vial, and the informational material may be contained in a plastic sleeve or package. In other embodiments, the individual elements of the kit are contained within a single, indivisible container. For example, the composition is contained in a bottle or vial having information material in the form of a label affixed thereto. In some embodiments, the kit comprises a plurality (e.g., a set) of individual containers, each container containing one or more unit dosage forms (e.g., dosage forms described herein) of the agent.

The kit optionally includes a device suitable for administering the composition or other suitable delivery device. The device may be preloaded with one or both reagents, or may be empty but suitable for loading. Such kits may optionally comprise a syringe to allow injection of the antibodies or protein complexes contained within the kit into an animal (e.g., human).

Interpretation of the terms

Nucleic acid or polynucleotide refers to a DNA molecule (such as, but not limited to, cDNA or genomic DNA) or an RNA molecule (such as, but not limited to, mRNA) and includes DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. The DNA or RNA molecule may include non-naturally occurring moieties such as modified bases, modified backbones, deoxyribonucleotides in RNA, and the like. The nucleic acid molecule may be single-stranded or double-stranded.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may include modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other modification, e.g., coupled to a labeling component. As used herein, the term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptidomimetics. These terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. Polypeptides and proteins may be produced by naturally occurring non-recombinant cells, or by genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a native protein, or molecules having the deletion, addition, and/or substitution of one or more amino acids of a native sequence. The terms "polypeptide" and "protein" specifically encompass antigen binding proteins, antibodies, or sequences having deletions, additions and/or substitutions of one or more amino acids of an antigen binding protein.

The term "polypeptide fragment" refers to a polypeptide having an amino terminal deletion, a carboxy terminal deletion, and/or an internal deletion as compared to the full-length protein. Such fragments may also contain modified amino acids as compared to the full-length protein. In certain embodiments, the fragment is about 5 to 500 amino acids in length. For example, a fragment may be at least 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) used to transfer protein-encoding information into a host cell. In some embodiments, a "vector" refers to a delivery vector capable of (a) promoting expression of a nucleic acid sequence encoding a polypeptide, (b) promoting the polypeptide produced thereby, (c) promoting transfection/transformation of a target cell, (d) promoting replication of the nucleic acid sequence, (e) promoting stability of the nucleic acid, (f) promoting detection of the nucleic acid and/or transformed/transfected cell, and/or (g) imparting other advantageous biological and/or physicochemical functions to the nucleic acid encoding the polypeptide. The vector may be any suitable vector, including chromosomal, nonchromosomal, and synthetic nucleic acid vectors (including nucleic acid sequences of a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmid and phage DNA, and viral nucleic acid (RNA or DNA) vectors.

The term "expression vector" or "expression construct" refers to a vector suitable for transformation of a host cell and containing a nucleic acid sequence that directs and/or controls (in conjunction with the host cell) the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, if an intron is present, RNA splicing of the coding region to which it is operably linked.

As used herein, "operably connected" means that the components to which the term applies are in a relationship that allows them to perform their inherent functions under the appropriate conditions. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence is linked thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

The term "host cell" refers to a cell that has been transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes progeny of a parent cell, whether or not the progeny are identical in morphology or genetic composition to the original parent cell, as long as the gene of interest is present.

The term "immune cell" refers to a cell of hematopoietic origin that is involved in antigen-specific recognition. Immune cells include Antigen Presenting Cells (APCs), such as dendritic cells or macrophages, B cells, T cells, NK cells (e.g., NK-92 cells), and the like. T cells include effector T cells (Teff cells) and regulatory T cells (Treg cells).

As used herein, "effector cells" refers to cells of the immune system that have been induced to differentiate into a form capable of producing a specific immune response, or cells that express one or more Fc receptors and mediate one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, T cells, B cells, large granular lymphocytes, langerhans cells, natural Killer (NK) cells, and γδ T cells, and may be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. As used herein, "effector function" refers to a biochemical event resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include fcγr mediated effector functions (e.g., ADCC and ADCP) and complement mediated effector functions (e.g., CDC).

The term "fusion polypeptide" or "fusion protein" refers to a protein produced by joining two or more polypeptide sequences together. Fusion polypeptides encompassed by the present invention include the translation product of a chimeric gene construct that links a nucleic acid sequence encoding a first polypeptide (e.g., targeting domain) to a nucleic acid sequence encoding a second polypeptide (e.g., effector domain) to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins linked by peptide bonds or by several peptides. The fusion protein may also comprise a peptide linker between the two domains.

The term "linker" refers to any means, entity, or moiety for connecting two or more entities. The linker may be a covalent linker or a non-covalent linker. Examples of covalent linkers include a covalent bond or a linker moiety covalently linked to one or more proteins or domains to be linked. The linker may also be non-covalent, for example an organometallic bond through a metal center such as a platinum atom. For covalent bonds, various functional groups may be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino groups, carbamates, ureas, and the like. To provide ligation, the domains may be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide coupling sites. Coupling methods are well known to those skilled in the art and include use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or, for example, peptide linker moieties (linker sequences). It will be appreciated that modifications that do not significantly reduce the function of the targeting domain and effector domain are preferred.

The term "conjugate" or "coupled" or "linked" as used herein refers to two or more entities joined to form one entity. Conjugates encompass peptide-small molecule conjugates and peptide-protein/peptide conjugates.

The terms "subject" and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, apes, humans, farm animals, sports animals, and pets. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also contemplated. In some embodiments, the subject may be an invertebrate, such as an insect or nematode, while in other cases the subject may be a plant or fungus.

As used herein, "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms refer to methods of achieving a beneficial or desired result, including but not limited to therapeutic benefit and/or prophylactic benefit. Therapeutic benefit refers to any treatment-related improvement or effect of one or more diseases, disorders or symptoms in treatment. For prophylactic benefit, the compositions can be administered to a subject at risk of developing a particular disease, disorder, or symptom, or to a subject reporting one or more physiological symptoms of the disease, even though the disease, disorder, or symptom may not have been manifested.

As used herein, the term "variant" refers to a second composition (e.g., a second molecule) that is associated with a first composition (e.g., a first molecule, also referred to as a "parent" molecule). Variant molecules may be derived from, isolated from, based on, or homologous to the parent molecule. The term variant may be used to describe a polynucleotide or polypeptide.

When applied to a polypeptide or protein (e.g., any of the immunoglobulin chains, targeting moieties/agents, effector moieties/agents, DDD moieties, and AD moieties as described herein), the variant polypeptide may have exactly the same amino acid sequence as the original parent polypeptide, or alternatively, may have less than 100% amino acid identity to the parent protein. For example, a variant of an amino acid sequence may be a second amino acid sequence that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity compared to the original amino acid sequence.

Reference to a peptide, polypeptide or functional variant or equivalent of a protein refers to a polypeptide derivative of the reference peptide, polypeptide or protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. It substantially retains the activity of the reference peptide, polypeptide or protein. In general, functional equivalents have 50% (e.g., any number between 50% and 100%, including endpoints such as 60%, 70%, 80%, 85%, 90%, 95%, and 99%) identity with a reference peptide, polypeptide, or protein.

Polypeptide variants include polypeptides that include the entire parent polypeptide and further include additional fusion amino acid sequences. Polypeptide variants also include polypeptides that are part or subsequences of a parent polypeptide, e.g., unique subsequences of a polypeptide disclosed herein (e.g., as determined by standard sequence comparison and alignment techniques) are also encompassed by the present disclosure.

