CN117545505A

CN117545505A - Molecules with engineered antibody constant region variants

Info

Publication number: CN117545505A
Application number: CN202280043658.3A
Authority: CN
Inventors: S·辛格; E·池; M·凯斯; H·M·周; L·玄; J·弗曼; A·拉科姆
Original assignee: Janssen Biotech Inc
Current assignee: Janssen Biotech Inc
Priority date: 2021-04-19
Filing date: 2022-04-18
Publication date: 2024-02-09

Abstract

A binding molecule comprising (i) a first polypeptide comprising a heavy chain variable region (VH) and a region derived from a first constant region (CH 1) of an antibody heavy chain; and (ii) a second polypeptide comprising a light chain variable region (VL) and a region derived from a constant region (CL) of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops.

Description

Molecules with engineered antibody constant region variants

Cross Reference to Related Applications

The present application claims the benefit of U.S. Ser. No. 63/176,718 submitted at month 4 of 2021, U.S. Ser. No. 63/176,720 submitted at month 4 of 2021, U.S. Ser. No. 63/176,725 submitted at month 4 of 2021, U.S. Ser. No. 63/176,731 submitted at month 4 of 2021, U.S. Ser. No. 63/176,736 submitted at month 4 of 2021, each of which is incorporated herein by reference in its entirety.

Electronically submitted reference sequence listing

The present application contains a sequence listing submitted electronically via EFS-Web as an ASCII formatted sequence listing, file name "14620-683-228_SEQ_LISTING. Txt", creation date 2022, 4 months 14 days, and size 18,124 bytes. This sequence listing submitted via EFS-Web is part of this specification and is incorporated by reference herein in its entirety.

1. Technical field

Provided herein are binding molecules comprising variants of antibody constant regions capable of binding to an antigen. Also provided herein are pharmaceutical compositions or kits comprising the binding molecules, methods of making and methods of using the same.

2. Background art

The present disclosure for the first time developed improved antibody structures with additional antigen binding sites outside the VH and VL regions and uses thereof. Antibodies have been successfully used to treat a variety of diseases and conditions. Conventional antibodies recognize their antigens through VH and VL regions. Various techniques have been developed to engineer proteins to bind to targets to which they do not normally bind. In order to expand the binding capacity of conventional antibodies to a variety of antigens and thus increase the therapeutic effect of antibodies, there is a need in the art for improved antibody structures with additional antigen binding sites outside the VH and VL regions.

3. Summary of the invention

In one aspect, provided herein is a binding molecule comprising a region derived from the CH1 region of an antibody heavy chain and/or a region derived from the CL region of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops.

In one aspect, provided herein is a binding molecule comprising: (i) A first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain; and (ii) a second polypeptide comprising a light chain variable region (VL) and a region derived from the CL region of the antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops.

In some embodiments, one or more antigen binding loops in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region. In some embodiments, one or more antigen binding loops in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CL region. In some embodiments, one or more antigen binding loops in the region derived from the CH1 region are located outside the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region. In some embodiments, one or more antigen binding loops in the region derived from the CL region are located outside the AB, BC, CD, DE, EF and/or FG loop region of the CL region. In some embodiments, one or more antigen binding loops in the region derived from the CH1 region are located in the A, B, C, D, E and/or fβ chain of the CH1 region. In some embodiments, one or more antigen binding loops in the region derived from the CL region are located in the A, B, C, D, E and/or fβ chain of the CL region.

In some embodiments, the region derived from the CH1 region comprises one or two antigen binding loops. In some embodiments, the region derived from the CL region comprises one or two antigen binding loops.

In some embodiments, the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region. In some embodiments, the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region. In some embodiments, the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region.

In some embodiments, the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region. In some embodiments, the region derived from the CL region comprises one antigen binding loop at the DE loop region of the CL region. In some embodiments, the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region.

In some embodiments, the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region; and the region derived from the CL region comprises an antigen binding loop at the CD loop region of the CL region. In some embodiments, the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region; and the region derived from the CL region comprises an antigen binding loop at the DE loop region of the CL region. In some embodiments, the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region; and the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region.

In some embodiments, the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and the region derived from the CL region comprises an antigen binding loop at the CD loop region of the CL region. In some embodiments, the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and the region derived from the CL region comprises an antigen binding loop at the DE loop region of the CL region. In some embodiments, the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region.

In some embodiments, the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and the region derived from the CL region comprises an antigen binding loop at the CD loop region of the CL region. In some embodiments, the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and the region derived from the CL region comprises an antigen binding loop at the DE loop region of the CL region. In some embodiments, the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region.

In some embodiments, the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1. In some embodiments, the region derived from the CL region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the region derived from the CL region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID No. 2. In some embodiments, the region derived from the CL region is a region derived from a human CL lambda region comprising the amino acid sequence of SEQ ID No. 3, and wherein the region derived from the CL region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 3.

In some embodiments, the antigen binding loop at the CD loop region of the CH1 region replaces the amino acid residue TSG of the CD loop of the human IgG1CH1 region. In some embodiments, the antigen binding loop at the DE loop region of the CH1 region replaces the amino acid residue QSS of the DE loop of the human IgG1CH1 region. In some embodiments, the antigen binding loop at the CD loop region of the CL region replaces the amino acid residue SGNS of the CD loop of the human clk region. In some embodiments, the antigen binding loop at the DE loop region of the CL region replaces the amino acid residue SKD of the DE loop of the human clk region.

In some embodiments, each of the one or more antigen binding loops comprises 7 to 15 amino acid residues.

In some embodiments, the VH region and the VL region bind to a first antigen; and the region derived from the CH1 region and/or the region derived from the CL region binds to the second antigen. In some embodiments, the first antigen and the second antigen are the same antigen. In some embodiments, the first antigen and the second antigen are two different antigens.

In another aspect, provided herein is a nucleic acid encoding a binding molecule.

In another aspect, provided herein is a vector comprising a nucleic acid encoding a binding molecule.

In yet another aspect, provided herein is a method of making a binding molecule, the method comprising expressing in a host cell a polynucleotide encoding the binding molecule. In some embodiments, the binding molecule comprises a region derived from the CH1 region of an antibody heavy chain and/or a region derived from the CL region of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops. In some embodiments, the binding molecule comprises: (i) A first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain; and (ii) a second polypeptide comprising a light chain variable region (VL) and a region derived from the CL region of the antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops.

In yet another aspect, provided herein is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises: (a) A binding molecule comprising a region derived from the CH1 region of an antibody heavy chain and/or a region derived from the CL region of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a binding molecule comprising: (i) A first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain; and (ii) a second polypeptide comprising a light chain variable region (VL) and a region derived from a CL region of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops; and (b) a pharmaceutically acceptable excipient.

In yet another aspect, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject a binding molecule or a nucleic acid encoding a binding molecule. In some embodiments, the binding molecule comprises a region derived from the CH1 region of an antibody heavy chain and/or a region derived from the CL region of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops. In some embodiments, the binding molecule comprises: (i) A first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain; and (ii) a second polypeptide comprising a light chain variable region (VL) and a region derived from the CL region of the antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops. In some embodiments, the disease or disorder is associated with an antigen of a binding molecule.

In one aspect, provided herein is a Constant Region Library (CRL) comprising a population of binding molecules, wherein each binding molecule in the binding molecules comprises: (i) A first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain; and (ii) a second polypeptide comprising a light chain variable region (VL) and a region derived from a CL region of an antibody light chain, wherein the population of binding molecules comprises different amino acid sequences in the region derived from the CH1 region and/or the region derived from the CL region. In another aspect, provided herein is a Constant Region Library (CRL) comprising a population of molecules each comprising a region derived from a CH1 region of an antibody and/or a region derived from a CL region of an antibody, wherein the population of molecules comprises different amino acid sequences in the region derived from the CH1 region and/or the region derived from the CL region.

In some embodiments, the different amino acid sequences in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region. In some embodiments, the different amino acid sequences in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CL region. In some embodiments, the different amino acid sequences in the region derived from the CH1 region are located outside the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region. In some embodiments, the different amino acid sequences in the region derived from the CL region are located outside the AB, BC, CD, DE, EF and/or FG loop region of the CL region. In some embodiments, the different amino acid sequences in the region derived from the CH1 region are located in the A, B, C, D, E and/or fβ chain of the CH1 region. In some embodiments, the different amino acid sequences in the region derived from the CL region are located in the A, B, C, D, E and/or fβ chain of the CL region.

In some embodiments, the population of molecules comprises different amino acid sequences in one or both loop regions in the region derived from the CH1 region. In some embodiments, the population of molecules comprises different amino acid sequences in one or both loop regions in the region derived from the CL region.

In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region. In some embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region. In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region.

In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CL region. In some embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CL region. In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region of the CL region. In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region. In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

In some embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region of the CL region. In some embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region. In some embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region and the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region of the CL region. In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region. In some embodiments, the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

In some embodiments, the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1. In some embodiments, wherein the region derived from the CL region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the region derived from the CL region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 2. In some embodiments, wherein the region derived from the CL region is a region derived from a human CL lambda region comprising the amino acid sequence of SEQ ID No. 3, and wherein the region derived from the CL region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 3.

In some embodiments, the amino acid residue TSG of the CD loop of the human IgG1 CH1 region is replaced by a different amino acid sequence in the molecule in the CRL. In some embodiments, the amino acid residue QSS of the DE loop of the human IgG1 CH1 region is replaced by a different amino acid sequence in the molecule in the CRL. In some embodiments, the amino acid residue SGNS of the CD loop of the human clk region is replaced by a different amino acid sequence in the molecule in the CRL. In some embodiments, the amino acid residue SKD of the DE loop of the human clk region is replaced by a different amino acid sequence in the molecule in the CRL.

In some embodiments, the different amino acid sequences comprise 7 to 15 amino acid residues. In some embodiments, each of the molecules further comprises a VH region and a VL region.

In some embodiments, the binding molecule or the molecule is a Fab fragment.

In some embodiments, the CRL having one loop region has a diversity ranging from 10 ⁷ To 10 ¹⁶ . In some embodiments, the CRL with two loop regions has a diversity in the range of 10 ¹⁸ To 10 ³³ 。

In another aspect, provided herein is a method for identifying a binding molecule comprising a first binding domain that binds a first antigen and a second binding domain that binds a second antigen, the method comprising screening CRLs to identify a binding molecule that binds a second antigen with an affinity that is higher than a reference level, wherein the first binding domain comprises a VH region and a VL region of an antibody, and wherein the second binding domain comprises an antibody constant region variant. In another aspect, provided herein is a method of producing a binding molecule, the method comprising: a first step for performing a function of identifying antibody constant region variants capable of binding to an antigen; and a second step of constructing a binding molecule comprising the antibody constant region variant. In some embodiments, the first step comprises screening the CRL. In yet another aspect, provided herein is a binding molecule produced according to the methods disclosed herein.

4. Description of the drawings

FIG. 1A shows an exemplary binding molecule comprising constant region variants provided herein. FIGS. 1B and 1C depict the construction of an exemplary Fab Constant Region Library (CRL). FIG. 1B shows the positions and original sequences of the CD and DE loops located within human CL kappa and human IgG1 CH 1. One or more of the positions highlighted in fig. 1B are replaced with a diverse library of new binding molecules for selection against the target of interest. Fig. 1C shows the different compositions and theoretical diversity of binding loops of Fab CRLs.

FIGS. 2A, 2B and 2C depict the selection of Fab constant region conjugates of anti-polyhistidine monoclonal antibodies. Specifically, figure 2A depicts the results of a polyclonal phage ELISA with anti-polyhistidine monoclonal antibody binding of three pools after one to six rounds of panning; FIG. 2B depicts the results of XO1B 1-bound polyclonal phage ELISA in three pools after one to six rounds of panning; figure 2C depicts the amino acid sequence of the binding loops of nine clones after four rounds of panning.

FIG. 3 depicts the overall process of selecting Fab constant region conjugates of mEphA2-Fc from Fab CRL.

FIG. 4A depicts the results of a mEphA2-Fc binding polyclonal phage ELISA of five enrichment pools after four to eight rounds of panning. The positive control for the mhha 2-Fc binding was an anti-mhha 2-Fc CH2 domain-phage fusion and the negative control was an anti-XO 1B1 parent Fab-phage fusion. The signal relative to the parental Fab was calculated as: (mEphA 2-Fc RLU of the panning pool)/(mEphA 2-Fc RLU of the parent Fab). Fig. 4B and 4C depict the results of monoclonal phage ELISA of 378 clones from five pools of enrichment, after six to eight rounds of panning, for binding of mhha 2-Fc (fig. 4B) and for binding of XO1B1 (fig. 4C). FIG. 4D depicts the results of a monoclonal phage ELISA of both mEphA2-Fc binding and XO1B1 binding of sixteen clones selected for further analysis by Sanger sequencing, all from P8 after six rounds of panning. FIG. 4E depicts a single amino acid sequence derived from a CH1 CD loop library, which was identified from all sixteen clones selected from P8.

FIG. 4F depicts size exclusion high performance liquid chromatography (SE-HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization of single Fab constant region conjugates isolated from mammalian expression in HEK Expi293 cells using single step Immobilized Metal Affinity Chromatography (IMAC) purification. Fig. 4F also depicts the determination of molecular weight of EPAXB1 using mass spectrometry. FIG. 4G depicts the binding kinetics and affinity of EPAXB1 for both mEphA2-Fc and XO1B1 using Surface Plasmon Resonance (SPR) analysis. Fig. 4G also depicts the binding kinetics and affinity of the anti-XO 1B1 parent Fab lacking an engineered CRL for both the mhha 2-Fc and XO1B 1.

FIG. 5 depicts novel fab constant region conjugates (EPAXB 17, EPAXB27 and EPAXB 28) identified from a subset of the panning pool by NGS and SPR.

FIG. 6A depicts pairing of the constant region of EPAXB1 with a new variable region and reformatting EPAXB1 into a monoclonal antibody. Fig. 6B depicts size exclusion high performance liquid chromatography (SE-HPLC) characterization of reformatted purified Fab and monoclonal antibodies. Fig. 6C depicts that reformatted bispecific Fab and monoclonal antibodies retain binding to their corresponding targets.

FIG. 7 depicts the purification yields of anti-IL 23R x anti-EphA 2 and anti-HER 2 x anti-EphA 2 Fab reformatted into standard monoclonal antibody form as observed by SE-HPLC.

Fig. 8A-8D depict binding data for HER2 x EphA2 and IL23 x EphA2 bispecific antibodies and corresponding monospecific control antibodies to untransfected HEK cells (fig. 8A), HEK cells stably expressing human HER2 (fig. 8B), HEK cells stably expressing human EphA2 (fig. 8C), and HEK cells stably expressing human IL23R (fig. 8D).

Fig. 9A-9C depict simultaneous binding of anti-HER 2 x anti-EphA 2 and anti-IL 23R x anti-EphA 2 bispecific Fab (fig. 9A and 9B, respectively) and anti-HER 2 x anti-EphA 2 bispecific in mAb form to their respective targets by Biological Layer Interferometry (BLI).

5. Detailed description of the preferred embodiments

The present disclosure is based in part on the surprising discovery that the CH1 and/or CL regions of a Fab can be modified such that the Fab can bind to a desired second antigen. Such modification is achieved by replacing certain amino acids originally present at the surface of the CH1 and/or CL regions with a diverse amino acid that can be selected to bind to the target of interest. Such modification is also achieved by introducing additional diversified amino acids at the surface of the CH1 and/or CL regions that can be selected to bind to the target of interest.

5.1 definition

Techniques and procedures described or referenced herein include those generally known to those skilled in the art and/or commonly employed using conventional methods, such as, for example, the widely used methods described in the following documents:Molecular Cloning:A Laboratory Manual(Sambrook et al, 3 rd edition, 2001);Current Protocols in Molecular Biology(Ausubel et al, 2003);Therapeutic Monoclonal Antibodies:From Bench to Clinic(An edit, 2009);Monoclonal Antibodies:Methods and Protocols(Albitar editing, 2010); andAntibody Engineeringvolumes 1 and 2 (Kontermann and Dubel editions, 2 nd edition, 2010).

Unless defined otherwise herein, technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. For the purposes of explaining the present specification, the following description of terms will be applied, and terms used in the singular will also include the plural, and vice versa, as appropriate. In the event that any description of an illustrated term conflicts with any document incorporated by reference, the description of the term set forth below will govern.

The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense and specifically cover, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions having multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, and multi-specific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), as described below. Antibodies can be human, humanized, chimeric and/or affinity matured, as well as antibodies from other species such as mice and rabbits, and the like. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin polypeptide class that are capable of binding to a particular molecular antigen and are composed of two pairs of identical polypeptide chains, wherein each pair has one heavy chain (about 50kDa-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain comprises a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain comprises a constant region. See for example, Antibody Engineering(Borrebaeck edition, 2 nd edition, 1995); and the sum of the values of Kuby,Immunology(3 rd edition, 1997). In particular embodiments, specific molecular antigens may be bound by antibodies provided herein, including polypeptides or epitopes. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, alpaca antibodies or humanized variants thereof, intracellular antibodies, and anti-unique (anti-Id) antibodies. As used herein, the term "antibody" also includes any binding molecule having an Fc region and a functional fragment of any of the above (e.g., an antigen binding fragment), which refers to a portion of an antibody heavy or light chain polypeptide and retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single chain Fv (scFv) (e.g., comprising monospecific, bispecific, etc.)) Fab fragment, F (ab') fragment, F (ab) ₂ Fragments, F (ab') ₂ Fragments, disulfide-linked Fv (dsFv), fd fragments, fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules (e.g., one or more CDRs of an antibody) that contain an antigen binding site that binds an antigen. Such antibody fragments can be found in, for example, harlow and Lane, Antibodies:A Laboratory Manual(1989)；Mol.Biology and Biotechnology:A Comprehensive Desk Reference(Myers editions, 1995); huston et al, 1993,Cell Biophysics22:189-224; pluckthun and Skerra,1989, meth. Enzymol.178:497-515; and the group consisting of Day,Advanced Immunochemistry(2 nd edition, 1990). Antibodies provided herein can be of any class (e.g., igG, igE, igM, igD and IgA) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) of immunoglobulin molecules. The antibody may be an agonistic antibody or an antagonistic antibody.

An "antigen" is a structure to which an antibody selectively binds. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. In some embodiments of each or any of the above or below embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with the cell, e.g., present on or in the cell.

An "intact" antibody is an antibody comprising an antigen binding site and constant domains (CL) and at least heavy chain constant regions CH1, CH2 and CH 3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, the intact antibody has one or more effector functions.

The term "binding" or "binding" refers to interactions between molecules, including, for example, the formation of complexes. The interactions may be, for example, non-covalent interactions including hydrogen bonding, ionic bonding, hydrophobic interactions, and/or van der Waals interactions. The complex may also include a covalent bond or non-covalent bond, an interaction or a force maintained in one Binding of two or more molecules. The strength of the total non-covalent interaction between a single antigen binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. Dissociation rate (k) of binding molecules (e.g., antibodies) from monovalent antigens _off ) With association rate (k) _on ) Ratio (k) _off /k _on ) Is dissociation constant K _D Which is inversely proportional to the affinity. K (K) _D The lower the value, the higher the affinity of the antibody. K (K) _D The values vary with different complexes of antibody and antigen, and depend on k _on And k _off Both of which are located in the same plane. Dissociation constant K of the antibodies provided herein _D May be determined using any of the methods provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen (such as a multivalent antigen) containing multiple repeat epitopes is contacted with an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at the second site. The strength of this multiple interaction between multivalent antibody and antigen is referred to as avidity.

Terms related to antibodies described herein, such as "bind," "specifically bind," and similar terms, are also used interchangeably herein and refer to antibodies that specifically bind to an antigen, such as an antigen binding domain of a polypeptide. Antibodies or antigen binding domains that bind or specifically bind to an antigen may cross-react with the associated antigen. In certain embodiments, an antibody or antigen binding domain that binds or specifically binds an antigen does not cross-react with other antigens. Antibodies or antigen binding domains that bind or specifically bind to an antigen can be detected, for example, by an immunoassay,Or other techniques known to those skilled in the art. In some embodiments of each or any of the above or below embodiments, e.g., using experimental techniques such as Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (elisa)Immunoadsorption assays (ELISA) determine that an antibody or antigen binding domain binds or specifically binds to an antigen when it binds to the antigen with higher affinity than any cross-reactive antigen. Typically, the specific or selective response will be at least twice the background signal or noise, and may be more than 10 times the background. See, for example Fundamental Immunology332-36 (Paul et al, 2 nd edition, 1989) in connection with the discussion of binding specificity. In certain embodiments, the extent of binding of an antibody or antigen binding domain to a "non-target" protein is less than about 10% of the binding of the antibody or antigen binding domain to its particular target antigen, e.g., as determined by Fluorescence Activated Cell Sorting (FACS) analysis or RIA. Terms such as "specifically bind," "specifically bind," or "specifically binds," mean binding that is significantly different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a similarly structured molecule that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., excess unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. An antibody or antigen binding domain that binds an antigen includes an antibody or antigen binding domain that is capable of binding an antigen with sufficient affinity such that the antibody can be used, for example, as a diagnostic or therapeutic agent for targeting the antigen. In certain embodiments, the antibody or antigen binding domain that binds to an antigen has a dissociation constant (K) of less than or equal to 1000nM, 800nM, 500nM, 250nM, 100nM, 50nM, 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM or 0.1nM _D ). In some embodiments of each or any of the above or below embodiments, the antibody or antigen binding domain that binds to the antigen has an dissociation constant (K _D ) Or have any of the aboveWhat two K _D Any K between the ranges defined by the values _D . In certain embodiments, the antibody or antigen binding domain binds to an epitope that is conserved among antigens from different species (e.g., between human and cynomolgus macaque species).

"binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as 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 members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y can generally be determined by the dissociation constant (K _D ) And (3) representing. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies typically bind antigen slowly and tend to dissociate easily, while high affinity antibodies typically bind antigen faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which may be used for the purposes of this disclosure. Specific exemplary embodiments include the following. In one embodiment, "K _D "or" K _D The value "may be measured by assays known in the art, for example by binding assays. K (K) _D Measurements can be made in RIA, for example, with Fab versions of the antibody of interest and its antigen (Chen et al, J.mol Biol,1999, 293:865-81). K (K) _D Or K _D The values may also be determined by using Biological Layer Interferometry (BLI) or Surface Plasmon Resonance (SPR) assaysUsing for example +.>Red96 System or->Red384 system, or by +.>Using for example +.>2000 or->3000 to make measurements. "binding Rate" or "rate of association" or "association Rate" or "k _on "the same Biological Layer Interferometry (BLI) or Surface Plasmon Resonance (SPR) techniques as described above can also be used, for example +.>Red96、/>2000、/>3000 System or->8000 system.

In certain embodiments, antibodies may comprise "chimeric" sequences in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA,1984, 81:6851-55).

In certain embodiments, antibodies may comprise portions of a "humanized" form of a non-human (e.g., murine) antibody that is a chimeric antibody and includes a human immunoglobulin (e.g., recipient antibody) in which natural CDR residues are replaced with corresponding CDR residues of desired specificity, affinity, and capacity from a non-human species such as a mouse, rat, rabbit, or non-human primate (e.g., donor antibody). In some cases, one or more FR region residues of a human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may comprise residues that are not present in the recipient antibody or the donor antibody. These modifications were made to further improve antibody performance. The humanized antibody heavy or light chain may comprise one or more variable regions in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones et al, nature,1986, 321:522-25; riechmann et al Nature,1988, 332:323-29; presta, curr.Op.struct.biol.,1992,2:593-96; carter et al, proc.Natl.Acad.Sci.USA,1992, 89:4285-89; us patent 6,800,738;6,719,971;6,639,055;6,407,213; and 6,054,297.

In certain embodiments, an antibody may comprise a "fully human antibody" or a portion of a "human antibody," wherein these terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. In particular embodiments, these terms refer to antibodies comprising variable and constant regions of human origin. In certain embodiments, "fully human" antibodies may also encompass antibodies that bind to polypeptides and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al (see Kabat et al (1991)Sequences of Proteins of Immunological InterestFifth edition, U.S. Pat. No. of Health and Human Services, NIH publication No. 91-3242). "human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and/or that has been prepared using any of the techniques used to prepare human antibodies. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol. Biol.,1991, 227:381; marks et al, 1991, J.mol. Biol.,1991, 222:581) and yeast display libraries (Chao et al, nature Protocols,2006, 1:755-68). Methods that can also be used to prepare human monoclonal antibodies are described in Cole et al, Monoclonal Antibodies and Cancer Therapy77 (1985); boerner et al, J.Immunol.,1991, 147 (1): 86-95; and van Dijk and van de Winkel, curr. Opin. Pharmacol.,2001,5:368-74. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, such as mice (see, e.g., jakobovits, curr. Opin. Biotechnol.,1995,6 (5): 561-66; bruggemann and Taussing, curr. Opin. Biotechnol.,1997,8 (4): 455-58; and in relation to XENOMOUSE se ^TM U.S. Pat. nos. 6,075,181 and 6,150,584 to the technology). See also, e.g., li et al, proc.Natl.Acad.Sci.USA,2006, 103:3557-62 for human antibodies generated via human B cell hybridoma technology.

In certain embodiments, the antibodies may comprise portions of a "recombinant human antibody," wherein the phrase includes human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, antibodies isolated from transgenic and/or transchromosomal animals of human immunoglobulin genes (e.g., mice or cattle) (see, e.g., taylor, l.d. et al, nucleic acids res.,1992, 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means that involves splicing a human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al (1991) Sequences of Proteins of Immunological InterestFifth edition, U.S. Pat. No. of Health and Human Services, NIH publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis(or, when using transgenic animals directed against human Ig sequences, in vivo somatic mutagenesis is performed), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally occur within the human antibody germline repertoire in vivo.

In certain embodiments, an antibody may comprise a portion of a "monoclonal antibody," wherein the term as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on the antigen. In particular embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for producing antibodies. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method described first by Kohler et al, 1975,Nature 256:495, or may be prepared using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al, nature,1991, 352:624-28 and Marks et al, J.mol.biol.,1991, 222:581-97. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art. See, for example Short Protocols in Molecular Biology(Ausubel et al, edition 5, 2002).

Typical 4-chain antibody units are heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) of each of the alpha and gamma chains and mu and epsilon isoformsFour CH domains. Each L chain has a variable domain (VL) at the N-terminus and then a constant domain (CL) at its other end. VL is aligned with VH, and CL is aligned with the first constant domain of the heavy chain (CH 1). It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains. Pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of antibodies of different classes, see e.gBasic and Clinical Immunology71 (Stites et al, edition, 8 th edition, 1994); andImmunobiology(Janeway et al, edition 5, 2001).

The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgG typically comprises two Fab regions, each residing on one of the two arms of a Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region for each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are VL and CL regions. VH, CH1, VL and CL in the Fab region may be arranged in various ways to confer antigen binding ability according to the present disclosure. For example, the VH and CH1 regions may be on one polypeptide, and the VL and CL regions may be on separate polypeptides, similar to the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL and CL regions may all be on the same polypeptide and oriented in different sequences, as described in more detail in the following section.

The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody's light or heavy chain that is typically located at the amino terminus of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is used for binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain fragments of the variable region vary widely in sequence from antibody to antibody. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acid span of the variable region.In contrast, the V region consists of an extension of about 15-30 amino acids called the Framework Region (FR) that is less variable (e.g., relatively constant), separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions", each about 9-12 amino acids long. The heavy and light chain variable regions each comprise four FR, principally in the β -sheet configuration, joined by three hypervariable regions, which form loops connecting the β -sheet structure and in some cases form part thereof. The hypervariable regions in each chain are held together by the FR and in close proximity to the hypervariable regions from the other chain, facilitating the formation of the antigen binding site of the antibody (see, e.g., kabat et al, Sequences of Proteins of Immunological Interest(5 th edition, 1991)). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits various effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence between different antibodies. In a specific embodiment, the variable region is a human variable region.

The term "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat" and variants thereof refers to the numbering system of the heavy chain variable region or the light chain variable region used in antibody compilation in Kabat et al (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion following residue 52 (residue 52a according to Kabat) and three insertion residues following residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat residue number of a given antibody can be determined by alignment of the homologous region of the antibody sequence with a "standard" Kabat numbering sequence. When referring to residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., kabat et al, supra). When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al, supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described by, for example, abM, chothia, contact, IMGT and AHon.

When used with reference to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50-70kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and the carboxy-terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isoforms), referred to as α (α), δ (δ), ε (ε), γ (γ), and μ (μ). Different heavy chains vary in size: alpha, delta and gamma contain about 450 amino acids, while mu and epsilon contain about 550 amino acids. When combined with light chains, these different types of heavy chains produce five well-known classes (e.g., isotypes) of antibodies, igA, igD, igE, igG and IgM, respectively, including the four subclasses of IgG, namely IgG1, igG2, igG3, and IgG4.

When used with reference to an antibody, the term "light chain" refers to a polypeptide chain of about 25kDa, wherein the amino-terminal portion comprises a variable region of about 100 to about 110 amino acids and the carboxy-terminal portion comprises a constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (kappa) or lambda (lambda).

As used herein, the terms "hypervariable region," "HVR," "complementarity determining region," and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH beta-sheet framework or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of an antibody VL beta-sheet framework. Thus, CDRs are variable region sequences interspersed with framework region sequences.

CDR regions are well known to those skilled in the art and have been defined by the well known numbering system. For example, kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (see, e.g., kabat et al, supra). Chothia conversely refers to the position of the structural loop (see, e.g., chothia and Lesk, J.mol.biol.,1987, 196:901-17). Chothia CD when numbered using the Kabat numbering conventionThe ends of the R-H1 loop vary between H32 and H34 depending on the length of the loop (since the Kabat numbering scheme will insert at H35A and H35B; loop ends at 32 if neither 35A nor 35B is present; loop ends at 33 if only 35A is present; loop ends at 34 if both 35A and 35B are present). The AbM hypervariable region represents a compromise between Kabat CDRs and Chothia structural loops and is used by Oxford Molecular AbM antibody modeling software (see, e.g.Antibody EngineeringVolume 2 (Kontermann and Dubel editions, 2 nd edition, 2010). The "contact" hypervariable region is based on analysis of the complex crystal structure available. Another common numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information(Lafranc et al, dev. Comp. Immunol.,2003, 27 (1): 55-77). IMGT is an integrated information system specifically studying Immunoglobulins (IG), T Cell Receptors (TCR) and Major Histocompatibility Complex (MHC) of humans and other vertebrates. Herein, CDRs are referenced according to both amino acid sequences and positions within the light chain or heavy chain. Since the "position" of CDRs within an immunoglobulin variable domain structure is conserved between species and exists in a structure called a loop, CDRs and framework residues are easily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from immunoglobulins of one species into the acceptor framework typically from human antibodies. Honyger and Pluckthun developed an additional numbering system (AHon), J.mol.biol.,2001, 309:657-70). Correspondence between numbering systems, including, for example, the Kabat numbering and IMGT unique numbering systems, are well known to those skilled in the art (see, for example, kabat, supra; chothia and Lesk, supra; martin, supra; lefranc et al, supra). Residues from each of these hypervariable regions or CDRs are indicated below.

TABLE 1.

The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise indicated, the terms "CDR" and "complementarity determining region" for a given antibody or region thereof (such as a variable region) are to be understood as encompassing complementarity determining regions as defined by any known scheme as described above for the individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or region thereof. In some cases, a scheme for identifying a particular CDR or CDRs is specified, such as the CDRs defined by the Kabat, chothia or Contact methods. In other cases, specific amino acid sequences of CDRs are given.

The hypervariable region may comprise the following "extended hypervariable region": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH.

The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding an antibody to an antigen, but that exhibit various effector functions, such as interactions with Fc receptors. The term refers to that portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to other portions of the immunoglobulin (variable regions comprising antigen binding sites). The constant region may comprise the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

The term "framework" or "FR" refers to those variable region residues flanking the CDRs. FR residues are present in, for example, chimeric, humanized, human domain antibodies, diabodies, linear antibodies and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues. Four FR regions are typically present in each of the VH and VL regions. The FR regions in VH are VH FR1, VH FR2, VH FR3 and VH FR4 (or FR H1, FR H2, FR H3 and FR H4). The FR regions in VL are VL FR1, VL FR2, VL FR3 and VL FR4 (or FR L1, FR L2, FRL3 and FRL 4).

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody or by recombinant engineering of nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibodies may include a population of antibodies that have all K447 residues removed, a population of antibodies that have no K447 residues removed, and a population of antibodies that have a mixture of antibodies with and without K447 residues. The "functional Fc region" has the "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions typically require the Fc region in combination with a binding region or binding domain (e.g., an antibody variable region or domain), and can be assessed using various assays known to those of skill in the art. A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, e.g., about one to about ten amino acid substitutions, or about one to about five amino acid substitutions, in the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc-regions herein may have at least about 80% homology with the native sequence Fc-region and/or the Fc-region of the parent polypeptide, or at least about 90% homology therewith, e.g., at least about 95% homology therewith.

The term "loop region" refers to the structural loop linking the β -strand, which in turn constitutes the β -sheet, a common motif of the secondary structure of conventional proteins. It is well known to those skilled in the art that the beta chain is labeled with letters in turn (A, B, C, D, E, F, etc.) in the order in which it appears in the primary amino acid sequence of the protein domain. Structural loops are labeled according to the β -strand to which they are attached. For example, the AB ring refers to the structural ring connecting β chains a and B; the BC ring refers to the structural ring connecting β chains B and C; CD ring refers to the structural ring linking the β chains C and D; DE ring refers to the structural ring connecting β chains D and E; etc.