In another aspect, polypeptide variants include polypeptides that contain minor, insignificant or insignificant changes to the parent amino acid sequence. For example, minor or insignificant changes include amino acid changes (including substitutions, deletions and insertions) that have little or no effect on the biological activity of the polypeptide, and result in functionally identical polypeptides, including the addition of nonfunctional peptide sequences. Those of skill in the art will appreciate that the present disclosure encompasses many variants of the disclosed polypeptides. In some aspects, polynucleotide or polypeptide variants of the present disclosure may include variant molecules that alter, add, or delete a small percentage of nucleotide or amino acid positions, e.g., typically less than about 10%, less than about 5%, less than 4%, less than 2%, or less than 1%.

As used herein, the term "conservative substitution" in a nucleotide or amino acid sequence refers to a change in the nucleotide sequence that either (i) does not result in any corresponding change in the amino acid sequence due to redundancy of the triplet codon code, or (ii) results in the replacement of the original parent amino acid with an amino acid having a chemically similar structure. Conservative substitution tables providing functionally similar amino acids are well known in the art, wherein one amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., an aromatic side chain or a positively charged side chain), and thus do not substantially alter the functional properties of the resulting polypeptide molecule.

The following are groupings of natural amino acids containing similar chemical properties, wherein substitutions within a group are "conservative" amino acid substitutions. The grouping indicated below is not strict, as these natural amino acids may be placed in different groupings when different functional properties are considered. Amino acids having nonpolar and/or aliphatic side chains include glycine, alanine, valine, leucine, isoleucine and proline. Amino acids having polar, uncharged side chains include serine, threonine, cysteine, methionine, asparagine and glutamine. Amino acids having aromatic side chains include phenylalanine, tyrosine, and tryptophan. Amino acids having positively charged side chains include lysine, arginine and histidine. Amino acids having negatively charged side chains include aspartic acid and glutamic acid.

When applied to a polynucleotide, the variant molecule may have exactly the same nucleotide sequence as the original parent molecule, or alternatively, may have less than 100% nucleotide sequence identity to the parent molecule. For example, a variant of a nucleotide sequence may be a second nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%,95%, 98%,99% or more nucleotide sequence identity compared to the first nucleotide sequence. Polynucleotide variants also include polynucleotides, which include the complete parent polynucleotide and further comprise additional fusion nucleotide sequences. Polynucleotide variants also include polynucleotides that are part or subsequences of the parent polynucleotide, e.g., unique subsequences of polynucleotides disclosed herein (e.g., determined by standard sequence comparison and alignment techniques) are also encompassed by the present disclosure.

In another aspect, a polynucleotide variant comprises a nucleotide sequence that contains minor, insignificant or insignificant changes to the parent nucleotide sequence. For example, minor or insignificant changes include changes to the nucleotide sequence that (i) do not change the amino acid sequence of the corresponding polypeptide, (ii) occur outside the protein-encoding open reading frame of the polynucleotide, (iii) result in deletions or insertions that may affect the corresponding amino acid sequence but have little or no effect on the biological activity of the polypeptide, (iv) nucleotide changes result in amino acid substitutions with chemically similar amino acids. In the case where the polynucleotide does not encode a protein (e.g., tRNA or crRNA or tracrrRNA), a variant of the polynucleotide can include a nucleotide change that does not result in loss of function of the polynucleotide. In another aspect, conservative variants of the disclosed nucleotide sequences that result in functionally identical nucleotide sequences are also encompassed by the present disclosure. Those of skill in the art will appreciate that the present disclosure encompasses many variants of the disclosed nucleotide sequences.

As disclosed herein, a plurality of numerical ranges are provided. It is to be understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is described in detail. Every smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the range or excluded from the range, and each range where neither or both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. The term "about" generally refers to plus or minus 10% of the specified number. For example, "about 10%" may represent a range of 9% to 11%, and "about 20" may represent 18-22. Other meanings of "about" may be apparent from the context, such as rounding, so that, for example, "about 1" may also mean 0.5 to 1.4.

The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment or single chain thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V _H) and a heavy chain constant region. The heavy chain constant region consists of three domains, C _H1、C_H and C _H. Each light chain consists of a light chain variable region (abbreviated herein as V _L) and a light chain constant region. The light chain constant region consists of one domain CL. The V _H and V _L regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each of V _H and V _L consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FR of the heavy chain variable region are HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4. The CDRs and FR of the light chain variable region are LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (CIq).

"Immunoglobulin (Ig)" refers to a protein consisting essentially of one or more polypeptides encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have a variety of structural forms including, but not limited to, full length antibodies, antibody fragments, and individual immunoglobulin domains.

The term "antigen-binding fragment or portion" of an antibody (or simply "antibody fragment or portion") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., CD 3). It has been shown that the antigen binding function of an antibody can be performed by fragments of full length antibodies. Examples of binding fragments encompassed by the term "antigen binding fragment or portion" of an antibody include (I) a Fab fragment, a monovalent fragment consisting of V _L、V_H、C_L and C _H I domains, (ii) a F (ab ') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge, (iii) a Fab' fragment, which is essentially a Fab with a partial hinge region (see, FUNDAMENTAL IMMUNOLOGY (Pailed., 3 ^rd ed.1993)), (iv) an Fd fragment consisting of V _H and C _H I domains, (V) a Fv fragment consisting of V _L and V _H I domains of a single arm of an antibody, (vi) a dAb fragment consisting of V _H domains (Ward et al., (1989) Nature 341:544-546), and (vii) a separate CDR, (viii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, V _L and V _H, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, enabling them to be made into a single protein chain, in which the V _L and V _H regions pair to form a monovalent molecule (known as a single chain Fv or scFv), see, e.g., bird et al (1988) Science 242:423-426, and Huston et al (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment or portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for use in the same manner as the whole antibody.

The "constant region" of an antibody as defined herein refers to the region of the antibody encoded by one of the light chain or heavy chain immunoglobulin constant region genes. As used herein, "constant light chain" or "light chain constant region" means an antibody region encoded by a kappa (ck) or lambda (cλ) light chain. Constant light chains typically comprise a single domain, and as defined herein refer to positions 108-214 of ck or cλ, where numbering is according to the EU index. As used herein, "constant heavy chain" or "heavy chain constant region" means the region of an antibody encoded by the μ, δ, γ, α or epsilon genes to define the isotype of the antibody as IgM, igD, igG, igA, or IgE, respectively. For full length IgG antibodies, the constant heavy chain as defined herein refers to the N-terminal end of the CH1 domain to the C-terminal end of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.

"Fab" or "Fab region" as used herein refers to a polypeptide that contains the immunoglobulin domains of V _H、CH1、V_H and C _L. Fab may refer to this region alone, or in the case of a full length antibody or antibody fragment.

As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising an antibody constant region (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, as well as the flexible hinge N-terminus of these domains. For IgA and IgM, the Fc may include the J chain. For IgG, fc comprises the immunoglobulin domains cγ2 and cγ3 (cγ2 and cγ3) and the hinge between cγ1 (cγ1) and cγ2 (cγ2). Although the boundaries of the Fc region may vary, for purposes herein, a human IgG heavy chain Fc region is defined starting from E216 to its carboxy-terminus, wherein numbering is according to the EU index as in Kabat. Fc may refer to this region alone, or in the context of an Fc polypeptide such as an antibody or immunoadhesin (e.g., an Fc fusion protein), as described below. It should be noted that for purposes described herein, an "Fc region" generally includes a hinge region that comprises residues 216-237 unless otherwise specified. Thus, an "Fc variant" may include variants of the hinge region with or without additional amino acid modifications in the cγ2 and cγ3 domains.

"Hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" herein refers to a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 215 and the IgG CH2 domain begins at residue EU position 238. Thus, for IgG, an antibody hinge is defined herein as comprising positions 216 (E216 in IgG 1) to 237 (G237 in IgG 1), wherein the numbering is according to the EU index as in Kabat. In some embodiments, for example in the context of an Fc region, a lower hinge is included, "lower hinge" generally referring to positions 231 through 237.

As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds a particular antigen that is substantially free of antibodies that specifically bind antigens other than the particular antigen). The isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules consisting of a single molecule. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues encoded by non-human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g. mouse) have been grafted onto human framework sequences.

The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity, which has variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody may be produced by a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

The term "recombinant human antibody" as used herein includes all human antibodies produced, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal human immunoglobulin genes or hybridomas produced therefrom (described further below), (b) antibodies isolated from host cells transformed to express human antibodies (e.g., from transfectomas), (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies produced, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when a human Ig sequence transgenic animal is used), and thus the amino acid sequence recombinant antibodies of the V _H and V _L regions of the recombinant human antibodies are sequences derived from and associated with human germline V _H and V _L sequences, but may not naturally occur in the human antibody germline repertoire in vivo.

The term "isotype" refers to the class of antibodies (e.g., igM or IgG 1) encoded by the heavy chain constant region gene. The phrases "antibody that recognizes an antigen" and "antigen-specific antibody" are used interchangeably herein with the term "antibody that specifically binds an antigen".

The term "human antibody derivative" refers to any modified form of a human antibody, such as a conjugate of an antibody with another agent or antibody. The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequence.

The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. The term may also refer to antibodies whose variable region sequences or CDRs are derived from one source (e.g., an IgA1 antibody), while the constant region sequences or fcs are derived from a different source (e.g., a different antibody, such as an IgG, igA2, igD, igE, or IgM antibody).

A "single chain antibody" or "scFv" is an Fv molecule in which the heavy and light chain variable regions have been joined by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. scFv is discussed in detail in WO88/01649 and U.S. Pat. nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference.

"Domain antibodies" or "single chain immunoglobulins" are immunologically functional immunoglobulin fragments that contain only heavy chain variable regions or light chain variable regions. Examples of domain antibodies include Nanobodies ^TM. In some cases, two or more VH regions are covalently linked to a peptide linker to produce a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

The term "antibody fusion protein" as used herein is a recombinantly produced antigen binding molecule in which an antibody or antibody fragment is linked to another protein or peptide, e.g., the same or a different antibody or antibody fragment or DDD or AD peptide. The fusion protein may comprise a single antibody component, a multivalent or multispecific combination of different antibody components, or multiple copies of the same antibody component. Fusion proteins may also include antibodies or antibody fragments and therapeutic agents. Examples of therapeutic agents suitable for use in such fusion proteins include immunomodulators and toxins. A preferred toxin comprises a ribonuclease (RNase), preferably a recombinant RNase. Preferred immunomodulators may be interferons, such as interferon-alpha, interferon-beta or interferon-lambda.

A "molecular complex" is a combination of two or more related moieties or molecules that are linked by covalent or non-covalent interactions. Examples of such molecular complexes include antibodies and antigen binding portions thereof.

A "multispecific" complex, protein, or antibody is a complex, protein, or antibody that can simultaneously bind to at least two targets having different structures, e.g., two different antigens, two different epitopes or haptens on the same antigen, and/or an antigen or epitope.

A "multivalent" complex, protein or antibody is a complex, protein or antibody that can bind at least two targets of the same or different structure simultaneously. Titers refer to how many binding arms or sites a complex, protein, or antibody has to a single antigen or epitope, i.e., monovalent, bivalent, trivalent, or multivalent. The multivalent nature of a complex, protein or antibody means that it can exploit multiple interactions with antigen binding, thereby increasing affinity for antigen binding. Specificity refers to how many antigens or epitopes a complex, protein or antibody is capable of binding to, i.e., monospecific, bispecific, trispecific, multispecific. Using these definitions, a natural antibody (e.g., igG) is bivalent in that it has two binding arms, but it is monospecific in that it binds to one epitope. Multispecific, multivalent antibodies are constructs that have multiple binding sites of different specificities.

A "multi-specific" complex, protein or antibody is a complex, protein or antibody that can bind simultaneously to a plurality of targets or epitopes having different structures, including but not limited to a "dual-specific" complex, protein or antibody. Such complexes, proteins or antibodies comprise two or more binding moieties with different specificities.

A "bispecific" complex, protein or antibody is a complex, protein or antibody that can bind two targets or epitopes of different structures simultaneously. T cell redirecting bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab or otherwise) can have at least one arm that specifically binds to, for example, T cells, and at least one other arm that specifically binds to an antigen produced by or associated with a diseased cell, tissue, organ, or pathogen (e.g., a tumor-associated antigen). Molecular engineering can be used to produce a variety of bispecific antibodies.

As used herein, the term "affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between binding pair members (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

As used herein, a protein that "specifically binds" an antigen refers to a protein that binds to an antigen when the dissociation constant (KD) is < 10 ^-6 M as measured by surface plasmon resonance techniques (e.g., BIACore, GE-HEALTHCARE UPPSALA, sweden) or kinetic exclusion assay (KinExA, sapidyne, boise, id.). Preferably, the protein (e.g., antibody) binds to the antigen with "high affinity", i.e., a KD of 1x 10 ^-7 M or less, more preferably 5x 10 ^-8 M or less, more preferably 3x10 ^-8 M or less, more preferably 1x 10 ^-8 M or less, more preferably 5x 10 ^-9 M or less, even more preferably 1x 10 ^-9 M or less. As used herein, the term "substantially does not bind" to a protein or cell means that the protein or cell is not bound or does not bind with high affinity, i.e., the KD of the bound protein or cell is 1x 10 ^-6 M or greater, more preferably 1x 10 ^-5 M or greater, more preferably 1x 10 ^-4 M or greater, more preferably 1x 10 ^-3 M or greater, even more preferably 1x 10 ^-2 M or greater.

The term "kasloc" or "Ka" as used herein refers to the rate of binding of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" as used herein refers to the rate of dissociation of a particular antibody-antigen interaction. The term "KD" as used herein refers to the dissociation constant, which is obtained from the ratio of KD to Ka (i.e. KD/Ka) and is expressed in molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such asThe system.

The term "epitope" as used herein refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B and/or T cells respond. It also refers to the region of antigen bound by an antibody. Epitopes may be defined as structural or functional epitopes. Functional epitopes are typically a subset of structural epitopes and have residues that directly contribute to interaction affinity. Epitopes can also be conformational, i.e. composed of non-linear amino acids. In certain embodiments, an epitope may comprise a determinant of a surface group of a chemically active molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments may have a particular three-dimensional structural feature, and/or a particular charge characteristic. Epitopes generally comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods of determining which epitopes a given antibody binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays in which overlapping or consecutive peptides from an antigen protein are tested for reactivity with a given antibody. Methods of determining epitope spatial conformation include those techniques described in the art and herein, such as X-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., epitope Mapping Protocols in Methods in Molecular Biology, vol.66, g.e.morris, ed. (1996)).

The term "binding epitope" or "recognition epitope" with respect to an antibody or antibody fragment refers to a continuous or discontinuous fragment of amino acids within an antigen. Those skilled in the art will appreciate that these terms do not necessarily mean that the antibody or antibody fragment is in direct contact with each amino acid within the epitope sequence.