The term "antigen binding loop" refers to a region of a polypeptide that specifically binds to an antigen. In some cases, the antigen binding loop is a CDR located within the VH and/or VL region. In other cases, the antigen binding loop is a polypeptide that is outside of the VH and/or VL regions. In some embodiments, the antigen binding loops provided herein are located in the CH1 region. In some embodiments, the antigen binding loops provided herein are located in the CL region. In some embodiments, the antigen binding loop is located in both the CH1 region and the CL region. In some embodiments, the antigen binding loop is located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region. In some embodiments, the antigen binding loop is located in the AB, BC, CD, DE, EF and/or FG loop region of the CL region. In some embodiments, the antigen binding loop is located outside the AB, BC, CD, DE, EF and/or FG loop regions of the CH1 region. In some embodiments, the antigen binding loop is located outside the AB, BC, CD, DE, EF and/or FG loop regions of the CL region. In some embodiments, the antigen binding loop is located in the A, B, C, D, E and/or fβ chain of the CL region. In some embodiments, the antigen binding loop is located in the A, B, C, D, E and/or fβ chain of the CH1 region.

The term "variant" when used in relation to an antigen or antibody may refer to a peptide or polypeptide comprising one or more (such as, for example, from about 1 to about 25, from about 1 to about 20, from about 1 to about 15, from about 1 to about 10, or from about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to the native or unmodified sequence.

The term "identity" refers to the relationship between sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by aligning and comparing sequences. "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, without regard to any conservative substitutions as part of the sequence identity after aligning the sequences and introducing gaps (if desired) to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megasign (DNAStar, inc.) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences.

"modification" of an amino acid residue/position refers to a change in the primary amino acid sequence as compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue/position. For example, typical modifications include substitution of a residue with another amino acid (e.g., conservative or non-conservative), insertion of one or more (e.g., typically less than 5, 4, or 3) amino acids adjacent to the residue/position, and/or deletion of the residue/position.

As used herein, an "epitope" is a term of art and refers to a localized region of an antigen to which an antibody can specifically bind. The epitope may be a linear epitope or a conformational, non-linear or discontinuous epitope. In the case of polypeptide antigens, for example, an epitope may be a contiguous amino acid of a polypeptide ("linear" epitope), or an epitope may comprise amino acids from two or more non-contiguous regions of a polypeptide ("conformational", "non-linear" or "discontinuous" epitope). Those skilled in the art will appreciate that in general, linear epitopes may or may not depend on secondary, tertiary or quaternary structure. For example, in some embodiments, an antibody binds to a set of amino acids, whether or not these amino acids are folded in the native three-dimensional protein structure. In some embodiments of each or any of the above or below embodiments, the antibody requires that the amino acid residues comprising the epitope exhibit a particular conformation (e.g., bending, twisting, flipping, or folding) in order to recognize and bind the epitope.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interspersed with non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes, for example, polypeptides that contain one or more amino acid analogs (including but not limited to unnatural amino acids) as well as other modifications known in the art. It will be appreciated that because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a "polypeptide" may appear as a single chain or two or more related chains.

The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid sequence encoding an antibody as described herein, in order to introduce the nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selection sequences or markers operable for stable integration into a host cell chromosome. In addition, the vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes may be included, for example, to provide resistance to antibiotics or toxins, to supplement auxotrophs, or to provide key nutrients that are not in the medium. Expression control sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, and the like, as are well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., antibody heavy and light chains or both antibody VH and VL), both nucleic acid molecules may be inserted into, for example, a single expression vector or into separate expression vectors. For single vector expression, the coding nucleic acids are operably linked to one common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of a nucleic acid molecule into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or Polymerase Chain Reaction (PCR) amplification of mRNA, immunoblot for expression of gene products, or other suitable analytical methods to test expression of the introduced nucleic acid sequences or their corresponding gene products. It will be appreciated by those skilled in the art that the nucleic acid molecules are expressed in amounts sufficient to produce the desired product, and that the expression levels may be optimized to obtain adequate expression using methods well known in the art.

As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

As used herein, the term "host cell" refers to a particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may differ from the parent cell transfected with the nucleic acid molecule, either due to mutations or environmental effects that may occur in the progeny or due to integration of the nucleic acid molecule into the host cell genome.

An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or a mixture of nucleic acids, that is substantially isolated from other genomic DNA sequences naturally accompanying the native sequence, and proteins or complexes such as ribosomes and polymerases. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term encompasses nucleic acid sequences that have been removed from their naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs that are biosynthesized by heterologous systems. A substantially pure molecule may include an isolated form of the molecule.

"Polynucleotide", "nucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base and/or analogue thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. As used herein, "oligonucleotide" refers to a short, usually single stranded, synthetic polynucleotide, typically, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides applies equally and entirely to oligonucleotides. Cells producing antibodies of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Unless otherwise indicated, the left end of any single stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand orientation of the double-stranded polynucleotide sequence is referred to as the 5' orientation. The 5 'to 3' addition direction of nascent RNA transcripts is referred to as the transcription direction; a region of sequence on the DNA strand having the same sequence as the RNA transcript, 5 'to 5' of the RNA transcript, referred to as the "upstream sequence"; the DNA strand has a sequence region of the same sequence as the RNA transcript, which is at the 3 'to 3' end of the RNA transcript, referred to as the "downstream sequence".

As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of antigen-binding sites, wherein a first antigen-binding site of the plurality of antigen-binding sites has binding specificity for a first epitope and a second antigen-binding site of the plurality of antigen-binding sites has binding specificity for a second epitope. In some embodiments of each or any of the above or below embodiments, the first epitope and the second epitope do not overlap or substantially do not overlap. In some embodiments of each or any of the above or below embodiments, the first epitope and the second epitope are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments of each or any of the above or below embodiments, the multispecific antibody comprises a third, fourth, or fifth antigen-binding site. In some embodiments of each or any of the embodiments above or below, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or two antigens. Bispecific antibodies are characterized by a first antigen binding site having binding specificity for a first epitope and a second antigen binding site having binding specificity for a second epitope. In some embodiments of each or any of the above or below embodiments, the first epitope and the second epitope are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments of each or any of the embodiments above or below, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In some embodiments of each or any of the embodiments above or below, the bispecific antibody comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a region derived from the CH1 region of an antibody heavy chain and/or a region derived from the CL region of an antibody light chain having binding specificity for a second epitope.

As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or used in animals, and more particularly in humansUnited states pharmacopoeia、European pharmacopoeiaOr other recognized pharmacopoeias.

By "excipient" is meant a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coatings, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizing agents, wetting agents and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., freund's adjuvant (complete or incomplete)), or vehicle.

In some embodiments of each or any of the above or below embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid; a low molecular weight (e.g., less than about 10 amino acid residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, e.g. TWEEN ^TM Polyethylene glycol (PEG) and PLURONICS ^TM . Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro,Remington's Pharmaceutical Sciences(18 th edition, 1990).

In some embodiments of each or any of the embodiments described above or below, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for contact with tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit/risk ratio. See, e.g., lippincott Williams & Wilkins: philiadelphia, PA,2005; handbook of Pharmaceutical Excipients, 6 th edition; rowe et al; the Pharmaceutical Press and the American Pharmaceutical Association:2009; handbook of Pharmaceutical Additives, 3 rd edition; ash and Ash editing; gower Publishing Company:2007; pharmaceutical Preformulation and Formulation, version 2; editing Gibson; CRC Press LLC, boca Raton, FL,2009. In some embodiments of each or any of the above or below embodiments, the pharmaceutically acceptable excipient is non-toxic to the cells or mammals exposed thereto at the dosage and concentration employed. In some embodiments of each or any of the above or below embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffer solution.

In some embodiments of each or any of the embodiments above or below, the excipient is a sterile liquid, such as water and oil, including those of petroleum, animal, vegetable or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary excipient. Saline and dextrose in water and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition can be in the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation, etc. Oral compositions, including formulations, may contain standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

For example, a composition comprising a pharmaceutical compound may contain an antibody, e.g., in isolated or purified form, together with a suitable amount of excipient.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an antibody or pharmaceutical composition provided herein sufficient to produce a desired result.

The terms "subject" and "patient" are used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In particular embodiments, the subject is a human. In some embodiments of each or any of the above or below embodiments, the subject is a mammal, e.g., a human, diagnosed as having a condition or disorder. In some embodiments of each or any of the above or below embodiments, the subject is a mammal, e.g., a human, at risk of suffering from a condition or disorder.

"administration" refers to the act of injecting or otherwise physically delivering a substance present in vitro into a patient, such as by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery, and/or any other physical delivery method described herein or known in the art.

As used herein, the term "treatment" refers to a reduction or improvement in the progression, severity, and/or duration of a disease or disorder caused by administration of one or more therapies. Treatment may be determined by assessing whether there has been a reduction, alleviation and/or relief of one or more symptoms associated with the underlying disorder such that an improvement in the patient is observed despite the patient possibly still being afflicted with the underlying disorder. The term "treatment" includes management and amelioration of the disease. The term "management" (manage, managing and management) refers to the beneficial effect a subject obtains from a therapy that does not necessarily result in cure of a disease.

The term "preventing" (prevention, preventing and presntation) refers to reducing the likelihood of a disease, disorder, condition, or associated symptom onset (or recurrence).

The terms "about" and "approximately" mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a given value or range.

As used in this disclosure and the claims, the singular forms "a," "an," and "the" include plural forms, unless the context clearly dictates otherwise.

It should be understood that wherever embodiments are described herein with the term "comprising," other similar embodiments described in terms of "consisting of … …" and/or "consisting essentially of … …" are also provided. It should also be understood that wherever embodiments are described herein with the phrase "consisting essentially of, other similar embodiments described in terms of" consisting of, … … "are also provided.

The term "between" as used in phrases such as "between a and B" or "between a-B" refers to a range that includes both a and B.

The term "and/or" as used in phrases such as "a and/or B" herein is intended to include both a and B; a or B; a (alone); and B (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

5.2 binding molecules

The binding molecules provided herein comprise at least one engineered antibody constant region variant (e.g., a CH1 region variant and/or a CL region variant), wherein the constant region variant comprises one or more antigen binding loops, so that the constant region variant in the molecules of the invention imparts antigen binding capacity. In some embodiments, the antigen binding loop in a constant region variant provided herein is located in one of the loop regions (e.g., CH1 region or CL region) in the antibody constant region. "loop region" of an antibody constant region refers to the structural loop that connects the β -strands (which in turn constitute the β -sheet, a common motif of the conventional protein secondary structure). It is well known to those skilled in the art that the beta chain is labeled with letters in turn (A, B, C, D, E, F, etc.) in the order in which it appears in the primary amino acid sequence of the protein domain. Structural loops are labeled according to the β -strand to which they are attached. For example, the AB ring refers to the structural ring connecting β chains a and B; the BC ring refers to the structural ring connecting β chains B and C; CD ring refers to the structural ring linking the β chains C and D; DE ring refers to the structural ring connecting β chains D and E; the EF ring refers to the structural ring connecting the beta strands E and F; FG ring refers to the structural ring connecting β chains F and G. Typical CH1 and CL regions contain seven beta-strands-A, B, C, D, E, F and G, and six loop regions-AB, BC, CD, DE, EF and FG loops (e.g., as shown in FIG. 1B).

In some embodiments, one or more antigen binding loops are introduced into and/or replace amino acid residues within the AB, BC, CD, DE, EF and/or FG loop regions of the CH1 region. In some embodiments, one or more antigen binding loops are introduced into and/or replace amino acid residues within the AB, BC, CD, DE, EF and/or FG loop regions of the CL region. In some embodiments, one or more antigen binding loops are introduced into and/or replace amino acid residues outside of the AB, BC, CD, DE, EF and/or FG loop regions of the CH1 region. In some embodiments, one or more antigen binding loops are introduced into and/or replace amino acid residues outside of the AB, BC, CD, DE, EF and/or FG loop regions of the CL region. In some embodiments, one or more antigen binding loops are introduced into and/or replace amino acid residues within the A, B, C, D, E and/or fβ chain region of the CH1 region. In some embodiments, one or more antigen binding loops are introduced into and/or replace amino acid residues within A, B, C, D, E and/or fβ chain regions of the CL region.

In some embodiments, the antigen binding loops provided herein can be inserted into the loop region. The antigen binding loop may be inserted into the AB loop region of the CH1 or CL region. The antigen binding loop may be inserted into the BC loop region of the CH1 or CL region. The antigen binding loop may be inserted into the CD loop region of the CH1 or CL region. The antigen binding loop may be inserted into the DE loop region of the CH1 or CL region. The antigen binding loop may be inserted into the EF loop region of the CH1 or CL region. The antigen binding loop may be inserted into the FG loop region of the CH1 or CL region.

In other embodiments, the antigen binding loops provided herein can replace a region within the loop region. The antigen binding loop may replace a region within the AB loop region of the CH1 or CL region. The antigen binding loop may replace a region within the BC loop region of the CH1 or CL region. The antigen binding loop may replace a region within the CD loop region of the CH1 or CL region. The antigen binding loop may replace a region within the DE loop region of the CH1 or CL region. The antigen binding loop may replace a region within the EF loop region of the CH1 or CL region. The antigen binding loop may replace a region within the FG loop region of the CH1 or CL region.

In some embodiments, the binding molecules provided herein comprise one antigen binding loop. In other embodiments, the binding molecules provided herein comprise two or more antigen binding loops. For example, the binding molecule may comprise a CH1 region variant comprising two or more antigen binding loops introduced into and/or replacing the AB, BC, CD, DE, EF and/or FG loop region amino acid fragments. In other embodiments, the binding molecule comprises a variant of the CL region comprising two or more antigen binding loops that are introduced into and/or replace the AB, BC, CD, DE, EF and/or FG loop region amino acid fragments of the CL region. In other embodiments, the binding molecules provided herein comprise: a CH1 region variant comprising one or more antigen binding loops introduced into and/or replacing the AB, BC, CD, DE, EF and/or FG loop region amino acid fragments; and a variant of the CL region, the variant comprising one or more antigen binding loops that are introduced into and/or replace the AB, BC, CD, DE, EF and/or FG loop region amino acid fragments of the CL region. In some embodiments, the antigen binding loop is located outside the AB, BC, CD, DE, EF and/or FG loop regions of the CH1 region. In some embodiments, the antigen binding loop is located outside the AB, BC, CD, DE, EF and/or FG loop regions of the CL region. In some embodiments, the antigen binding loop is located in the A, B, C, D, E and/or fβ chain of the CL region. In some embodiments, the antigen binding loop is located in the A, B, C, D, E and/or fβ chain of the CH1 region.

In some embodiments, the CH1 region is a human IgG1 CH1 region comprising the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSC (SEQ ID NO: 1). In some embodiments, the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO. 1.

In some embodiments, the CL region is a human CL kappa region, which comprises the amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO: 2). In some embodiments, the CL region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO. 2.

In some embodiments, the CL region is a human CL lambda region, which comprises the amino acid sequence of GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVK AGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS (SEQ ID NO: 3). In some embodiments, the CL region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO. 3.

In some embodiments, the binding molecules provided herein comprise one or two antigen binding loops in the CH1 region. In some embodiments, the binding molecules provided herein comprise one or two antigen binding loops in the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CH1 region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the DE loop region of the CH1 region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the DE loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the CD loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CH1 region, one antigen binding loop at the CD loop region of the CL region, and one antigen binding loop at the DE loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the DE loop region of the CH1 region and one antigen binding loop at the CD loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the DE loop region of the CH1 region and one antigen binding loop at the DE loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the DE loop region of the CH1 region, one antigen binding loop at the CD loop region of the CL region, and one antigen binding loop at the DE loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CH1 region, one antigen binding loop at the DE loop region of the CH1 region, and one antigen binding loop at the CD loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CH1 region, one antigen binding loop at the DE loop region of the CH1 region, and one antigen binding loop at the DE loop region of the CL region. In some embodiments, the binding molecules provided herein comprise one antigen binding loop at the CD loop region of the CH1 region, one antigen binding loop at the DE loop region of the CH1 region, one antigen binding loop at the CD loop region of the CL region, and one antigen binding loop at the DE loop region of the CL region.

In some specific embodiments, the antigen binding loop at the CD loop region of the CH1 region replaces the amino acid residue TSG of the CD loop of the human IgG1 CH1 region. In some specific embodiments, wherein the antigen binding loop at the DE loop region of the CH1 region replaces the amino acid residue QSS of the DE loop of the human IgG1 CH1 region. In some specific embodiments, the antigen binding loop at the CD loop region of the CL region replaces the amino acid residue SGNS of the CD loop of the human clk region. In some embodiments, the antigen binding loop at the DE loop region of the CL region replaces the amino acid residue SKD of the DE loop of the human clk region.

In some embodiments, the binding molecule comprises a region derived from the CH1 region of an antibody heavy chain and/or a region derived from the CL region of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops.

The binding molecules provided herein may be antibodies (including any antigen binding fragments thereof). In some embodiments, the binding molecules provided herein are multispecific or multivalent binding molecules comprising an antigen binding domain formed from one or more constant region variants provided herein.

In some embodiments, the binding molecules provided herein are in the form of conventional antibodies except that they comprise one or more constant region variants having one or more antigen binding loops, thereby introducing additional antigen binding domains into the antibody to generate a multispecific/multivalent antibody. Such an exemplary binding molecule is shown in fig. 1A. In some embodiments, the multi-specific/multivalent binding molecules provided herein comprise a binding domain comprising a VH region and a VL region capable of binding to a first antigen. In addition, the multispecific binding molecules provided herein comprise an additional binding domain comprising a region derived from the CH1 region and/or a region derived from the CL region that is capable of binding to the second antigen.

In some embodiments, the binding molecules provided herein are multispecific antibodies. In other embodiments, the binding molecules provided herein are multivalent antibodies. Antibodies provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, and the like.

In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. The immunoglobulin molecules provided herein can be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or subclass of immunoglobulin molecule. In a specific embodiment, the antibodies provided herein are IgG antibodies, such as IgG1 antibodies, igG2 antibodies, or IgG4 antibodies (e.g., igG4 null and variants of IgG4 antibodies). In a specific embodiment, the IgG antibody is an IgG1 antibody.

In some embodiments, various binding molecules provided herein comprise variants and/or derivatives of antibodies, including antibody fragments, that retain the ability to specifically bind to an epitope. In other embodiments of the various binding molecules provided herein, the first binding domain and/or the second binding domain is a variant and/or derivative of an antibody, including antibody fragments, that retain the ability to specifically bind to an epitope. Exemplary fragments include Fab fragments (antibody fragments containing an antigen binding domain and comprising a light chain and a portion of a heavy chain bridged by a disulfide bond); fab' (antibody fragment containing a single anti-binding domain comprising Fab and an additional portion of the heavy chain through the hinge region); f (ab ') 2 (two Fab ' molecules linked by an interchain disulfide linkage in the hinge region of the heavy chain; the Fab ' molecules may be directed against the same or different epitopes); bispecific Fab (Fab molecules with two antigen binding domains, where each antigen binding domain may be directed against a different epitope). The derivatives of the antibodies also include one or more CDR sequences of the antigen binding site of the antibody. When two or more CDR sequences are present, the CDR sequences may be linked together on a scaffold.

In some embodiments, the antibodies provided herein are bispecific antibodies. In some embodiments, the antibody is a trispecific antibody. In some embodiments, the antibody is a tetraspecific antibody. In some embodiments, the antibodies provided herein are diabodies. In some embodiments, the antibody is a trivalent antibody. In some embodiments, the antibody is a tetravalent antibody.

In one embodiment, the antibody comprises: (a) A first binding domain that binds to a first antigen, and (b) a second binding domain that binds to a second antigen. In one embodiment, the multispecific antibody comprises: (a) a first binding domain that binds a first antigen, (b) a second binding domain that binds a second antigen, and (c) a third binding domain that binds a third antigen. In one embodiment, the multispecific antibody comprises: (a) a first binding domain that binds a first antigen, (b) a second binding domain that binds a second antigen, (c) a third binding domain that binds a third antigen, and (d) a fourth binding domain that binds a fourth antigen. In some embodiments, two or more of the first antigen, the second antigen, the third antigen, and/or the fourth antigen are the same. In some embodiments, two or more of the first antigen, the second antigen, the third antigen, and/or the fourth antigen are different.

In another aspect, provided herein is an antibody comprising: (a) A first binding domain comprising a VH region and a VL region capable of binding to a first antigen; and (b) a second binding domain comprising a region derived from the CH1 region and/or a region derived from the CL region capable of binding to a second antigen. In some embodiments, the first antigen and the second antigen are the same antigen. In some embodiments, the first antigen and the second antigen are two different antigens.

In some embodiments, the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1.

In some embodiments, the region derived from the CL region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the region derived from the CL region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID No. 2.

In some embodiments, the region derived from the CL region is a region derived from a human CL lambda region comprising the amino acid sequence of SEQ ID No. 3, and wherein the region derived from the CL region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 3.

In some specific embodiments, provided herein are bispecific antibodies generated in the following section 7.

Antibodies provided herein can be from any animal source, including birds and mammals (e.g., humans, monkeys, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens). In certain embodiments, the antibodies provided herein are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of human immunoglobulins, and include antibodies isolated from a human immunoglobulin library or from mice expressing antibodies from human genes.

In certain embodiments, the antibody is a whole mouse antibody. In certain embodiments, the antibody is a mouse-human chimeric antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a fully human antibody. In other embodiments, the antibodies provided herein are humanized antibodies (e.g., comprise human constant regions and framework regions). Antibodies provided herein can be bispecific, trispecific, or more multispecific.

In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 1000nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 100nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein is provided with a K of less than 50nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 40nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 30nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 20nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 10nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 9nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 8nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 7nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 6nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 5nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 4nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 3nM _D Binding to the antigen. In some embodiments, the antibodies provided hereinOr antigen binding fragments with a K of less than 2nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 1nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 0.1nM _D Binding to the antigen. In some embodiments, an antibody or antigen binding fragment provided herein has a K of less than 0.01nM _D Binding to the antigen. K (K) _D Or K _D The value may also be measured by any method known in the art, for example, by using a Biological Layer Interferometry (BLI) or Surface Plasmon Resonance (SPR) assayUsing for example +.>Red96 systems, or byUsing for example +.>TM-2000 or->TM-3000. "binding Rate" or "associated Rate" or "kon" may also be used with the same Biological Layer Interferometry (BLI) or Surface Plasmon Resonance (SPR) techniques described above using, for example- >Red96、/>TM-2000 or->TM-3000 system. In a specific embodiment, K _D By->And (3) determining by measurement. In some embodiments, the antigen is a human antigen. In some embodiments, the antigen is a macaque antigen. In some embodiments, the antigen is a rat antigen. In other embodiments, the antigen is a mouse antigen.

In some embodiments, provided herein are antibodies that specifically bind to an antigen and can modulate antigen activity and/or expression (e.g., inhibit antigen-mediated signaling). In certain embodiments, provided herein is an antigen antagonist that is an antibody described herein that specifically binds to an antigen and inhibits (including partially inhibits) the activity of the antigen. In some embodiments, the antibodies provided herein inhibit (including partially inhibit or reduce) binding of an antigen to its ligand. Antigen activity may relate to any activity of an antigen, such as those known or described in the art. In certain embodiments, antigen activity and antigen signaling (or antigen-mediated signaling) are used interchangeably herein.

In certain embodiments, an antibody described herein reduces (e.g., partially reduces) antigen activity. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 10%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 20%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 30%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 40%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 50%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 60%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 70%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 80%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 90%. In some embodiments, the antibodies provided herein attenuate antigen activity by at least about 95%. In certain embodiments, an antibody described herein can attenuate (e.g., partially attenuate) antigen activity by at least about 15% to about 65%. In certain embodiments, an antibody described herein can attenuate (e.g., partially attenuate) antigen activity by at least about 20% to about 65%. In certain embodiments, an antibody described herein can attenuate (e.g., partially attenuate) antigen activity by at least about 30% to about 65%.

In specific embodiments, attenuation of antigen activity is assessed by the methods described herein. In specific embodiments, the attenuation of antigen activity is assessed by methods known to those of skill in the art. In certain embodiments, the reduction in antigen activity is relative to antigen activity in the absence of antigen antibody stimulation. In certain embodiments, the attenuation of antigen activity is relative to antigen activity in the absence of an associated antibody (e.g., an antibody that does not specifically bind to an antigen).

A non-limiting example of antigen activity is antigen-mediated signal transduction. Thus, in certain embodiments, an antibody described herein reduces (e.g., partially reduces) antigen-mediated signaling. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 10%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 20%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 30%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 40%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 50%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 60%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 70%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 80%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 90%. In some embodiments, the antibodies provided herein attenuate antigen-mediated signaling by at least about 95%. In certain embodiments, an antibody described herein can attenuate (e.g., partially attenuate) antigen-mediated signaling by at least about 15% to about 65%. In certain embodiments, an antibody described herein can attenuate (e.g., partially attenuate) antigen-mediated signaling by at least about 20% to about 65%. In certain embodiments, an antibody described herein can attenuate (e.g., partially attenuate) antigen-mediated signaling by at least about 30% to about 65%.

In some embodiments, one antigen bound by a binding molecule of the invention is an antigen on the surface of a target cell, such as a cancer cell. In some embodiments, the antigen is a tumor-specific antigen, a tumor-associated antigen, or a neoantigen.

In some embodiments, the target cell is a cancer cell, e.g., a cell of an adrenal cancer, anal cancer, appendiceal cancer, cholangiocarcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gall bladder cancer, gestational trophoblastic cancer, head and neck cancer, hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumor, non-hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer. In some embodiments, the cancer is adrenal cancer, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gall bladder cancer, gestational trophoblastic cancer, head and neck cancer, hodgkin's lymphoma, intestinal cancer, renal cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumor, non-hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer. In some embodiments, the cancer is an adrenal cancer. In some embodiments, the cancer is anal cancer. In some embodiments, the cancer is appendiceal cancer. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is bone cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is gallbladder cancer. In some embodiments, the cancer is gestational trophoblastic cancer. In some embodiments, the cancer is a head and neck cancer. In some embodiments, the cancer is hodgkin's lymphoma. In some embodiments, the cancer is a bowel cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is a neuroendocrine tumor. In some embodiments, the cancer is non-hodgkin's lymphoma. In some embodiments, the cancer is oral cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is a sinus cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is a soft tissue sarcoma, a spinal carcinoma. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is laryngeal cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is uterine cancer, endometrial cancer. In some embodiments, the cancer is vaginal cancer. In some embodiments, the cancer is vulvar cancer.

In some embodiments, the adrenal cancer is adrenocortical carcinoma (ACC), pararenal cortical carcinoma, pheochromocytoma, or neuroblastoma. In some embodiments, the anal cancer is squamous cell carcinoma, one-hole anorectal carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma. In some embodiments, the appendiceal cancer is a neuroendocrine tumor (NET), mucous adenocarcinoma, goblet cell carcinoid, intestinal adenocarcinoma, or seal ring cell adenocarcinoma. In some embodiments, the bile duct cancer is extrahepatic bile duct cancer, adenocarcinoma, porta hepatica bile duct cancer, periportal bile duct cancer, distal bile duct cancer, or intrahepatic bile duct cancer. In some embodiments, the bladder cancer is Transitional Cell Carcinoma (TCC), papillary carcinoma, squamous carcinoma, adenocarcinoma, small cell carcinoma or sarcoma. In some embodiments, the bone cancer is a primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, bone giant cell tumor, chordoma, or metastatic bone cancer. In some embodiments, the brain cancer is astrocytoma, brain stem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary adenocarcinoma, pituitary adenoma, craniopharyngeal tube tumor, germ cell tumor, pineal gland tumor, medulloblastoma, or primary CNS lymphoma. In some embodiments, the breast cancer is breast adenocarcinoma, invasive breast cancer, non-invasive breast cancer, breast sarcoma, metastatic cancer, adenoid cystic cancer, phylloxera, angiosarcoma, HER2 positive breast cancer, triple negative breast cancer, or inflammatory breast cancer. In some embodiments, the cervical cancer is squamous cell carcinoma or adenocarcinoma. In some embodiments, the colorectal cancer is colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucous adenocarcinoma, seal ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma. In some embodiments, the esophageal cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments, the gall bladder cancer is adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma or sarcoma. In some embodiments, the Gestational Trophoblastic Disease (GTD) is grape embryo, gestational trophoblastic tumor (GTN), choriocarcinoma, placental Site Trophoblastic Tumor (PSTT), or epithelial-like trophoblastic tumor (ETT). In some embodiments, the head and neck cancer is laryngeal, nasopharyngeal, hypopharyngeal, nasal, sinus, salivary gland, oral, oropharyngeal, or tonsil cancer. In some embodiments, the hodgkin lymphoma is classical hodgkin lymphoma, nodular sclerosis, mixed cell type, enriched lymphocyte type, lymphocyte depletion type, or nodular lymphocyte type based hodgkin lymphoma (NLPHL). In some embodiments, the intestinal cancer is small intestine cancer (small intestine cancer), small intestine cancer (small bowel cancer), adenocarcinoma, sarcoma, gastrointestinal stromal tumor, carcinoid tumor, or lymphoma. In some embodiments, the renal cancer is Renal Cell Carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting tube RCC, unclassified RCC, transitional cell carcinoma, urothelial carcinoma, renal pelvis carcinoma, or renal sarcoma. In some embodiments, the leukemia is Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), hairy Cell Leukemia (HCL), or myelodysplastic syndrome (MDS). In a specific embodiment, the leukemia is AML. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), fiberboard layer-like HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis. In some embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large cell undifferentiated carcinoma, lung nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland type lung cancer, lung carcinoid, mesothelioma, lung sarcoidosis, or malignant granulocytic lung tumor. In some embodiments, the melanoma is superficial diffuse melanoma, nodular melanoma, acro-lentigo melanoma, malignant lentigo melanoma, leucomatous melanoma, pro-fibroproliferative melanoma, ocular melanoma, or metastatic melanoma. In some embodiments, the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In some embodiments, the multiple myeloma is active myeloma or smoky myeloma. In some embodiments, the neuroendocrine tumor is a gastrointestinal neuroendocrine tumor, a pancreatic neuroendocrine tumor, or a lung neuroendocrine tumor. In some embodiments, the non-hodgkin lymphoma is anaplastic large cell lymphoma, lymphoblastic lymphoma, peripheral T cell lymphoma, follicular lymphoma, cutaneous T cell lymphoma, lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, MALT lymphoma, small cell lymphocytic lymphoma, burkitt lymphoma, chronic Lymphocytic Leukemia (CLL), small Lymphocytic Lymphoma (SLL), precursor T lymphoblastic leukemia/lymphoma, acute Lymphoblastic Leukemia (ALL), adult T cell lymphoma/leukemia (ATLL), hairy cell leukemia, B cell lymphoma, diffuse Large B Cell Lymphoma (DLBCL), primary mediastinal B cell lymphoma, primary Central Nervous System (CNS) lymphoma, mantle Cell Lymphoma (MCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (stand) lymphoma, lymph node marginal zone B cell lymphoma, splenic marginal zone B cell lymphoma, lymphoplasmacytic lymphoma, B cell non-hodgkin lymphoma, T cell non-lymphoblastic leukemia/lymphoma, acute Lymphoblastic Leukemia (ALL), adult T cell lymphoma/leukemia (ALL), hairy cell leukemia (head-cell lymphoma), diffuse large cell lymphoma (ALL), diffuse large cell lymphoma, anaplastic tumor of the skin, and the like. In some embodiments, the oral cancer is squamous cell carcinoma, warty carcinoma, small salivary gland carcinoma, lymphoma, benign oral tumor, eosinophilic granuloma, fibroma, granuloma, keratoacanthoma, smooth myoma, osteochondrima, lipoma, schwannoma, neurofibroma, papilloma, condyloma acuminatum, warty xanthoma, suppurative granuloma, rhabdomyoma, odontogenic tumor, white spots, erythema, squamous cell lip carcinoma, basal cell lip carcinoma, oral cancer, gum carcinoma, or tongue carcinoma. In some embodiments, the ovarian cancer is ovarian epithelial cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, clear cell epithelial ovarian cancer, undifferentiated epithelial ovarian cancer, ovarian low malignant potential tumors, primary peritoneal cancer, fallopian tube cancer, germ cell tumor, teratoma, asexual cell tumor, ovarian germ cell cancer, endoembryo sinus tumor, sex cord-stromal tumor, sex cord-gonadal stromal tumor, ovarian stromal tumor, granulosa cell tumor, granulosa-follicular cell tumor, seltoli-Leydig cell tumor, ovarian sarcoma, ovarian carcinoma sarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, kuken b tumor, or ovarian cyst. In some embodiments, the pancreatic cancer is pancreatic exocrine adenocarcinoma, pancreatic endocrine adenocarcinoma, or pancreatic adenocarcinoma, islet cell tumor, or neuroendocrine tumor. In some embodiments, the prostate cancer is a prostate cancer, a prostate sarcoma, transitional cell carcinoma, small cell carcinoma, or a neuroendocrine tumor. In some embodiments, the sinus cancer is squamous cell carcinoma, mucosal cell carcinoma, adenoid cystic cell carcinoma, acinar cell carcinoma, sinus undifferentiated carcinoma, nasal cavity carcinoma, paranasal sinus carcinoma, maxillary sinus carcinoma, ethmoid sinus carcinoma, or nasopharyngeal carcinoma. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, melanoma, merkel cell carcinoma, kaposi's Sarcoma (KS), actinic keratosis, cutaneous lymphoma, or keratoacanthoma. In some embodiments, the soft tissue carcinoma is angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), kaposi's sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated Liposarcoma (DL), myxoid/round cell liposarcoma (MRCL), hyperdifferentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma. In some embodiments, the spinal cancer is spinal metastasis. In some embodiments, the gastric cancer is gastric adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumor, carcinoid tumor, gastric carcinoid tumor, ECL cell carcinoid type I, ECL cell carcinoid type II, or ECL cell carcinoid type III. In some embodiments, the testicular cancer is a seminoma, a non-seminoma, an embryo cancer, a yolk sac cancer, a choriocarcinoma, a teratoma, a gonadal stromal tumor, a testicular stromal tumor, or a testicular supportive cell tumor. In some embodiments, the laryngeal carcinoma is squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal carcinoma, pharyngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, lymphoepithelial tumor, spindle cell carcinoma, warty carcinoma, undifferentiated carcinoma, or lymph node carcinoma. In some embodiments, the thyroid cancer is papillary carcinoma, follicular carcinoma, hurthle cell carcinoma, medullary thyroid carcinoma, or undifferentiated carcinoma. In some embodiments, the uterine cancer is endometrial cancer, endometrial adenocarcinoma, endometrioid carcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinoma sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, or undifferentiated sarcoma. In some embodiments, the vaginal cancer is squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In some embodiments, the vulvar cancer is squamous cell carcinoma or adenocarcinoma.