The term "binding pair" refers to a first molecule and a second molecule that specifically bind to each other. Exemplary binding pairs include any hapten or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., digoxin and anti-digoxin) and non-immune binding pair (e.g., biotin-avidin, biotin-streptavidin, biotin-neutravidin, hormones (e.g., thyroxine and cortisol-hormone binding protein), receptor-receptor agonists, receptor-receptor antagonists (e.g., acetylcholine receptor-acetylcholine or analogs thereof), igG-protein A, igG-protein G, igG-synthetic protein AG, lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme inhibitor, and complementary oligonucleotide pair capable of forming a nucleic acid duplex), and the like. The binding pair may also include a negatively charged first molecule and a positively charged second molecule.

As used herein, the term "immune response" refers to a biological response within a vertebrate against foreign substances, cancer cells, or other abnormal cells that protects the organism from these substances and diseases caused by them. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which result in selective targeting, binding, injury, destruction, and/or elimination of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or other abnormal cells in vertebrates, or normal human cells or tissues in the case of autoimmune or pathological inflammation. Immune responses include, for example, activation or suppression of T cells (e.g., effector T cells or Th cells, such as cd4+ or cd8+ T cells), or suppression of Treg cells.

An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate symptoms and/or root causes, prevent the occurrence of symptoms and/or root causes thereof, and/or ameliorate or repair damage caused by or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. When applied to a single active ingredient administered alone, the term refers to the amount of that ingredient alone. When applied to a combination, the term refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, serially or simultaneously.

A "therapeutically effective amount" is an amount sufficient to treat a disease state or condition, particularly a state or condition associated with a disease state, or otherwise prevent, hinder, retard, or reverse the progression of a disease state or any other undesirable condition associated with a disease state in any way.

A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have a desired prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of a disease state, or reducing the likelihood of the onset (or recurrence) of a disease state or related symptoms. The complete therapeutic or prophylactic effect is not necessarily achieved by administration of one dose, and may be achieved only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations.

The term "pharmaceutically acceptable carrier or excipient" as used herein refers to a carrier medium or excipient that does not interfere with the effectiveness of the biological activity of the active ingredient of the composition and that is not overly toxic to the host at its concentration at which it is administered. In the context of the present invention, a pharmaceutically acceptable carrier or excipient is preferably suitable for topical formulations. The term includes, but is not limited to, solvents, stabilizers, solubilizers, tonicity enhancing agents, structure-forming agents, suspending agents, dispersing agents, chelating agents, emulsifying agents, anti-foaming agents, ointment bases, emollients, skin protectants, gel-forming agents, thickening agents, pH adjusting agents, preservatives, permeation enhancers, complexing agents, lubricants, demulcents, viscosity enhancing agents, bioadhesive polymers, or combinations thereof. The use of such agents to formulate pharmaceutically active substances is well known in the art (see, e.g., "Remington's Pharmaceutical Sciences", e.w. martin,18th Ed.,1990,Mack Publishing Co: easton, PA, the entire contents of which are incorporated herein by reference).

As used herein, the term "agent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule (e.g., a nucleic acid, antibody, protein, or portion thereof, e.g., a peptide), or an extract made from biological material, e.g., bacterial, plant, fungal, or animal (particularly mammalian) cells or tissues. The activity of such agents may make them suitable as "therapeutic agents" which are biologically, physiologically or pharmacologically active substance(s) that act locally or systemically in a subject.

As used herein, the terms "therapeutic agent," "therapeutically effective agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that imparts some beneficial effect upon administration to a subject. Benefits include achieving diagnostic determinations, amelioration of a disease, symptom, disorder, or pathological condition, reducing or preventing occurrence of a disease, symptom, disorder, or condition, and combating a disease, symptom, disorder, or pathological condition in general.

The following examples illustrate the present disclosure. These examples should not be construed as limiting the scope of the application. These examples are included for illustrative purposes and the present application determines the priority date of the intended global patent rights covering the present disclosure.

Example

EXAMPLE 1 Generation of cell lines expressing anti-CD 3 Single chain-AD modules

To prepare T cell redirected IgG-scFv bispecific antibodies, three primary cell lines were first generated to express different anti-CD 3 scFv-AD modules. The huα3sc-AD2 (SEQ ID NO 8) and huα3sc-AD7 (SEQ ID NO 9) modules were designed from humanized SP34 monoclonal antibodies (SP 34 mAbs) against CD3, with the complementarity determining regions of huα3 listed in Table 1. The 3scFv module (SEQ ID NO 32) was designed from a humanized Okt3 monoclonal antibody against CD 3.

TABLE 1 Complementarity Determining Regions (CDRs) of hu alpha 3 (anti-CD 3)

Module 1 hu α3sc-AD2

This module was designed from an anti-CD 3 humanized SP34 monoclonal antibody and added with the anchoring domain of AKAP protein (CG and GC added at the N-and C-terminus, respectively, designated AD 2) and assembled in the form of V _k-L1-V_H -L2-AD2-GS-6H, where the variable (V) domain of the humanized SP34 monoclonal antibody was fused by a flexible peptide linker followed by the AD2 and 6-His tag. The sequences of the leader peptide, anti-CD 3 variable domain, linker and AD2 are shown below.

Leader peptide (SEQ ID NO 28)

MGWSCIILFLVATATGVHS

Vk sequence of anti-CD 3 single chain huα3sc (SEQ ID NO 4)

QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL

L1 joint (SEQ ID NO 27)

GGGGSGGGGSGGGGS

VH sequence of anti-CD 3 single chain huα3sc (SEQ ID NO 5)

EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

L2 joint (SEQ ID NO 6)

GGGSGGGSGGGS

AD2 peptide (SEQ ID NO 2)

CGQIVYLAKQIVDNAIQQAGC

GS-6-His(SEQ ID NO 7)

GS-HHHHHH

CDNA sequences encoding the leader peptide (SEQ ID NO 28) and huα3sc-AD2 (SEQ ID NO 4, 27, 5-6, 2, 7) were synthesized and cloned into the pcDNA3.4 vector (G418 ^R). A Kozak sequence "GCCGCCACC" (SEQ ID NO: 39) was added near the ATG start codon to prepare the final expression vector. Expression vectors were transfected into CHO or Sp2/0 cells using a Neon electroporation transfection system. Clones were selected in medium containing 0.25mg/ml G418 and screened for protein expression by spot hybridization. The supernatant was captured on nitrocellulose membrane and detected with HRP-labeled anti-His monoclonal antibody. Clones with the highest protein expression levels were selected for further culture and selection until stable subclones were established as the master cell line. The AD 2-linked anti-CD 3 single-chain module was designated huα3sc-AD2 (SEQ ID NO 8). The master cell line was designated huα3sc-AD2-SC34.

Huα3sc-AD2 Vk-L1-VH-L2-AD2-GS-6-His (SEQ ID NO 8)

QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGSGGGSGGGSCGQIVYLAKQIVDNAIQQAGCGSHHHHHH

Module 2 hu α3sc-AD7

This module was designed from a humanized SP34 monoclonal antibody against CD3 and added with an anchor domain from AKAP7 (also known as AKAP18 or AKAP 15) protein (CG and GC added at the N-and C-terminus, respectively, designated AD 7) and assembled in the form of V _k-L1-V_H -L2-AD7-GS-6H, where the variable (V) domain of the humanized SP34 monoclonal antibody was fused by a flexible peptide linker followed by the AD7 and 6-His tag. The sequences of the leader peptide, anti-CD 3 variable domain, linker and AD7 are shown below.