In some embodiments, one antigen bound by a binding molecule of the invention is a cancer antigen. In some embodiments, the cancer antigen is angiogenin, BCMA, CD19, CD20, CD22, CD25 (IL 2-R), CD30, CD33, CD37, CD38, CD52, CD56, CD123 (IL-3R), cMET, DLL/Notch, EGFR, epCAM, FGF, FGF-R, GD2, HER2, mesothelin, fibronectin-4, PDGFRα, RANKL, SLAMF7, TROP2, VEGF, or VEGF-R. In some embodiments, the cancer antigen is angiogenin. In some embodiments, the cancer antigen is BCMA. In some embodiments, the cancer antigen is CD19. In some embodiments, the cancer antigen is CD20. In some embodiments, the cancer antigen is CD22. In some embodiments, the cancer antigen is CD25 (IL 2-R). In some embodiments, the cancer antigen is CD30. In some embodiments, the cancer antigen is CD33. In some embodiments, the cancer antigen is CD37. In some embodiments, the cancer antigen is CD38. In some embodiments, the cancer antigen is CD52. In some embodiments, the cancer antigen is CD56. In some embodiments, the cancer antigen is CD123 (IL-3R). In some embodiments, the cancer antigen is cMET. In some embodiments, the cancer antigen is DLL/Notch. In some embodiments, the cancer antigen is EGFR. In some embodiments, the cancer antigen is EpCAM. In some embodiments, the cancer antigen is FGF. In some embodiments, the cancer antigen is FGF-R. In some embodiments, the cancer antigen is GD2. In some embodiments, the cancer antigen is HER2. In some embodiments, the cancer antigen is mesothelin. In some embodiments, the cancer antigen is fibronectin-4. In some embodiments, the cancer antigen is pdgfrα. In some embodiments, the cancer antigen is RANKL. In some embodiments, the cancer antigen is SLAMF7. In some embodiments, the cancer antigen is TROP2. In some embodiments, the cancer antigen is VEGF. In some embodiments, the cancer antigen is VEGF-R.

In some embodiments, the cancer antigen is CEA, an immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium activated chloride channel 2, cyclin-B1, 9D7, epCAM, ephA3, her2/neu, telomerase, mesothelin, SAP-1, surviving, BAGE family antigen, CAGE family antigen, GAGE family antigen, MAGE family antigen, SAGE family antigen, XAGE family antigen, NY-ESO-1/LAGE-1, PRAME, SSX-2, melan-A, MART-1, gp100, pmel17, tyrosinase, TRP-1, TRP-2, P.polypeptide, MC1R, prostate specific antigen, β -catenin, BRCA1, BRCA2, CDK4, CML66, fibronectin, MART-2, p53, ras, TGF- βRII, or MUC1. In some embodiments, the cancer antigen is CEA. In some embodiments, the cancer antigen is an immature laminin receptor. In some embodiments, the cancer antigen is TAG-72. In some embodiments, the cancer antigen is HPV E6. In some embodiments, the cancer antigen is HPV E7. In some embodiments, the cancer antigen is BING-4. In some embodiments, the cancer antigen is calcium activated chloride channel 2. In some embodiments, the cancer antigen is cyclin-B1. In some embodiments, the cancer antigen is 9D7. In some embodiments, the cancer antigen is EpCAM. In some embodiments, the cancer antigen is EphA3. In some embodiments, the cancer antigen is Her2/neu. In some embodiments, the cancer antigen is telomerase. In some embodiments, the cancer antigen is mesothelin. In some embodiments, the cancer antigen is SAP-1. In some embodiments, the cancer antigen is surviving. In some embodiments, the cancer antigen is a BAGE family antigen. In some embodiments, the cancer antigen is a mage family antigen. In some embodiments, the cancer antigen is a GAGE family antigen. In some embodiments, the cancer antigen is a MAGE family antigen. In some embodiments, the cancer antigen is a SAGE family antigen. In some embodiments, the cancer antigen is a XAGE family antigen. In some embodiments, the cancer antigen is NY-ESO-1/LAGE-1. In some embodiments, the cancer antigen is PRAME. In some embodiments, the cancer antigen is SSX-2. In some embodiments, the cancer antigen is Melan-A. In some embodiments, the cancer antigen is MART-1. In some embodiments, the cancer antigen is Gp100. In some embodiments, the cancer antigen is pmel17. In some embodiments, the cancer antigen is tyrosinase. In some embodiments, the cancer antigen is TRP-1. In some embodiments, the cancer antigen is TRP-2. In some embodiments, the target antigen is a p. In some embodiments, the cancer antigen is MC1R. In some embodiments, the cancer antigen is a prostate specific antigen. In some embodiments, the cancer antigen is β -catenin. In some embodiments, the cancer antigen is BRCA1. In some embodiments, the cancer antigen is BRCA2. In some embodiments, the cancer antigen is CDK4. In some embodiments, the cancer antigen is CML66. In some embodiments, the cancer antigen is fibronectin. In some embodiments, the cancer antigen is MART-2. In some embodiments, the cancer antigen is p53. In some embodiments, the cancer antigen is Ras. In some embodiments, the cancer antigen is TGF-beta RII. In some embodiments, the cancer antigen is MUC1.

In some embodiments, the binding molecules of the invention bind to B cell antigens. In some embodiments of the present invention, in some embodiments, B cell antigens are CD1a, CD1B, CD1c, CD1d, CD2, CD5, CD6, CD9, CD11a, CD11B, CD11c, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD29, CD30, CD31, CD32a, CD32B, CD35, CD37, CD38, CD39, CD40, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49B, CD49c, CD49d, CD50, CD52, CD53, CD54, CD55 CD58, CD60a, CD62L, CD, CD68, CD69, CD70, CD72, CD73, CD74, CD75S, CD, CD79a, CD79B, CD80, CD81, CD82, CD83, CD84, CD85E, CD85I, CD85J, CD86, CD92, CD95, CD97, CD98, CD99, CD100, CD102, CD108, CD119, CD120a, CD120B, CD121B, CD122, CD124, CD125, CD126, CD130, CD132, CD137, CD138, CD139, CD147, CD148, CD150, CD152, CD162, CD CD58, CD60a, CD62L, CD63, CD68, CD69, CD70, CD72, CD73, CD74, CD75S, CD77, CD79a, CD79B, CD80, CD81, CD82, CD83, CD84, CD85E, CD85I, CD J, CD86, CD92, CD95, CD97, CD CD98, CD99, CD100, CD102, CD108, CD119, CD120a, CD120B, CD121B, CD122, CD124, CD125, CD126, CD130, CD132, CD137, CD138, CD139, CD147, CD148, CD150, CD152, CD162, CD126, CD130, CD132, CD137, CD138, CD139, CD 147. In some embodiments, the B cell antigen is a CD1a antigen. In some embodiments, the B cell antigen is a CD1B antigen. In some embodiments, the B cell antigen is a CD1c antigen. In some embodiments, the B cell antigen is a CD1d antigen. In some embodiments, the B cell antigen is a CD2 antigen. In some embodiments, the B cell antigen is a CD5 antigen. In some embodiments, the B cell antigen is a CD6 antigen. In some embodiments, the B cell antigen is a CD9 antigen. In some embodiments, the B cell antigen is a CD11a antigen. In some embodiments, the B cell antigen is a CD11B antigen. In some embodiments, the B cell antigen is a CD11c antigen. In some embodiments, the B cell antigen is a CD17 antigen. In some embodiments, the B cell antigen is a CD18 antigen. In some embodiments, the B cell antigen is a CD19 antigen. In some embodiments, the B cell antigen is a CD20 antigen. In some embodiments, the B cell antigen is a CD21 antigen. In some embodiments, the B cell antigen is a CD22 antigen. In some embodiments, the B cell antigen is a CD23 antigen. In some embodiments, the B cell antigen is a CD24 antigen. In some embodiments, the B cell antigen is a CD25 antigen. In some embodiments, the B cell antigen is a CD26 antigen. In some embodiments, the B cell antigen is a CD27 antigen. In some embodiments, the B cell antigen is a CD29 antigen. In some embodiments, the B cell antigen is a CD30 antigen. In some embodiments, the B cell antigen is a CD31 antigen. In some embodiments, the B cell antigen is a CD32a antigen. In some embodiments, the B cell antigen is a CD32B antigen. In some embodiments, the B cell antigen is a CD35 antigen. In some embodiments, the B cell antigen is a CD37 antigen. In some embodiments, the B cell antigen is a CD38 antigen. In some embodiments, the B cell antigen is a CD39 antigen. In some embodiments, the B cell antigen is a CD40 antigen. In some embodiments, the B cell antigen is a CD45 antigen. In some embodiments, the B cell antigen is a CD45RA antigen. In some embodiments, the B cell antigen is a CD45RB antigen. In some embodiments, the B cell antigen is a CD45RC antigen. In some embodiments, the B cell antigen is a CD45RO antigen. In some embodiments, the B cell antigen is a CD46 antigen. In some embodiments, the B cell antigen is a CD47 antigen. In some embodiments, the B cell antigen is a CD48 antigen. In some embodiments, the B cell antigen is a CD49B antigen. In some embodiments, the B cell antigen is a CD49c antigen. In some embodiments, the B cell antigen is a CD49d antigen. In some embodiments, the B cell antigen is a CD50 antigen. In some embodiments, the B cell antigen is a CD52 antigen. In some embodiments, the B cell antigen is a CD53 antigen. In some embodiments, the B cell antigen is a CD54 antigen. In some embodiments, the B cell antigen is a CD55 antigen. In some embodiments, the B cell antigen is a CD58 antigen. In some embodiments, the B cell antigen is a CD60a antigen. In some embodiments, the B cell antigen is a CD62L antigen. In some embodiments, the B cell antigen is a CD63 antigen. In some embodiments, the B cell antigen is a CD68 antigen. In some embodiments, the B cell antigen is a CD69 antigen. In some embodiments, the B cell antigen is a CD70 antigen. In some embodiments, the B cell antigen is a CD72 antigen. In some embodiments, the B cell antigen is a CD73 antigen. In some embodiments, the B cell antigen is a CD74 antigen. In some embodiments, the B cell antigen is a CD75 antigen. In some embodiments, the B cell antigen is a CD75S antigen. In some embodiments, the B cell antigen is a CD77 antigen. In some embodiments, the B cell antigen is a CD79a antigen. In some embodiments, the B cell antigen is a CD79B antigen. In some embodiments, the B cell antigen is a CD80 antigen. In some embodiments, the B cell antigen is a CD81 antigen. In some embodiments, the B cell antigen is a CD82 antigen. In some embodiments, the B cell antigen is a CD83 antigen. In some embodiments, the B cell antigen is a CD84 antigen. In some embodiments, the B cell antigen is a CD85E antigen. In some embodiments, the B cell antigen is a CD85I antigen. In some embodiments, the B cell antigen is a CD85J antigen. In some embodiments, the B cell antigen is a CD86 antigen. In some embodiments, the B cell antigen is a CD92 antigen. In some embodiments, the B cell antigen is a CD95 antigen. In some embodiments, the B cell antigen is a CD97 antigen. In some embodiments, the B cell antigen is a CD98 antigen. In some embodiments, the B cell antigen is a CD99 antigen. In some embodiments, the B cell antigen is a CD100 antigen. In some embodiments, the B cell antigen is a CD102 antigen. In some embodiments, the B cell antigen is a CD108 antigen. In some embodiments, the B cell antigen is a CD119 antigen. In some embodiments, the B cell antigen is a CD120a antigen. In some embodiments, the B cell antigen is a CD120B antigen. In some embodiments, the B cell antigen is CD121B antigen. In some embodiments, the B cell antigen is CD122 antigen. In some embodiments, the B cell antigen is a CD124 antigen. In some embodiments, the B cell antigen is a CD125 antigen. In some embodiments, the B cell antigen is a CD126 antigen. In some embodiments, the B cell antigen is a CD130 antigen. In some embodiments, the B cell antigen is a CD132 antigen. In some embodiments, the B cell antigen is a CD137 antigen. In some embodiments, the B cell antigen is a CD138 antigen. In some embodiments, the B cell antigen is a CD139 antigen. In some embodiments, the B cell antigen is a CD147 antigen. In some embodiments, the B cell antigen is a CD148 antigen. In some embodiments, the B cell antigen is a CD150 antigen. In some embodiments, the B cell antigen is a CD152 antigen. In some embodiments, the B cell antigen is a CD162 antigen. In some embodiments, the B cell antigen is a CD164 antigen. In some embodiments, the B cell antigen is a CD166 antigen. In some embodiments, the B cell antigen is CD167a antigen. In some embodiments, the B cell antigen is a CD170 antigen. In some embodiments, the B cell antigen is a CD171 antigen. In some embodiments, the B cell antigen is CD175 antigen. In some embodiments, the B cell antigen is CD175s antigen. In some embodiments, the B cell antigen is a CD180 antigen. In some embodiments, the B cell antigen is a CD184 antigen. In some embodiments, the B cell antigen is CD185 antigen. In some embodiments, the B cell antigen is a CD192 antigen. In some embodiments, the B cell antigen is CD196 antigen. In some embodiments, the B cell antigen is a CD197 antigen. In some embodiments, the B cell antigen is a CD200 antigen. In some embodiments, the B cell antigen is CD205 antigen. In some embodiments, the B cell antigen is CD201a antigen. In some embodiments, the B cell antigen is CDw210B antigen. In some embodiments, the B cell antigen is a CD212 antigen. In some embodiments, the B cell antigen is CD213a1 antigen. In some embodiments, the B cell antigen is CD213a2 antigen. In some embodiments, the B cell antigen is a CD215 antigen. In some embodiments, the B cell antigen is a CD217 antigen. In some embodiments, the B cell antigen is CD218a antigen. In some embodiments, the B cell antigen is CD218B antigen. In some embodiments, the B cell antigen is a CD220 antigen. In some embodiments, the B cell antigen is CD221 antigen. In some embodiments, the B cell antigen is CD222 antigen. In some embodiments, the B cell antigen is a CD224 antigen. In some embodiments, the B cell antigen is a CD225 antigen. In some embodiments, the B cell antigen is a CD226 antigen. In some embodiments, the B cell antigen is CD227 antigen. In some embodiments, the B cell antigen is a CD229 antigen. In some embodiments, the B cell antigen is a CD230 antigen. In some embodiments, the B cell antigen is a CD232 antigen. In some embodiments, the B cell antigen is a CD252 antigen. In some embodiments, the B cell antigen is a CD252 antigen. In some embodiments, the B cell antigen is a CD254 antigen. In some embodiments, the B cell antigen is a CD255 antigen. In some embodiments, the B cell antigen is a CD256 antigen. In some embodiments, the B cell antigen is a CD257, CD258 antigen. In some embodiments, the B cell antigen is CD259 antigen. In some embodiments, the B cell antigen is a CD260 antigen. In some embodiments, the B cell antigen is a CD261 antigen. In some embodiments, the B cell antigen is a CD262 antigen. In some embodiments, the B cell antigen is a CD263 antigen. In some embodiments, the B cell antigen is a CD264 antigen. In some embodiments, the B cell antigen is CD267 antigen. In some embodiments, the B cell antigen is a CD268 antigen. In some embodiments, the B cell antigen is CD269 antigen. In some embodiments, the B cell antigen is a CD270 antigen. In some embodiments, the B cell antigen is a CD272 antigen. In some embodiments, the B cell antigen is CD274 antigen. In some embodiments, the B cell antigen is a CD275 antigen. In some embodiments, the B cell antigen is a CD277 antigen. In some embodiments, the B cell antigen is CD279 antigen. In some embodiments, the B cell antigen is CD283 antigen. In some embodiments, the B cell antigen is CD289 antigen. In some embodiments, the B cell antigen is a CD290 antigen. In some embodiments, the B cell antigen is a CD295 antigen. In some embodiments, the B cell antigen is CD298 antigen. In some embodiments, the B cell antigen is a CD300 antigen. In some embodiments, the B cell antigen is a CD300c antigen. In some embodiments, the B cell antigen is CD305 antigen. In some embodiments, the B cell antigen is a CD306 antigen. In some embodiments, the B cell antigen is a CD307a antigen. In some embodiments, the B cell antigen is a CD307B antigen. In some embodiments, the B cell antigen is a CD307c antigen. In some embodiments, the B cell antigen is a CD307d antigen. In some embodiments, the B cell antigen is a CD307e antigen. In some embodiments, the B cell antigen is a CD314 antigen. In some embodiments, the B cell antigen is a CD215 antigen. In some embodiments, the B cell antigen is a CD316 antigen. In some embodiments, the B cell antigen is CD317 antigen. In some embodiments, the B cell antigen is a CD319 antigen. In some embodiments, the B cell antigen is CD321 antigen. In some embodiments, the B cell antigen is a CD327 antigen. In some embodiments, the B cell antigen is a CD328 antigen. In some embodiments, the B cell antigen is a CD329 antigen. In some embodiments, the B cell antigen is CD338 antigen. In some embodiments, the B cell antigen is a CD351 antigen. In some embodiments, the B cell antigen is CD352 antigen. In some embodiments, the B cell antigen is a CD353 antigen. In some embodiments, the B cell antigen is a CD354 antigen. In some embodiments, the B cell antigen is a CD355 antigen. In some embodiments, the B cell antigen is a CD356 antigen. In some embodiments, the B cell antigen is CD357 antigen. In some embodiments, the B cell antigen is a CD358 antigen. In some embodiments, the B cell antigen is a CD360 antigen. In some embodiments, the B cell antigen is CD361 antigen. In some embodiments, the B cell antigen is a CD362 antigen. In some embodiments, the B cell antigen is CD363 antigen.