Leader peptide (SEQ ID NO 28)

MGWSCIILFLVATATGVHS

Vk sequence of anti-CD 3 single chain huα3sc (SEQ ID NO 4)

QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL

L1 joint (SEQ ID NO 27)

GGGGSGGGGSGGGGS

VH sequence of anti-CD 3 single chain huα3sc (SEQ ID NO 5)EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

L2 joint (SEQ ID NO 6)

GGGSGGGSGGGS

AD7 peptide (SEQ ID NO 3)

CGPEDAELVRLSKRLVENAVLKAVQQYGC

GS-6-His(SEQ ID NO 7)

GS-HHHHHH

CDNA sequences encoding the leader peptide (SEQ ID NO 28) and huα3sc-AD7 (SEQ ID NO 4, 27, 5-6, 3, 7) were synthesized and cloned into the pcDNA3.4-P vector (puromycin ^R). A Kozak sequence "GCCGCCACC" (SEQ ID NO: 39) was added near the ATG start codon to prepare the final expression vector. Expression vectors were transfected into CHO or Sp2/0 cells using a Neon electroporation transfection system. Clones were selected in medium containing 10. Mu.g/ml puromycin and screened for protein expression by spot hybridization. The supernatant was captured on nitrocellulose membrane and detected with HRP-labeled anti-His monoclonal antibody. Clones with the highest protein expression levels were selected for further culture and selection until stable subclones were established as the master cell line. The AD 7-linked anti-CD 3 single-chain module was designated huα3sc-AD7 (SEQ ID NO 9). The master cell line was designated huα3sc-AD7-SC7.

Huα3sc-AD7 Vk-L1-VH-L2-AD7-GS-6-His (SEQ ID NO 9)

QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGSGGGSGGGSCGPEDAELVRLSKRLVENAVLKAVQQYGCGSHHHHHH

Module 3:3scFv

This module was designed from a humanized Okt3 monoclonal antibody against CD3 and added with AD2 (SEQ ID NO 2) and assembled in the form of V _H-L1-V_K -L2-AD2-GS-6H, where the variable (V) domains of the humanized Okt3 monoclonal antibody are fused by a flexible peptide linker followed by the AD2 and 6-His tags. The sequences of the leader peptide, anti-CD 3 variable domain, linker and AD2 are shown below.

Leader peptide (SEQ ID NO 28)

MGWSCIILFLVATATGVHS

V _H sequence of anti-CD 3 scFv (SEQ ID NO 29)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS

L1a linker (SEQ ID NO 30)

VEGGSGGSGGSGGSGGVD

V _K sequence of anti-CD 3 scFv (SEQ ID NO 31)

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

L2 joint (SEQ ID NO 6)

GGGSGGGSGGGS

AD2 peptide (SEQ ID NO 2)

CGQIVYLAKQIVDNAIQQAGC

GS-6-His(SEQ ID NO 7)

GS-HHHHHH

CDNA sequences encoding the leader peptide (SEQ ID NO 28) and 3scFv (SEQ ID NO 29-31, 6, 2, 7) were synthesized and cloned into the pcDNA3.4 vector. A Kozak sequence "GCCGCCACC" (SEQ ID NO: 39) was added near the ATG start codon to prepare the final expression vector. Expression vectors were transfected into CHO or Sp2/0 cells using a Neon electroporation transfection system. Clones were selected in medium containing 0.25mg/ml G418 and screened for protein expression by spot hybridization. The supernatant was captured on nitrocellulose membrane and detected with HRP-labeled anti-His monoclonal antibody. Clones with the highest protein expression levels were selected for further culture and selection until stable subclones were established as the master cell line. The AD 2-linked anti-CD 3 single chain module was designated as 3scFv (SEQ ID NO 32). The master cell line was designated 3scFv-C21.

V _H-L1a-V_K -L2-AD2-GS-6H (SEQ ID NO 32) of 3scFv

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS-VEGGSGGSGGSGGSGGVD-DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK-GGGSGGGSGGGS-CGQIEYLAKQIVDNAIQQAGC-GS-HHHHHH

EXAMPLE 2 Generation of cell lines expressing anti-CD 3 Fab-AD

To prepare T cell redirected IgG-Fab bispecific antibodies, two master cell lines were generated to express different anti-CD 3-Fab-AD modules. In these modules, the V _K and V _H domains were interchanged to connect with C _H1 and C _K, respectively, to avoid light chain mismatches.

Module 1 huα3cm-Fab-AD2 (Ck)

This module was designed from the Fab fragment of the humanized SP34 monoclonal antibody against CD3, in which the V _K and V _H domains were cross-exchanged, the C _K domain fused to a flexible peptide linker, followed by AD2 and 6-His tags. The two chains with domain cross-exchange are assembled in the form of Vk-SS-CH1 and VH-Ck-L2-AD2, respectively. The sequences of leader peptide, anti-CD 3 variable and constant domains, linker and AD2 are shown below.

huα3cm:Vk-SS-CH1

Leader peptide (SEQ ID NO 28)

MGWSCIILFLVATATGVHS

Vk(SEQ ID NO 4)

QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL

SS-CH1(SEQ ID NO 33)

SS-STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

huα3cm:VH-Ck-L2-AD2

Leader peptide (SEQ ID NO 28)

MGWSCIILFLVATATGVHS

VH(SEQ ID NO 5)

EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

Ck(SEQ ID NO 34)

ASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

L2 joint (SEQ ID NO 6)

GGGSGGGSGGGS

AD2(SEQ ID NO 2)

CGQIEYLAKQIVDNAIQQAGC

Two cDNA sequences encoding the leader peptide (SEQ ID NO 28) -Vk-SS-CH1 (SEQ ID NO 4 and 33) and the leader peptide (SEQ ID NO 28) -VH-Ck-L2-AD2 (SEQ ID NO 5, 34, 6 and 2) were synthesized and cloned into human IgG expression vectors, respectively, wherein the original CH and Ck sequences were replaced by the two synthesized cDNA sequences. In addition, the methotrexate resistance gene (DHFR) in the vector was replaced with puromycin resistance gene to prepare the final huα3cm-Fab-AD2 (Ck) expression vector. The AD2 linked anti-CD 3 Fab module was designated huα3cm-Fab-AD2 (Ck) (SEQ ID NO 35 and 36).

Huα3cm-Fab-AD2 Vk-CH1 (SEQ ID NO 35)

QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL-SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

VH-CK-GS-AD2 of huα3cm-Fab-AD2 (SEQ ID NO 36)

EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS-ASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-GGGSGGGSGGGS-CGQIEYLAKQIVDNAIQQAGC

Module 2 huα3cm-Fab-AD2 (CH 1)

This module was designed from the Fab fragment of the humanized SP34 monoclonal antibody against CD3, in which the V _K and V _H domains were cross-exchanged, the C _H1 domain fused to a flexible peptide linker, followed by AD2 and 6-His tags. The two chains with domain cross-exchange are assembled in the form of Vk-SS-CH1-AD2 and VH-Ck-L2, respectively. The AD2 linked anti-CD 3Fab module was designated huα3cm-Fab-AD2 (CH 1) (SEQ ID NO 37 and 38).

Huα3cm-Fab-AD2 Vk-CH1-GS-AD2 (SEQ ID NO 37)

QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGGSGGGSGGGSCGQIEYLAKQIVDNAIQQAGC

VH-Ck-GS-His of huα3cm-Fab-AD2 (SEQ ID NO 38)

EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSHHHHHH

Example 3 production of humanized anti-Trop 2 antibody hL0125

Mice were immunized with recombinant human Trop2 extracellular fragments (AA 1-275). Five mice received four consecutive immunizations. Blood was taken and serum was prepared for determination of antibody titer by enzyme-linked immunosorbent assay (ELISA). The highest titer mice were selected for booster immunization and monoclonal antibodies were isolated by hybridoma technology (i.e., spleen cells fused with mouse Sp2/0 myeloma cells).

The supernatants of a number of hybridoma cell colonies were screened by ELISA (where Maxisorp plates immobilized human Trop2 protein) and 40 positive supernatants were selected for further screening. Clones were tested for binding to recombinant human Trop2 protein (amino acid residues 1-275) by ELISA and clones were tested for binding to Trop2 positive and negative cell lines by flow cytometry (FACS). Clone L0125 was selected for subcloning and cDNA sequencing based on binding affinity and specificity to generate recombinant and humanized IgG.

The complementarity determining regions of L0125 are listed in Table 2. Chimeric and humanized anti-Trop 2 antibodies (cL 012 and hL 0125) were generated based on the amino acid sequences of the V _L and V _H domains of murine anti-L0125 antibodies. Humanization based on sequence alignment, IGBLAST (a tool for Immunoglobulin (IG) and T cell receptor (TR) V domain sequences) was used as well as BLAST to query the human protein database and therapeutic antibody database. The amino acid sequence of the humanized V _L variant is shown as SEQ ID NO.47, and the amino acid sequence of the humanized V _H variant is shown as SEQ ID NO.48.

The humanized amino acid sequences of the light and heavy chain variable regions of hL0125 were reverse translated into DNA sequences that were synthesized and cloned into human IgG expression vectors as fusion proteins. Recombinant IgG antibodies were produced and purified from cell culture supernatants by standard antibody preparation methods.

TABLE 2 Complementarity Determining Regions (CDRs) of hL0125-Cm (anti-Trop 2)

VL of hL0125 (SEQ ID NO. 47)

DIQLTQSPAIMSASPGERVTMTCRASSSVSSSYLHWYQQRSGQSPKLLIYSTSNLASGVPARFSGSGSGTDYSLTISSLEAEDAATYYCQQYSGSPLTFGSGTKLEIKR

VH of hL0125 (SEQ ID No. 48)

QVQLQESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPDKRLEWVAEISSDGFYTYYPDTVTGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARDGNYVDYAMDYWGQGTSVTVSS EXAMPLE 4 construction of expression vector for preparing IgG-Cm

Unlike the design of trivalent DNL complexes previously binding anti-CD 3scFv to anti-TAA F (ab) 2 (patent US9,315,567B2), the present invention relates to compositions and methods of novel constructs for grafting monovalent scFv to full-length IgG by intracellular or extracellular bioconjugation. As shown in fig. 10A-10D and 11A-11D, four forms of scFv x IgG bispecific complexes were generated, wherein scFv x IgG-C forms (e.g. 3scFv x hL 0125-Cm) were well prepared with optimal quality and were suitable for T cell redirection. Thus, the scFv XIgG-C form was selected for further investigation.

4.1 Construction of hL0125-Cm

In this construct, the dimerization/docking domain of the riiα regulatory subunit of protein kinase a (DDD 2) is inserted into the hinge region of the hL0125 IgG Heavy Chain (HC) through two GS peptide linkers. The sequences of LC, GS-DDD2-GS and modified HC (V _H-C_H1 -hinge-GS-DDD 2-GS-hinge-C _H2-C_H3) are shown below (SEQ ID NOS 10-11, 49). The hL0125 IgG with V _H-C_H1 -hinge-GS-DDD 2-GS-hinge-C _H2-C_H3 was designated hL0125-C.

VL-CL of hL0125-Cm (SEQ ID NO 10)

DIQLTQSPAIMSASPGERVTMTCRASSSVSSSYLHWYQQRSGQSPKLLIYSTSNLASGVPARFSGSGSGTDYSLTISSLEAEDAATYYCQQYSGSPLTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

GS-DDD2-GS(SEQ ID NO 11)

GGGGSGGGGSGGG-CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA-GGGGSGGGGSGGG

VH-CH1--GS-DDD2-GS--CH2-CH3(SEQ ID NO 49)

One key component of Fc-containing T cell redirecting bispecific antibodies is the elimination of Fc region binding to fcγr, thereby avoiding off-target T cell activation by fcγr expressing cells and potential T cell lysis mediated by fcγr expressing effector cells. Based on previous studies (Moore et al, methods.2019, 154:38-50), fcgammaR interacts with antibodies primarily through contact with hinge and CH2 domains, and it is known that IgG2 antibodies have much lower affinity for FcgammaR than human IgG1. In this example, the construct of hL0125-C was further modified to prepare an IgG1 variant (hL 0125-Cm) that combines the substitution of the hinge and the additional CH2 substitution of the surface residues (SEQ ID NO 12) under IgG 2.

V _H-C_H1 of hL 0125-Cm--GS-DDD2-GS--C_H2-C_H3(SEQ ID NO 12)

The cDNA sequence encoding GS-DDD2-GS (SEQ ID NO 11) was synthesized and inserted into the hinge region of the hL0125 IgG heavy chain to prepare the IgG expression vector IgG V-hL0125-C. The vector was further modified to prepare an expression vector IgG V-hL0125-Cm with a mutated heavy chain (V _H-C_H1 -hinge-GS-DDD 2-GS-hinge-C _H2-C_H3, SEQ ID NO 12).

4.2 Construction of T-Cm

The construction of hL0125-Cm was extended to other IgG moieties targeting multiple disease-related antigens to make IgG-DDD2 modules, commonly abbreviated as IgG-Cm. For example, V _H and V _L of hL0125-Cm were replaced with V _H and V _L of Trastuzumab, a humanized anti-HER 2 monoclonal antibody whose complementarity determining regions (SEQ ID NOs 21-26) are shown in table 3, to prepare T-Cm modules. Specifically, in the IgG expression vector of IgG V-hL0125-Cm, the cDNA sequence encoding V _H of hL0125-Cm was replaced with the cDNA sequence encoding V _H of Trastuzumab, and the cDNA sequence encoding V _L of hL0125-Cm was replaced with the cDNA sequence encoding V _L of Trastuzumab, thereby obtaining the IgG V-T-Cm vector expressing T-Cm heavy and light chains (SEQ ID NO 13-14).

TABLE 3 Complementarity Determining Regions (CDRs) of T-Cm (anti-HER 2)

VL-CL of T-Cm (SEQ ID NO 13)

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

VH-CH1--GS-DDD2-GS-CH2-CH3(SEQ ID NO 14)

EXAMPLE 5 preparation of bispecific antibodies by intracellular Assembly of IgG and scFv

In the present application, three exemplary bispecific antibodies were prepared, including a combination of anti-CD 3 single chain huα3sc-AD2 or huα3sc-AD7 with hL0125-Cm (a humanized full-length IgG that specifically targets Trop 2), and a combination of huα3sc-AD2 with T-Cm (a humanized full-length IgG that specifically targets HER 2). To examine the yield and quality, the expression vectors of IgG V-hL0125-Cm were transfected into two main cell lines huα3sc-AD2-SC34 and huα3sc-AD7-SC7, respectively, to prepare two bispecific antibodies huα3sc-AD2 XhL 0125-Cm and huα3sc-AD7 XhL 0125-Cm, respectively. The expression vector of IgG V-T-Cm was transfected into the master cell line huα3sc-AD2-SC34 to prepare the bispecific antibody huα3sc-AD2×T-Cm. For comparison, 2.8X10 ⁶ cells in 100. Mu.l of transfection buffer containing 20. Mu.g of IgG V-X-Cm DNA were transfected by electroporation, recovered in 60ml of medium and then dispensed into six 96-well plates. Clones were selected in medium containing 0.2. Mu.M Methotrexate (MTX) and 1.0. Mu.g/ml puromycin and screened for human IgG expression. The anti-CD 3 single chain AD2 or AD7 modules were grafted to IgG-Cm modules inside the cell to form trivalent (1+2) bispecific antibodies. For each antibody, the three clones with the highest yield were selected and expanded to 100ml in T175 flasks and bispecific antibodies were purified from the supernatant by affinity chromatography using MabSelect ^TM resin. Depending on yield, purity and cell health, some clones were further expanded to 500ml cultures, followed by purification of bispecific antibodies by MabSelect ^TM and HisPur Ni-NTA resin, followed by analysis by HPLC and SDS-PAGE.