In one embodiment, the binding molecules of the invention bind to a pathogen. In some embodiments, the pathogen causes an infectious disease selected from the group consisting of: acute myelitis (AFM), anabrosis, anthrax, babesiosis, botulism, brucellosis, campylobacter, carbapenem-resistant infections, chancre, chikungunya virus infections, chlamydia, fish botulinum, clostridium difficile infections, clostridium perfringens, coccidioidomycosis, coronavirus infections, covid-19 (SARS-CoV-2), creutzfeldt-Jakob disease/infectious spongiform encephalopathy, cryptosporidiosis (Crypto), cyclosporin, dengue 1, 2, 3 or 4, diphtheria, escherichia coli infection/shiga toxin production (STEC), eastern equine encephalitis, hemorrhagic fever (Ebola), epstein-Barr disease, encephalitis, arbovirus or paraminosis, non-polio enterovirus, D68 enterovirus (EV-D68), giardiasis, meldonia disease, gonococcal infection, inguinal granuloma, haemophilus influenzae type B (Hib or H-influenza), hantavirus Pulmonary Syndrome (HPS), hemolytic Uremic Syndrome (HUS), hepatitis A (hepA), hepatitis B (hepB), hepatitis C (hepC), hepatitis D (hepD), hepatitis E (hepE), herpes zoster, histoplasmosis infection, human immunodeficiency virus/AIDS (HIV/AIDS), influenza (Legioma), human influenza (Legioma), influenza (HPV (Legioma), leprosy (hansen's disease), leptospirosis, listeriosis (listeria), lyme disease, venereal lymphogranulomatosis infection (LGV), malaria, measles, meliosis, meningitis (viral), meningococcal disease (meningitis (bacterial)), middle east respiratory syndrome coronavirus (MERS-CoV), mumps, norovirus, pediculosis, pelvic Inflammatory Disease (PID), pertussis (wheezing), plague (inguinal adenitis, septicaemia, pneumonitis), pneumococosis (pneumonia), poliomyelitis (poliomyelitis), bovaccina, psittacosis, pubic lice, impetigo (smallpox, monkey pox, vaccinia), Q fever, rabies, pneumonitis rickettsia (falling mountain zebra fever), rubella (german measles), salmonella gastritis (salmonella), scabies, mackerel toxin, septicemia, severe Acute Respiratory Syndrome (SARS), shigella gastroenteritis (shigella), smallpox, methicillin-resistant staphylococcal infection (MRSA), staphylococcal food poisoning enterotoxin B poisoning (staphylococcal food poisoning), staphylococcal infection Vancomycin Intermediate (VISA), staphylococcal infection Vancomycin Resistant (VRSA), streptococcal disease group a (invasive) (group a streptococcus (invasive)), streptococcal disease group B (group B streptococcus), streptococcal midbody STSS toxic shock, syphilis (primary, secondary, early latent, late latent, congenital), tetanus infection, trichomonas vaginalis, trichomonas infection, tuberculosis (TB), latent Tuberculosis (LTBI), rabbit fever, typhoid group D, vaginosis, varicella virus (varicella), vibrio cholerae, vibriosis (vibrio), ebola hemorrhagic fever, lassa hemorrhagic fever, marburg hemorrhagic fever, west nile virus, yellow fever, yersinia, and zika virus infection. In some embodiments, the infectious disease is acute myelitis weakness (AFM). In some embodiments, the infectious disease is an intangible disease. In some embodiments, the infectious disease is anthrax. In some embodiments, the infectious disease is babesiosis. In some embodiments, the infectious disease is botulism. In some embodiments, the infectious disease is brucellosis. In some embodiments, the infectious disease is campylobacteriosis. In some embodiments, the infectious disease is a carbapenem-resistant infection. In some embodiments, the infectious disease is chancre. In some embodiments, the infectious disease is a chikungunya virus infection. In some embodiments, the infectious disease is chlamydia. In some embodiments, the infectious disease is fish toxicity. In some embodiments, the infectious disease is clostridium difficile infection. In some embodiments, the infectious disease is clostridium perfringens. In some embodiments, the infectious disease is a coccidioidomycosis fungal infection. In some embodiments, the infectious disease is a coronavirus. In some embodiments, the infectious disease is Covid-19 (SARS-CoV-2). In some embodiments, the infectious disease is creutzfeldt-jakob disease/transmissible spongiform encephalopathy. In some embodiments, the infectious disease is Cryptosporidiosis (Crypto). In some embodiments, the infectious disease is cyclosporin. In some embodiments, the infectious disease is dengue 1, 2, 3, or 4. In some embodiments, the infectious disease is diphtheria. In some embodiments, the infectious disease is escherichia coli infection/shiga toxin production (STEC). In some embodiments, the infectious disease is eastern equine encephalitis. In some embodiments, the infectious disease is hemorrhagic fever (ebola). In some embodiments, the infectious disease is irinotecan's disease. In some embodiments, the infectious disease is encephalitis. In some embodiments, the infectious disease is arbovirus or parainfectious. In some embodiments, the infectious disease is a non-polio enterovirus. In some embodiments, the infectious disease is D68 enterovirus (EV-D68). In some embodiments, the infectious disease is giardiasis. In some embodiments, the infectious disease is a gangrene disease. In some embodiments, the infectious disease is gonococcal infection. In some embodiments, the infectious disease is inguinal granuloma. In some embodiments, the infectious disease is haemophilus influenzae type B (Hib or H-influenza). In some embodiments, the infectious disease is hantavirus lung syndrome (HPS). In some embodiments, the infectious disease is Hemolytic Uremic Syndrome (HUS). In some embodiments, the infectious disease is hepatitis a (hepa). In some embodiments, the infectious disease is hepatitis B (Hep B). In some embodiments, the infectious disease is hepatitis C (Hep C). In some embodiments, the infectious disease is hepatitis D (Hep D). In some embodiments, the infectious disease is hepatitis E (Hep E). In some embodiments, the infectious disease is herpes. In some embodiments, the infectious disease is shingles (shingles). In some embodiments, the infectious disease is a histoplasmosis infection. In some embodiments, the infectious disease is human immunodeficiency virus/AIDS (HIV/AIDS). In some embodiments, the infectious disease is Human Papilloma Virus (HPV). In some embodiments, the infectious disease is influenza (Flu). In some embodiments, the infectious disease is legionellosis (legionnaires disease). In some embodiments, the infectious disease is leprosy (hansen's disease). In some embodiments, the infectious disease is leptospirosis. In some embodiments, the infectious disease is listeriosis (listeria). In some embodiments, the infectious disease is lyme disease. In some embodiments, the infectious disease is a venereal lymphogranulomatous infection (LGV). In some embodiments, the infectious disease is malaria. In some embodiments, the infectious disease is measles. In some embodiments, the infectious disease is a melioidosis. In some embodiments, the infectious disease is meningitis (viral). In some embodiments, the infectious disease is meningococcal disease (meningitis (bacterial)). In some embodiments, the infectious disease is the middle east respiratory syndrome coronavirus (MERS-CoV). In some embodiments, the infectious disease is mumps. In some embodiments, the infectious disease is a norovirus. In some embodiments, the infectious disease is pediculosis. In some embodiments, the infectious disease is Pelvic Inflammatory Disease (PID). In some embodiments, the infectious disease is pertussis (asthma). In some embodiments, the infectious disease is plague (inguinal adenotis, in some embodiments, the infectious disease is septicemic, in some embodiments, the infectious disease is pneumonic). In some embodiments, the infectious disease is pneumococcal disease (pneumonia). In some embodiments, the infectious disease is poliomyelitis (poliomyelitis). In some embodiments, the infectious disease is bovalson. In some embodiments, the infectious disease is psittacosis. In some embodiments, the infectious disease is pubic lice. In some embodiments, the infectious disease is impetigo (smallpox, in some embodiments, the infectious disease is monkey pox, in some embodiments, the infectious disease is vaccinia). In some embodiments, the infectious disease is Q fever. In some embodiments, the infectious disease is rabies. In some embodiments, the infectious disease is rickettsia (fever of the hiking mountain spot). In some embodiments, the infectious disease is rubella (german measles). In some embodiments, the infectious disease is salmonella gastroenteritis (salmonella). In some embodiments, the infectious disease is scabies. In some embodiments, the infectious disease is mackerel toxin. In some embodiments, the infectious disease is sepsis. In some embodiments, the infectious disease is Severe Acute Respiratory Syndrome (SARS). In some embodiments, the infectious disease is shigella gastroenteritis (shigella). In some embodiments, the infectious disease is smallpox. In some embodiments, the infectious disease is methicillin-resistant staphylococcus infection (MRSA). In some embodiments, the infectious disease is staphylococcal food poisoning enterotoxin B poisoning (staphylococcal food poisoning). In some embodiments, the infectious disease is staphylococcal Vancomycin Intermediate (VISA). In some embodiments, the infectious disease is staphylococcal Vancomycin Resistance (VRSA). In some embodiments, the infectious disease is streptococcal group a (invasive) (group a streptococci (invasive)). In some embodiments, the infectious disease is streptococcal disease. In some embodiments, the infectious disease is group B (group B streptococcus). In some embodiments, the infectious disease is streptococcal toxic shock syndrome STSS toxic shock. In some embodiments, the infectious disease is syphilis (primary, in some embodiments, the infectious disease is secondary, in some embodiments, the infectious disease is early latency, in some embodiments, the infectious disease is late latency, in some embodiments, the infectious disease is congenital). In some embodiments, the infectious disease is tetanus infection. In some embodiments, the infectious disease is trichomonas vaginalis. In some embodiments, the infectious disease is a trichomonas infection. In some embodiments, the infectious disease is Tuberculosis (TB). In some embodiments, the infectious disease is Latent Tuberculosis (LTBI). In some embodiments, the infectious disease is rabbit fever. In some embodiments, the infectious disease is typhoid D group. In some embodiments, the infectious disease is a vaginal disease. In some embodiments, the infectious disease is varicella virus (varicella), vibrio cholerae (cholera). In some embodiments, the infectious disease is vibriosis (vibrios). In some embodiments, the infectious disease is ebola virus hemorrhagic fever. In some embodiments, the infectious disease is lassa virus hemorrhagic fever. In some embodiments, the infectious disease is marburg hemorrhagic fever. In some embodiments, the infectious disease is west nile virus. In some embodiments, the infectious disease is yellow fever. In some embodiments, the infectious disease is yersinia. In some embodiments, the infectious disease is a zika virus infection.

In some embodiments, the pathogen is a virus. In some embodiments, the virus is a virus of the adenoviridae, arenaviridae, astroviridae, bunaeae, caliciviridae, coronaviridae, filoviridae, flaviviridae, hepadnaviridae, hepaciviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae, poxviridae, reoviridae, retrovirus, rhabdoviridae, or togaviridae families. In some embodiments, the virus is a virus of the family adenoviridae. In some embodiments, the virus is a virus of the arenaviridae family. In some embodiments, the virus is a virus of the astroviridae family. In some embodiments, the virus is a virus of the family euphorbiaceae. In some embodiments, the virus is a virus of the caliciviridae family. In some embodiments, the virus is a virus of the family coronaviridae. In some embodiments, the virus is a virus of the family filoviridae. In some embodiments, the virus is a flaviviridae virus. In some embodiments, the virus is a hepadnaviridae virus. In some embodiments, the virus is a virus of the hepaciviridae family. In some embodiments, the virus is a virus of the orthomyxoviridae family. In some embodiments, the virus is a virus of the papillomaviridae family. In some embodiments, the virus is a virus of the family paramyxoviridae. In some embodiments, the virus is a virus of the parvoviridae family. In some embodiments, the virus is a virus of the picornaviridae family. In some embodiments, the virus is a virus of the family polyomaviridae. In some embodiments, the virus is a virus of the poxviridae family. In some embodiments, the virus is a virus of the reoviridae family. In some embodiments, the virus is a virus of the retrovirus family. In some embodiments, the virus is a virus of the Rhabdoviridae family. In some embodiments, the virus is a virus of the togaviridae family.

In some embodiments, the virus is adenovirus, coronavirus, coxsackie virus, epstein-barr virus, hepatitis a virus, hepatitis b virus, hepatitis c virus, herpes simplex virus type 2, cytomegalovirus, human herpes virus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human papilloma virus, parainfluenza virus, polio virus, rabies virus, respiratory syncytial virus, rubella virus, or varicella-zoster virus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is Covid-19 (SARS-CoV-2). In some embodiments, the virus is a coxsackievirus. In some embodiments, the virus is epstein-barr virus. In some embodiments, the virus is hepatitis a virus. In some embodiments, the virus is hepatitis b virus. In some embodiments, the virus is hepatitis c virus. In some embodiments, the virus is herpes simplex virus type 2. In some embodiments, the virus is a cytomegalovirus. In some embodiments, the virus is human herpesvirus 8. In some embodiments, the virus is a human immunodeficiency virus. In some embodiments, the virus is an influenza virus. In some embodiments, the virus is measles virus. In some embodiments, the virus is mumps virus. In some embodiments, the virus is a human papilloma virus. In some embodiments, the virus is a parainfluenza virus. In some embodiments, the virus is a poliovirus. In some embodiments, the virus is rabies virus. In some embodiments, the virus is a respiratory syncytial virus. In some embodiments, the virus is a rubella virus. In some embodiments, the virus is varicella zoster virus.

In some embodiments, the pathogen is a bacterium. In some embodiments, the bacterium is a bacterium of the genus bacillus, bartonella, bordetella, brucella, campylobacter, chlamydia, chlamydophila, clostridium, corynebacterium, enterococcus, escherichia, franciscensis, haemophilus, helicobacter, legionella, leptospira, listeria, mycobacterium, mycoplasma, neisseria, pseudomonas, rickettsia, salmonella, shigella, staphylococcus, streptococcus, treponema, uroplasma, vibrio, or yersinia. In some embodiments, the bacteria are bacteria of the genus bacillus. In some embodiments, the bacterium is a bacterium of the genus bartonella. In some embodiments, the bacterium is a bacterium of the genus bordetella. In some embodiments, the bacterium is a bacterium of the genus borrelia. In some embodiments, the bacterium is a bacterium of the genus brucella. In some embodiments, the bacteria are campylobacter bacteria. In some embodiments, the bacterium is a chlamydia bacterium. In some embodiments, the bacterium is a chlamydophila bacterium. In some embodiments, the bacterium is a clostridium bacterium. In some embodiments, the bacterium is a coryneform bacterium. In some embodiments, the bacterium is a bacterium of the genus enterococcus. In some embodiments, the bacterium is a bacterium of the genus escherichia. In some embodiments, the bacterium is a bacterium of the genus franciscensis. In some embodiments, the bacterium is a bacterium of the genus haemophilus. In some embodiments, the bacteria are helicobacter bacteria. In some embodiments, the bacteria are bacteria of the genus legionella. In some embodiments, the bacteria are leptospira bacteria. In some embodiments, the bacterium is a listeria bacterium. In some embodiments, the bacterium is a mycobacterium genus bacterium. In some embodiments, the bacteria are mycoplasma bacteria. In some embodiments, the bacterium is a neisseria species bacterium. In some embodiments, the bacterium is a pseudomonas bacterium. In some embodiments, the bacterium is a bacterium of the genus rickettsia. In some embodiments, the bacteria are salmonella bacteria. In some embodiments, the bacterium is a shigella bacterium. In some embodiments, the bacterium is a staphylococcus bacterium. In some embodiments, the bacterium is a streptococcus bacterium. In some embodiments, the bacterium is a treponema bacterium. In some embodiments, the bacteria are bacteria of the genus uroplasms. In some embodiments, the bacteria are bacteria of the genus vibrio. In some embodiments, the bacterium is a bacterium of the genus yersinia.