As shown in table 4, all three bispecific antibodies from different clones showed good yields in T175 flasks, with purities between 73.8% and 92.29%, after selection by MabSelect ^TM resin. Some clones were expanded to 500ml cultures and bispecific antibodies were purified by MabSelect ^TM followed by HisPur Ni-NTA resin, both in more than 90% purity (91.44-96.94%, see Table 5 and FIG. 6). In SDS-PAGE, all three antibodies and their modules showed high purity under both reducing and non-reducing conditions (FIG. 5). The antibody huα3sc-AD7 XhL 0125-Cm showed better yields and purity than AD2 XhL 0125-Cm (Table 4), indicating that AD7 can be better coupled and prepared in intracellular IgG-scFv than AD 2. On the other hand, the purity of hua3sc-AD2 XT-Cm was superior to hua3sc-AD2 XhL 0125-Cm (tables 4 and 5), indicating that the variable domain of the IgG module can also have an effect on the quality of these bispecific antibodies.

TABLE 4 preparation of bispecific antibodies in T175 flasks

TABLE 5 preparation of bispecific antibodies in roller bottles

Example 6 cell surface binding of bispecific antibodies

The relative cell binding strength was assessed using flow cytometry. For CD3 binding, jurkat cells were dispensed into 96-well plates, 2×10 ⁵ cells per well, and incubated with serial dilutions of 3-fold bispecific antibody or its associated monospecific monoclonal antibody (mAb) for 45 minutes at 4 ℃. After washing with PBS, cells were incubated with AF 488-labeled goat anti-mouse or goat anti-human IgG Fc secondary for 45 minutes at 4 ℃. After two washing steps, the cells were resuspended in PBS and analyzed using Attune NxT flow cytometer. The results demonstrated that both anti-CD 3 monoclonal antibody (SP 34) and bispecific antibody (AD 2 XhL 0125-Cm, AD7 XhL 0125-Cm and hua3sc-AD 2X T-Cm) were able to bind to human CD3 on the Jurkat cell surface and were detected by AF 488-labeled goat anti-mouse or goat anti-human IgG Fc secondary antibodies (FIGS. 7A and 7B). In contrast, the detection signal of hL0125-Cm was very weak due to the lack of anti-CD 3 domain (fig. 7B).

For Trop2 binding, MDA-MB-468, HCC 1806 or BT-474 cells were isolated from culture, dispensed into 96-well plates, 2×10 ⁵ cells per well, and incubated for 45 min at 4 ℃ with serially diluted 3-fold bispecific antibodies (AD 2×hl0125-Cm, AD7×hl0125-Cm or hua3sc-AD2×t-Cm) or their related monospecific monoclonal antibodies (hL 0125 or trastuzumab). After washing with PBS, cells were incubated with AF 488-labeled goat anti-human IgG Fc secondary antibody for 45 minutes at 4 ℃. After two washing steps, binding was analyzed using Attune NxT flow cytometry. The results indicate that whether an AD2 XhL 0125-Cm or AD7 XhL 0125-Cm bispecific antibody binds with similar affinity to human Trop2 on MDA-MB-468 or HCC 1806 cells as the anti-Trop 2 monoclonal antibody hL0125 (FIGS. 8A and 8B), whereas hua3sc-AD2 XT-Cm bispecific antibody binds with similar affinity to human HER2 as the anti-HER 2 monoclonal antibody trastuzumab (FIG. 8C).

EXAMPLE 7 in vitro cytotoxicity

Cancer cells were mixed with human Peripheral Blood Mononuclear Cells (PBMCs) and dispensed into 96-well plates at 200 μl per well to provide 1.1x10 ⁴ tumor cells and 8.8x10 ⁴ PBMC cells per well (PBMC to target cell ratio of 8:1). Beginning at 20nmol/L, bispecific antibodies or their related monospecific monoclonal antibodies were serially diluted 4-fold to treat mixed cells. After 60 hours incubation, the medium was removed and replaced with fresh medium to flush the PBMC cells and dead cancer cells twice. Cell viability was determined by MTS reagent. For MDA-MB-468 and HCC1806 cells, three replicates were performed using bispecific antibodies huα3sc-AD2 XhL 0125-Cm, huα3sc-AD7 XhL 0125-Cm, hua3sc-AD2 XT-Cm, and 3scFv XhL 0125-Cm, respectively (FIGS. 9A and 9B). For HCT-116 cells, three replicates were performed using the bispecific antibodies huα3sc-AD2 XhL 0125-Cm and hua3sc-AD2 XT-Cm, respectively, and the two related monospecific monoclonal antibodies SP34 (anti-CD 3) and hL0125 (anti-Trop 2) (FIG. 9C). For BT-474 cells, three replicates were performed using the bispecific antibodies huα3sc-AD2 XhL 0125-Cm and hua3sc-AD2 XT-Cm, respectively, and the two related monospecific monoclonal antibodies SP34 (anti-CD 3) and trastuzumab (anti-HER 2) (FIG. 9D).

As shown in fig. 9A-9B and table 6, the bispecific antibody huα3sc-AD7 xhl 0125-Cm induced strong cytotoxicity in all four Trop2 positive cell lines. Based on a 60 hour MTS viability assay, the IC ₅₀ of huα3sc-AD7 XhL 0125-Cm was about 0.33pM in MDA-MB-468 cells, about 4.79pM in HCC1806 cells, about 2.28pM in HCT-116 cells, and about 5.99pM in BT-474 cells (Table 6). huα3sc-AD2 XhL 0125-Cm showed similar efficacy to huα3sc-AD7 XhL 0125-Cm in the tested MDA-MB-468 and HCC1806 cell lines. In contrast, the efficacy of antibody 3scFv xhl 0125-Cm was relatively low, with an IC ₅₀ of 2.45pM in the MDA-MB-468 cell line and an IC ₅₀ of 18.3pM in the HCC1806 cell line, suggesting that SP34 may activate T cells more effectively and mediate dose-dependent tumor cell killing than Okt3 when grafted as scFv onto targeted IgG (fig. 9A-9B and table 6). The antibody hua3sc-AD2×T-Cm showed strong toxicity in two HER2 positive cell lines, with IC ₅₀ of 3.3pM and 1.92pM for the HCT-116 cell line (HER 2 low expression) and BT-474 cell line (HER 2 high expression), respectively, while toxicity was relatively low in the MDA-MB-468 cell line (35.76 pM) and HCC1806 cell line (19.71 pM). All three related monospecific monoclonal antibodies, including SP34, hL0125 and trastuzumab, showed little or no detectable toxicity in the cell lines tested (fig. 9C-9D and table 6).

TABLE 6 in vitro cytotoxicity of bispecific antibodies against cancer cells

ND not detected, NA not applicable

The foregoing description of the examples and preferred embodiments should be taken as illustrating the invention rather than as limiting the invention as defined by the claims. It will be readily appreciated that various changes and combinations of the features described above can be made without departing from the invention as set forth in the claims. Such variations are not to be regarded as a departure from the scope of the disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims (30)

1. A protein complex comprising:

A first portion comprising two immunoglobulin light chains and two immunoglobulin heavy chains, wherein the two light chains or the two heavy chains are linked to two dimerization/docking domain (DDD) portions, respectively, and

A second moiety comprising (i) an Anchor Domain (AD) moiety and (ii) an agent linked to said AD moiety, said AD moiety comprising a sequence having at least 70% identity to the sequence of SEQ ID NO. 3 or 2 or 46,

Wherein the two DDD moieties form a dimer that binds to the AD moiety.

2. The protein complex according to claim 1, wherein the protein complex is,

(A) The first moiety is a targeting moiety that specifically binds an antigen or epitope, and

(B) The second moiety is an effector moiety and the agent is an effector agent.

3. The protein complex according to claim 1, wherein the protein complex is,

(A) The first moiety is an effector moiety comprising an effector agent, and

(B) The second moiety is a targeting moiety and the agent is a targeting agent that specifically binds an antigen or epitope.

4. The protein complex according to claim 1, wherein the protein complex is,

(A) The first moiety and the second moiety are two targeting moieties that specifically bind to two antigens or epitopes, or

(B) The first moiety and the second moiety are two effector moieties and the agent is an effector.

5. A fusion protein comprising:

(i) Dimerization/docking domain (DDD) moiety, and

(Ii) An immunoglobulin light chain fused to the DDD moiety, or an immunoglobulin light chain fragment fused to the DDD moiety, or an immunoglobulin heavy chain fragment fused to the DDD moiety.

6. The protein complex or fusion protein of any one of claims 1-5, wherein each DDD moiety is inserted into each immunoglobulin heavy chain.

7. The protein complex or fusion protein of claim 6, wherein the DDD moiety is inserted into the hinge region of the immunoglobulin heavy chain or a flanking region thereof.

8. The protein complex or fusion protein of any one of claims 1-5, wherein each DDD moiety is fused to the C-terminus of each immunoglobulin light chain.

9. The protein complex or fusion protein of claim 8, wherein the DDD moiety is fused to the C-terminus of the immunoglobulin light chain via a linker sequence.

10. The protein complex or fusion protein of claim 9, wherein the linker sequence comprises at least one cysteine and the protein complex comprises a disulfide bond between two linker sequences.

11. The protein complex or fusion protein of any one of claims 1-5, wherein each DDD moiety is fused to the C-terminus of each immunoglobulin heavy chain.

12. The protein complex of any one of claims 2-4 and 6-11, wherein the targeting moiety specifically binds to a tumor-associated antigen or a disease-associated antigen.

13. The protein complex of claim 12, wherein the tumor-associated antigen or the disease-associated antigen is Trop2、EpCAM、GPRC5、FcRH5、ROR1、BCMA、CD15、CD16、CD19、CD20、CD22、CD27、CD30、CD33、CD40、CD47、CD40L、CD66、CD70、CD74、CD79b、CD80、CD95、CD133、CD160、CD166、CD229、MUC1、MUC5、MUC16、IGF-1R、EGFR、HER2、HER3、EGP2、HLA-DR、TNF-α、TRAIL receptor, ICOS, ICOSL, VEGF, VEGFR, hypoxia-inducible factor (HIF), flt-3, folate receptor, TDGF1, tfR, mesothelin 、PSMA、CEACAM5、CEACAM6、B7、IFN-α、IFN-β、IFN-γ、IFN-λ、IL-1β、IL2、IL6、IL-6R、IL-15、IL-15R、IL-17、IL-17R、IL-12、C1r、C1s、C2、C3、C5、C5a、C5aR1、C6、MASPs、MSAP2、MASP3、FB、FD、 properdin 、Lag-3、CTLA-4、PD-1、PD-L1、TIM3、SIRPα、TIGIT、OX40、OX40L、4-1BB、NKG2A、NKG2B、BTLA、GITR、GITRL、TCR、Nectin-4、c-Met、LIV1、 mesothelin, DLL3, DLL4, tissue factor, TGF- β receptor, DKK1, or CLDN18.2.

14. The protein complex or fusion protein of any one of claim 1 to 13,

The immunoglobulin light chain comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the immunoglobulin heavy chain comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and

Wherein said LCDR1, LCDR2 and LCDR3 comprise sequences of SEQ ID NOS: 21-23 and/or said HCDR1, HCDR2 and HCDR3 comprise sequences of SEQ ID NOS: 24-26, or

Wherein said LCDR1, LCDR2 and LCDR3 comprise the sequences of SEQ ID NOS: 40-42 and/or said HCDR1, HCDR2 and HCDR3 comprise the sequences of SEQ ID NOS: 43-45.

15. The protein complex or fusion protein of claim 14, wherein the protein complex or fusion protein is a protein that,

The immunoglobulin light chain comprises the sequence of SEQ ID NO. 13, and/or

The immunoglobulin heavy chain comprises the sequence of SEQ ID NO. 14.

16. The protein complex or fusion protein of claim 14, wherein the protein complex or fusion protein is a protein that,

The immunoglobulin light chain comprises the sequence of SEQ ID NO. 10 or 47, and/or

The immunoglobulin heavy chain comprises the sequence of SEQ ID NO. 12 or 48 or 49.

17. The protein complex of any one of claims 2-4 and 6-16, wherein the effector comprises an antibody or antigen-binding fragment thereof, an aptamer, a ligand, a cytotoxin, a chemotherapeutic agent, a detectable label or tag, a drug, a prodrug, a toxin, an enzyme, an immunomodulator, a checkpoint inhibitor, an anti-angiogenic agent, a pro-apoptotic agent, a cytokine, a growth factor, a hormone, a cytokine, a radioisotope, a protein, a peptide, a peptidomimetic, a polynucleotide, an RNAi oligosaccharide, a natural or synthetic polymer, a nanoparticle, a quantum dot, an organic compound or an inorganic compound.

18. The protein complex of claim 17, wherein the antibody or antigen-binding fragment thereof specifically binds to a marker on an immune cell.

19. The protein complex of claim 18, wherein said antibody or antigen binding fragment thereof specifically binds to a T cell specific marker.

20. The protein complex of claim 19, wherein said T cell specific marker is CD3.

21. The protein complex of claim 20, wherein the antibody or antigen-binding fragment comprises:

(A) 15-20 of SEQ ID NO, or

(B) SEQ ID NO. 4 and 5, or

(C) One or more sequences selected from SEQ ID NOS.8, 9, 29 and 31-38.

22. The protein complex or fusion protein of any one of claims 1-21, or the antibody or antigen-binding fragment of any one of claims 17-21, further comprising a variant Fc constant region.

23. The protein complex or fusion protein of any one of claims 1-22, wherein the DDD moiety comprises the sequence of SEQ ID No. 1.

24. One or more nucleic acid sequences encoding a protein complex or fusion protein according to any one of claims 1-23.

25. An expression vector comprising one or more nucleic acid sequences according to claim 24.

26. A host cell comprising one or more nucleic acid sequences according to claim 24, or an expression vector according to claim 25.

27. A method of preparing a protein complex or fusion protein comprising:

Obtaining a cultured host cell comprising one or more nucleic acid sequences encoding a protein complex or fusion protein according to any one of claims 1-23,

Culturing the cell under conditions permitting expression of (i) the fusion protein or (ii) the protein complex, and intracellular or extracellular assembly of the protein complex, and

Purifying the protein complex or fusion protein from the cultured cells or the cell culture medium.

28. The method of claim 27, wherein the assembling is performed intracellularly.

29. A pharmaceutical composition comprising the protein complex or fusion protein of any one of claims 1-23 and a pharmaceutically acceptable carrier.

30. A method of treating cancer or a disease in a subject in need thereof, comprising administering to the subject an effective amount of the protein complex or fusion protein of any one of claims 1-23 or the pharmaceutical composition of claim 29.