In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is a protozoa, helminth, or ectoparasite. In some embodiments, the protozoa are amoeba, giardia, leishmania, intestinal bag worm, plasmodium, or cryptosporidium. In some embodiments, the worm is a trematode, tapeworm, acanthocellate or roundworm. In some embodiments, the ectoparasite is an arthropod.

The constant region variants can be introduced into any existing multispecific antibody platform or format known in the art, including any bispecific antibody format known in the art, to provide one or more additional antigen binding domains.

Such known multispecific antibody forms include multispecific antibodies obtained via controlled Fab arm exchange. Multispecific antibodies include IgG-like molecules having complementary CH3 domains that promote heterodimerization; a recombinant IgG-like dual targeting molecule, wherein each of the two sides of the molecule comprises a portion of a Fab fragment or Fab fragment of at least two different antibodies; an IgG fusion molecule, wherein a full length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment; an Fc fusion molecule, wherein a single chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, fc region, or portion thereof; fab fusion molecules in which different Fab fragments are fused together; heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and diabodies, wherein different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

In some embodiments, igG-like molecules with complementary CH3 domain molecules include Triomab/Quadroma (Trion Pharma/Fresenius Biotech), knob-in-hole antibodies (Genentech), crossMAbs (Roche), and electrostatic counterparts (amben), LUZ-Y (Genentech), strand-exchange engineering domain (Strand Exchange Engineered Domain body) (SEEDbody) (EMD Serono), biclonic (Merus), and DuoBody (Genmab A/S).

In some embodiments, recombinant IgG-like dual targeting molecules include Dual Targeting (DT) -Ig (GSK/domanis), diabodies (Genentech), cross-links Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX bodies (CovX/Pfizer).

In some embodiments, igG fusion molecules include Dual Variable Domains (DVD) -Ig (Abbott), igG-like bispecific antibodies (ImClone/Eli Lilly), ts2 abs (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).

In some embodiments, the Fc fusion molecules can include ScFv/Fc fusions (Academic Institution), SCORPHON (Emergent BioSolutions/Trubion, zymogenetics/BMS), dual affinity re-targeting technology (Fc-DART) (MacroGenics), and bis (ScFv) 2-Fab (National Research Center for Antibody Medicine- -China).

In some embodiments, fab fusion bispecific antibodies include F (ab) 2 (Medarex/AMGEN), dual-Action or Bis-Fab (Genentech), dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific antibodies (Biotech), and Fab-Fv (UCB-Celltech). ScFv-based, diabody-based domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE) (Micromet), tandem diabodies (Tandab) (affied), amphipathic retargeting techniques (DART) (macrographics), single chain diabodies (Academic), TCR-like antibodies (AIT, receptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), double targeting nanobodies (Ablynx), double targeting heavy chain domain-only antibodies.

Other examples may be generated, for example, using Fab arm exchange (or half molecular exchange) between two monospecific bivalent antibodies by: substitution is introduced at the heavy chain CH3 junction in each half-molecule to facilitate heterodimer formation of two antibody half-molecules with different specificities in an in vitro cell-free environment or using co-expression. Fab arm exchange reactions are the result of disulfide isomerization reactions and CH3 domain dissociation-association. The heavy chain disulfide bond in the hinge region of the parent monospecific antibody is reduced. The resulting free cysteine of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine residue of the second parent monospecific antibody molecule while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm can be engineered to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a different epitope. Other methods of preparing multispecific antibodies are known and contemplated.

As used herein, "homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, "homodimer" refers to an antibody having two heavy chains containing the same CH3 amino acid sequence.

As used herein, "heterodimerization" refers to the interaction of two heavy chains having different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains containing different CH3 amino acid sequences.

The "knob and hole structure" strategy (see, e.g., PCT publication WO 2006/028936) can be used to generate full length bispecific antibodies. In short, selected amino acids that form the interface of CH3 domains in human IgG may be mutated at positions that affect CH3 domain interactions, thereby promoting heterodimer formation. Amino acids with small side chains (knob) are introduced into the heavy chain of an antibody that specifically binds a first antigen, and amino acids with large side chains (knob) are introduced into the heavy chain of an antibody that specifically binds a second antigen. After co-expression of the two antibodies, heterodimers are formed due to preferential interaction of the heavy chain with the "mortar" with the heavy chain with the "pestle". Exemplary CH3 substitution pairs forming a knob structure (expressed as modification positions in the first CH3 domain of the first heavy chain/modification positions in the second CH3 domain of the second heavy chain) are: T366Y/F405A, T366W/F405W, F W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F W/T394S and T366W/T366S_L368A_Y407V.

Other strategies may also be used, such as promoting heavy chain heterodimerization using electrostatic interactions by replacing positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, as in U.S. patent publication No. US 2010/0015133; U.S. patent publication No. US 2009/0182127; U.S. patent publication No. US2010/028637; or as described in U.S. patent publication No. US 2011/0123032. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modification position in the first CH3 domain of the first heavy chain/modification position in the second CH3 domain of the second heavy chain): l351Y_F405AY407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K F, L351Y 407A/T366V K409F Y407A/T366A_K409F or T350V_L351Y_F405A Y407V/T350V_T366L_K392L_T394W as described in U.S. patent publication No. 2012/0149876 or U.S. patent publication No. 2013/0195849.

In addition to the above-described methods, another known bispecific antibody format can also be produced according to the method described in international patent publication No. WO2011/131746 in an in vitro cell-free environment by: asymmetric mutations were introduced in the CH3 region of both monospecific homodimeric antibodies, and bispecific heterodimeric antibodies were formed from both parental monospecific homodimeric antibodies under reducing conditions to allow disulfide isomerization. In the method, the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; incubating the antibodies under reducing conditions sufficient to disulfide isomerize cysteines in the hinge region; thereby generating bispecific antibodies by Fab arm exchange. The incubation conditions may optionally be restored to non-reducing conditions. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably reducing agents selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. For example, the following conditions may be used: incubation is carried out for at least 90 minutes at a temperature of at least 20℃in the presence of at least 25mM 2-MEA or in the presence of at least 0.5mM dithiothreitol at a pH of from 5 to 8, for example at a pH of 7.0 or at a pH of 7.4.

5.2.1 monoclonal antibodies

Antibodies (including multispecific or multivalent antibodies) of the present disclosure may be monoclonal antibodies or derived from monoclonal antibodies. Monoclonal antibodies can be prepared using the hybridoma method described first by Kohler et al, 1975,Nature 256:495-97, or can be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Following immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, monoclonal Antibodies: principles and Practice 59-103 (1986)).

The hybridoma cells thus prepared are inoculated and cultured in a suitable medium that, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells (also referred to as fusion partners). For example, if the parent myeloma cell lacks the enzyme hypoxanthine guanine phosphoribosyl enzyme (HGPRT or HPRT), the selective medium for the hybridoma will typically comprise hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of cells that lack HGPRT.

Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable high level production of antibodies by selected antibody-producing cells, and are sensitive to selective media selected for unfused parent cells. Exemplary myeloma cell lines are murine myeloma cell lines such as SP-2 and derivatives, e.g., X63-Ag8-653 cells available from the American type culture Collection (Manassas, va.) and those derived from MOPC-21 and MPC-11 mouse tumors available from the Sork institute cell distribution center (San Diego, calif.). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, 1984, immunol.133:3001-05; and Brodeur et al, 1987,Monoclonal Antibody Production Techniques and Applications,51-63).

The medium in which the hybridoma cells are cultured for the production of monoclonal antibodies directed against the antigen is determined. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as RIA or ELISA. The binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard assay described by Munson et al, 1980, anal biochem. 107:220-39.

Once hybridoma cells producing antibodies of the desired specificity, affinity and/or activity are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, supra). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, for example, by injecting cells into the abdominal cavity of a mouse, the hybridoma cells can be cultured in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein a or protein G-agarose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or the like.

DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells can be used as a source of such DNA. After isolation, the DNA may be placed into expression vectors, which are then transfected into host cells such as e.coli cells, simian COS cells, chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody proteins, to effect synthesis of the monoclonal antibodies in the recombinant host cells. A review article on the recombinant expression of DNA encoding antibodies in bacteria includes Skerra et al 1993,Curr.Opinion in Immunol.5:256-62 and Pluckthun, 1992, immunol. Revs.130:151-88.

In another embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques such as those described in Antibody Phage Display: methods and Protocols (O' Brien and Aitken editors, 2002). In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry polynucleotide sequences encoding the functional antibody domains. Examples of phage display methods that can be used to prepare the antibodies described herein include those disclosed in the following: brinkman et al, 1995, J.Immunol. Methods182:41-50; ames et al, 1995,J.Immunol.Methods 184:177-186; kettlebough et al, 1994, eur. J. Immunol.24:952-958; persic et al, 1997, gene 187:9-18; burton et al, 1994,Advances in Immunology 57:191-280; PCT application PCT/GB91/O1 134; international publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401 and WO97/13844; and U.S. Pat. nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

In principle, synthetic antibody clones are selected by screening phage libraries containing phages displaying the antigen binding sites of antibodies fused to phage coat proteins. Such phage libraries are screened against the desired antigen. Clones expressing antigen binding sites capable of binding to the desired antigen are adsorbed to the antigen and thus isolated from non-binding clones in the library. The binding clone is then eluted from the antigen and may be further enriched by additional antigen adsorption/elution cycles.

The antigen binding site may be within the variable domain of an antibody. The variable domains may be displayed functionally as Fab fragments (where each is fused to a constant domain and interacts non-covalently) on phage as described, for example, in Winter et al, 1994, ann. Rev. Immunol. 12:433-55.

The repertoire of VH and VL genes were cloned individually by PCR and randomly recombined in phage libraries, and then antigen binding clones could be searched for, as described in Winter et al, supra. Libraries from immunogens provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self antigens as well as self antigens without any immunization, as described by Griffiths et al 1993,EMBO J12:725-34. Finally, the original library can also be prepared synthetically by cloning unrearranged V-gene fragments from stem cells and using PCR primers containing random sequences for encoding highly variable CDR3 regions and completing the rearrangement in vitro as described, for example, in Hoogenboom and Winter,1992, J.mol.biol.227:381-88.

The antigen binding site may be outside the variable domain of the antibody. For example, the antigen binding site may be in the constant region of an antibody. A constant region library can be constructed by replacing the structural loops of the constant region with one or more highly variable antigen binding loops. Such constant region libraries are described in more detail in section 5.4 below.

Screening of the library may be accomplished by a variety of techniques known in the art. For example, a specific antigen (e.g., a polypeptide, fragment, or epitope of an antigen) can be used to coat the wells of an adsorption plate, expressed on host cells attached to an adsorption plate or used for cell sorting, conjugated to biotin for streptavidin-coated bead capture, or used in any other method to panning a display library. Antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by using long washes and monovalent phage display as described in Bass et al, 1990,Proteins 8:309-14 and WO 92/09690, and by using low antigen coating densities as described in Marks et al, 1992, biotechnol.10:779-83.

Antibodies can be obtained by: appropriate antigen screening programs are designed to select phage clones of interest, and then full-length antibody clones are constructed using VH and/or VL sequences (e.g., fv sequences), various CDR sequences from VH and VL sequences, or other antigen-binding sequences from phage clones of interest, and appropriate constant region (e.g., fc) sequences as described in Kabat et al (supra).

Antibodies described herein may also include, for example, chimeric antibodies. Chimeric antibodies are molecules in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, e.g., morrison,1985,Science 229:1202; oi et al, 1986,BioTechniques 4:214; gilles et al 1989,J.Immunol.Methods 125:191-202; and U.S. Pat. nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

Antibodies or antigen binding fragments (e.g., fab) produced using techniques such as those described herein can be isolated using well known standard techniques. For example, the antibody or antigen binding fragment may be suitably separated from, for example, a culture medium, ascites fluid, serum, cell lysates, synthetic reaction materials, and the like, by conventional immunoglobulin purification procedures such as, for example, protein a-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. As used herein, an "isolated" or "purified" antibody is substantially free of cellular material or other proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.

5.2.2 antibody fragments

The present disclosure provides multispecific antibodies comprising antibody fragments that bind to more than one antigen. In some embodiments, provided herein is a binding molecule comprising one or more antibody fragments and one or more constant region variants provided herein.

Various techniques have been developed to generate antibody fragments. Traditionally, these fragments are derived via proteolysis of the intact antibody (see, e.g., morimoto et al, 1992,J.Biochem.Biophys.Methods 24:107-17; and Brennan et al, 1985,Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, fv and scFv antibody fragments are all expressed and secreted in e.coli or yeast cells, allowing for easy production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab') 2 fragments (Carter et al, 1992, bio/Technology 10:163-67). According to another method, the F (ab') 2 fragment may be isolated directly from the recombinant host cell culture. Fab and F (ab') 2 fragments comprising salvage receptor binding epitope residues with increased in vivo half-life are described, for example, in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. scFv fusion proteins can be constructed to produce fusion of a binding molecule provided herein at the amino or carboxy terminus of an scFv (see, e.g., borrebaeck editions, supra). An antibody fragment may also be a "linear antibody", for example, as described in the references cited above. Such linear antibodies may be monospecific or multispecific, such as bispecific.

The smaller antibody-derived binding structure is a separate variable domain (V domain), also known as a single variable domain antibody (sdAb). Certain types of organisms, alpaca and cartilaginous fish, have a single V-like domain with high affinity that is mounted on an Fc equivalent domain structure that is part of their immune system. (Woolven et al 1999,Immunogenetics 50:98-101; and Streltsov et al 2004,Proc Natl Acad Sci USA.101:12444-49). The V-like domain (called VhH in alpaca and V-NAR in shark) generally shows a long surface loop that allows for luminal penetration of the target antigen. They also stabilize the isolated VH domains by masking hydrophobic surface plaques.

These VhH and V-NAR domains have been used to engineer sdabs. Human V domain variants have been designed using other methods for selection from phage libraries and generation of stable, highly binding VL and VH derived domains. The sdAb fusion proteins can be constructed to produce fusion of the binding molecules provided herein at the amino or carboxy terminus of an sdAb.

Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. The immunoglobulin molecules provided herein may be of any class (e.g., igG, igE, igM, igD and IgA) or subclass (e.g., igG1, igG2, igG3, igG4, igA1 and IgA 2) of immunoglobulin molecules. In a specific embodiment, the antibodies provided herein are IgG antibodies, such as IgG1 antibodies, igG2 antibodies, or IgG4 antibodies (e.g., igG4 null and variants of IgG4 antibodies). In a specific embodiment, the IgG antibody is an IgG1 antibody. In some embodiments, the IgG antibody comprises a mutated Fc region with enhanced Fc effector function.

Variants and derivatives of antibodies include functional fragments of antibodies that retain the ability to bind to a specific antigen. Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen binding domain and comprising a light chain and a portion of a heavy chain bridged by a disulfide bond); fab' (e.g., antibody fragments containing a single antigen binding domain comprising Fab and an additional portion of the heavy chain through the hinge region); f (ab ') 2 (e.g., two Fab ' molecules linked by an interchain disulfide linkage in the hinge region of the heavy chain; fab ' molecules may be directed against the same or different epitopes); bispecific Fab (e.g., fab molecules having two antigen binding domains, wherein each antigen binding domain can be directed against a different epitope).

5.2.3 humanized antibodies

Antibodies described herein can, for example, include humanized antibodies (e.g., deimmunized or complex human antibodies).

Humanized antibodies may comprise human framework regions and human constant region sequences. For example, a humanized antibody may comprise human constant region sequences. In certain embodiments, the humanized antibody may be selected from any class of immunoglobulin, including IgM, igG, igD, igA and IgE, and any isotype, including IgG1, igG2, igG3, and IgG4 (e.g., variants of IgG4 and IgG4 null). In certain embodiments, the humanized antibody may comprise kappa or lambda light chain constant sequences.

Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European patent EP 239,400; international publication WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), veneering or resurfacing (European patent EP 592,106 and EP 519,596;Padlan,1991,Molecular Immunology 28 (4/5): 489-498; studnica et al, 1994,Protein Engineering 7 (6): 805-814; and Roguska et al, 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniques such as those disclosed in the following references: U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 93/17105, tan et al, J.Immunol.169:111925 (2002), caldas et al, protein Eng.13 (5): 353-60 (2000), morea et al, methods 20 (3): 26779 (2000), baca et al, J.biol. Chem.272 (16): 10678-84 (1997), roguska et al, protein Eng.9 (10): 895 904 (1996), couto et al, cancer Res.55 (23 supplements): 5973s-5977s (1995), couto et al, cancer Res.55 (8): 1717-22 (1995), sandhu JS, gene 150 (2): 409-10 (1994), and Pedersen et al, J.mol.biol.235 (3): 959-73 (1994). See also U.S. patent publication No. US 2005/0042664A1 (24.2.2005), each of which is incorporated herein by reference in its entirety.

Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization may be, for example, according to Jones et al, 1986,Nature 321:522-25; riechmann et al 1988,Nature 332:323-27; and Verhoeyen et al, 1988,Science 239:1534-36) by substituting hypervariable region sequences for the corresponding sequences of human antibodies.

In some cases, humanized antibodies are constructed by CDR grafting, wherein the amino acid sequences of the six CDRs of a parent non-human (e.g., rodent) antibody are grafted onto a human antibody framework. For example, padlan et al determined that only about one third of the residues in the CDRs actually contacted the antigen, and these residues were referred to as "specificity determining residues" or SDR (Padlan et al 1995,FASEB J.9:133-39). In SDR grafting techniques, only SDR residues are grafted onto human antibody frameworks (see, e.g., kashmiri et al, 2005,Methods 36:25-34).

The choice of human variable domains (both light and heavy chains) used to make humanized antibodies is important for reducing antigenicity. For example, the sequences of the variable domains of non-human (e.g., rodent) antibodies are screened against an entire library of known human variable domain sequences according to a so-called "best fit" method. The human sequence closest to the rodent may be selected as the human framework for the humanized antibody (Sims et al, 1993, J.Immunol.151:2296-308; and Chothia et al, 1987, J.mol. Biol. 196:901-17). Another approach uses a specific framework of consensus sequences from all human antibodies of a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al, 1992,Proc.Natl.Acad.Sci.USA 89:4285-89; and Presta et al, 1993, J.Immunol.151:2623-32). In some cases, the framework is derived from the most abundant human subgroup VL6 subgroup I (VL 6I) and VH subgroup III (VHIII) consensus sequences. In another approach, human germline genes are used as a source of framework regions.

In an alternative example of CDR-based comparison, known as superhumanization, FR homology is irrelevant. The method consists of comparison of non-human sequences with a functional human germline genomic library. Those genes encoding the same or closely related canonical structures were then selected for the murine sequences. Next, among genes sharing a canonical structure with a non-human antibody, a gene having the highest homology within CDRs is selected as an FR donor. Finally, non-human CDRs are grafted onto these FRs (see, e.g., tan et al, 2002, J. Immunol. 169:1119-25).

It is also generally desirable that antibodies be humanized while retaining their affinity for antigens and other advantageous biological properties. To achieve this objective, according to one method, humanized antibodies are prepared by an analytical process of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees,2000,Protein Eng.13:819-24), modeller (Sali and Bluntell, 1993, J.Mol. Biol. 234:779-815), swiss PDB Viewer (Guex and Peitsch,1997,Electrophoresis 18:2714-23). These displayed assays allow for analysis of the likely role of the residues in the functional functioning of the candidate immunoglobulin sequence, e.g., analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the receptor and input sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. Typically, the hypervariable region residues are directly and substantially mostly involved in influencing antigen binding.

Another method for antibody humanization is based on a measure of antibody humanization known as human chain content (HSC). The method compares mouse sequences to a repertoire of human germline genes, and differences are scored as HSCs. The target sequence was then humanized by maximizing its HSC, rather than using global identity measurements, to generate a number of different humanized variants (Lazar et al, 2007, mol. Immunol. 44:1986-98).

In addition to the methods described above, empirical methods can also be used to generate and select humanized antibodies. These methods include methods based on large gene libraries for the generation of humanized variants and selection of optimal clones using enrichment techniques or high throughput screening techniques. Antibody variants can be isolated from phage, ribosome and yeast display libraries and isolated by bacterial colony screening (see, e.g., hoogenboom,2005, nat. Biotechnol.23:1105-16; dufner et al 2006,Trends Biotechnol.24:523-29; feldhaus et al 2003, nat. Biotechnol.21:163-70; and Schlapschy et al 2004,Protein Eng.Des.Sel.17:847-60).

In the FR library approach, a collection of residue variants is introduced at specific positions in the FR, and the library is then screened to select the FR that best supports the grafted CDRs. Residues to be substituted may include some or all of the "Vernier" residues identified as potentially contributing to the CDR structure (see, e.g., foote and Winter,1992, J. Mol. Biol. 224:487-99), or a more limited set of target residues identified by Baca et al (1997, J. Biol. Chem. 272:10678-84).

In FR shuffling, intact FRs are combined with non-human CDRs, rather than generating a combinatorial library of selected residue variants (see, e.g., dall' Acqua et al, 2005,Methods 36:43-60). The method can be carried out in two steps: the VL is first humanized and then the humanized VH is screened for libraries for binding. Alternatively, a one-step FR shuffling method may be used. This approach has proven to be more efficient than two-step screening because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity and thermostability (see, e.g., damschromar et al, 2007, mol. Immunol. 44:3049-60).

The "human engineering" method is based on experimentally identifying the Minimum Specificity Determinant (MSD) necessary and on sequential substitution of non-human fragments into a human FR library and assessing binding. It starts at the CDR3 region of the non-human VH and VL chains and gradually replaces other regions of the non-human antibody with human FR, including CDR1 and CDR2 of both VH and VL. This approach generally results in epitope retention and identification of antibodies with multiple subclasses of different human V-segment CDRs. Human engineering allows isolation of antibodies 91% -96% homologous to human germline gene antibodies (see, e.g., alfenito, cambridge Healthtech Institute's Third Annual PEGS, the Protein Engineering Summit, 2007).

The "human engineering" method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody having reduced immunogenicity in humans, which antibody still retains the desired binding properties of the original non-human antibody. Generally, the technology involves classifying amino acid residues of a non-human (e.g., mouse) antibody as "low-risk", "medium-risk", or "high-risk" residues. Classification is performed using an overall risk/return calculation that evaluates the predictive benefit of making a specific substitution (e.g., for immunogenicity in humans) for the risk that the substitution will affect the resulting antibody fold. The particular human amino acid residues to be substituted at a given position (e.g., low or medium risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning the amino acid sequence from the non-human antibody variable region with the corresponding region of the particular or consensus human antibody sequence. Amino acid residues at low or moderate risk positions in non-human sequences may be substituted for corresponding residues in human antibody sequences, depending on the alignment. In Studnica et al, 1994,Protein Engineering 7:805-14; U.S. Pat. nos. 5,766,886, 5,770,196, 5,821,123 and 5,869,619; and PCT publication WO 93/11794 describes in more detail techniques for preparing an ergonomic protein.

For example, complex human antibodies can be used ^TM The technology (anti ltd., cambridge, united Kingdom) produces complex human antibodies. To generate a composite human antibody, variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody. Such antibodies may comprise human constant region sequences, such as human light and/or heavy chain constant regions.

Deimmunized antibodies are antibodies in which T cell epitopes have been removed. Methods for preparing deimmunized antibodies have been described. See, for example, jones et al, methods Mol biol 2009;525:405-23, xiv, and De Groot et al, cell. Immunol.244:148-153 (2006)). The deimmunized antibodies comprise variable regions depleted of T cell epitopes and human constant regions. Briefly, VH and VL of antibodies are cloned, and T cell epitopes are then identified by testing overlapping peptides derived from VH and VL of antibodies in a T cell proliferation assay. T cell epitopes are identified electronically to identify peptides that bind to human MHC class II. Mutations were introduced into VH and VL to eliminate binding to human MHC class II. The mutated VH and VL are then used to generate deimmunized antibodies.

5.2.4 human antibodies

In specific embodiments, the antibodies provided herein comprise fully human antibodies or fragments thereof. Fully human antibodies may be produced by any method known in the art. The human antibodies provided herein can be constructed by combining Fv clone variable domain sequences selected from phage display libraries of human origin with known human constant domain sequences. Alternatively, the human monoclonal antibodies of the present disclosure may be prepared by a hybridoma method. Human myeloma and murine-human heterologous myeloma cell lines for the preparation of human monoclonal antibodies have been described, for example by Kozbor,1984, j.immunol.133:3001-05; brodeur et al Monoclonal Antibody Production Techniques and Applications,51-63 (1987); and Boerner et al, 1991, J.Immunol.147:86-95.

It is also possible to produce transgenic animals (e.g., mice) that are capable of producing an entire repertoire of human antibodies after immunization in the absence of endogenous immunoglobulin production. Transgenic mice expressing a repertoire of human antibodies have been used to generate high affinity human sequence monoclonal antibodies against a variety of potential drug targets (see, e.g., jakobovits, a.,1995, curr. Opin. Biotechnol.6 (5): 561-66; bruggemann and Taussing,1997, curr. Opin. Biotechnol.8 (4): 455-58; U.S. Pat. nos. 6,075,181 and 6,150,584; and Lonberg et al, 2005,Nature Biotechnol.23:1117-25).

Alternatively, human antibodies may be prepared via immortalization of human B lymphocytes that produce antibodies to the target antigen (e.g., such B lymphocytes may be recovered from the individual or may have been immunized in vitro) (see, e.g., cole et al, monoclonal Antibodies and Cancer Therapy (1985); boerner et al, 1991, J. Immunol.147 (1): 86-95; and U.S. Pat. No. 5,750,373).

Gene shuffling can also be used to derive human antibodies from non-human (e.g., rodent) antibodies, where the human antibodies have similar affinity and specificity as the starting non-human antibodies. According to this method, which is also referred to as "epitope imprinting" or "guided selection", the heavy or light chain variable regions of the non-human antibody fragments obtained by phage display techniques as described herein are replaced by a human V domain gene library, resulting in a population of non-human/human Fab chimeras. The selection with antigen results in the isolation of a non-human chain/human chain chimeric Fab wherein the human chain reverts to the antigen binding site destroyed after removal of the corresponding non-human chain in the primary phage display clone (e.g., epitope directs (imprints) selection of human chain partners). When this process is repeated to displace the remaining non-human chains, human antibodies are obtained (see, e.g., PCT WO 93/06213; and Osbourn et al, 2005,Methods 36:61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies that do not have FR or CDR residues of non-human origin. Examples of pilot selections for humanization of mouse antibodies to cell surface antigens include folate binding proteins present on ovarian cancer cells (see, e.g., figini et al, 1998,Cancer Res.58:991-96) and CD147 highly expressed on hepatocellular carcinoma (see, e.g., bao et al, 2005,Cancer Biol.Ther.4:1374-80).

A potential disadvantage of the guided selection method is that shuffling of one antibody chain while keeping the other constant may lead to epitope drift. In order to maintain epitopes recognized by non-human antibodies, CDR retention may be used (see, e.g., klimka et al, 2000, br. J. Cancer.83:252-60; and Beiboer et al, 2000, J. Mol. Biol. 296:833-49). In this approach, the non-human VH CDR3 is typically retained because this CDR may be centered at the antigen binding site and may be the most important region of the antibody for antigen recognition. However, in some cases, VH CDR3 and VL CDR3, and VH CDR2, VL CDR2, and VL CDR1 of the non-human antibody may be retained.

5.2.5 Fc engineering

It may be desirable to modify the antibodies provided herein by Fc engineering. In certain embodiments, modification of the Fc region of an antibody results in a reduction or elimination of effector function of the antibody. In certain embodiments, the effector function is ADCC, ADCP and/or CDC. In some embodiments of each or any of the above or below embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector functions are ADCC and ADCP. In one embodiment, the effector functions are ADCC and CDC. In one embodiment, the effector functions are ADCP and CDC. In one embodiment, the effector functions are ADCC, ADCP and CDC. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody.

In certain embodiments, modification of the Fc region of an antibody results in enhancement of effector function of the antibody. In certain embodiments, the effector function is ADCC, ADCP and/or CDC. In some embodiments of each or any of the above or below embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector functions are ADCC and ADCP. In one embodiment, the effector functions are ADCC and CDC. In one embodiment, the effector functions are ADCP and CDC. In one embodiment, the effector functions are ADCC, ADCP and CDC. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. In some embodiments of each or any of the embodiments above or below, the antibody is engineered using knob-in-holes (KIH) technology.

To extend the serum half-life of antibodies, salvage receptor binding epitopes can be incorporated into antibodies (particularly antibody fragments), for example, as described in U.S. Pat. No. 5,739,277. The term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., igG1, igG2, igG3, or IgG 4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

5.2.6 alternative conjugates

The present disclosure includes non-immunoglobulin binding agents that specifically bind to the same epitope as antibodies disclosed herein. In some embodiments of each or any of the above or below embodiments, the non-immunoglobulin conjugate is identified as an agent that replaces or is replaced by an antibody of the present disclosure in a competitive binding assay. These alternative binding agents may include, for example, any engineered protein scaffold known in the art. Such scaffolds include, for example, anti-cargo proteins based on a lipocalin scaffold, which is a protein structure characterized by a rigid β -barrel supporting four hypervariable loops forming a ligand binding site. The novel binding specificities can be engineered by targeted random mutagenesis in the loop region in combination with functional display and guided selection (see, e.g., skerra,2008, FEBS J., volume 275: pages 2677-2683). Other suitable scaffolds may include, for example, an adnectin or monomer based on the tenth extracellular domain of human fibronectin III (see, e.g., koide and Koide,2007,Methods Mol.Biol. Vol. 352: pages 95-109); an affibody based on the Z domain of staphylococcal protein A (see, e.g., nygren et al, 2008, FEBS J.,. 275:2668-2676); DARPin based on ankyrin repeat protein (see, e.g., stumpp et al, 2008, drug. Discovery. Today, volume 13: pages 695-701); fynomer based on the SH3 domain of human Fyn protein kinase (see, e.g., grablovski et al, 2007, J.biol. Chem. Volume 282: pages 3196-3204); africins based on Sac7d from sulfolobus acidophilus (Sulfolobus acidolarius) (see, e.g., krehhenbrink et al, 2008, J.mol. Biol. Volume 383: pages 1058-1068); affilin based on human y-B-crystallin (see, e.g., ebersbach et al, 2007, J.mol. Biol. Vol. 372:172-185); high affinity multimers based on the A domain of membrane receptor proteins (see, e.g., silverman et al 2005, biotechnol. 23:1556-1561); cysteine-rich knotting element peptides (see, e.g., kolmar,2008, febs J., volume 275: pages 2684-2690); and engineered Kunitz-type inhibitors (see, e.g., nixon and Wood,2006, curr. Opin. Drug. Discovery. Dev. Volume 9: pages 261-268). For reviews, see, e.g., gebauer and Skerra,2009, curr. Opin. Chem. Biol.13:245-55.

5.2.7 antibody variants

In some embodiments of each or any of the above or below embodiments, amino acid sequence modifications of the antibody or antigen binding fragment are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity or solubility. Thus, it is contemplated that antibody variants may be prepared in addition to the antibodies described herein. For example, antibody variants may be prepared by introducing appropriate nucleotide changes into the encoding DNA and/or by synthesizing the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter the post-translational processes of the antibody, such as altering the number or position of glycosylation sites or altering membrane anchoring characteristics.

In some embodiments of each or any of the embodiments above or below, the antibodies provided herein are chemically modified, for example, by covalently linking any type of molecule to the antibody. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In addition, antibodies may contain one or more atypical amino acids.

A variant may be a substitution, deletion, or insertion of one or more codons encoding an antibody or polypeptide that results in a change in the amino acid sequence as compared to the native sequence antibody or polypeptide. Amino acid substitutions may be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as replacing leucine with serine, e.g., a conservative amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution at one or more predicted nonessential amino acid residues. The allowed variants can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full length or mature native sequence.

Amino acid sequence insertions include amino 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 fusions of the N-terminus or C-terminus of an antibody with an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or polypeptide, which increases the serum half-life of the antibody.

A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. The art has defined families of amino acid residues with similarly charged side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations may be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants may be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein may be expressed and the activity of the protein may be determined.

Basic modifications in the biological properties of antibodies are achieved by selection of substitutions that maintain the structure of the (a) polypeptide backbone in the substitution region, e.g., as a folded or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the effect of the bulk of the side chain is significantly different. Alternatively, conservative substitutions (e.g., within amino acid groups having similar properties and/or side chains) may be made in order to maintain or not significantly alter the properties. Amino acids may be grouped according to their similarity in side chain characteristics (see, e.g., lehninger,Biochemistry73-75 (2 nd edition 1975)): (1) nonpolar: ala (A), val (V), leu (L), ile (I), pro (P), phe (F), trp (W), met (M); (2) uncharged polarity: gly (G), ser (S), thr (T), cys (C), tyr (Y), asn (N), gln (Q); (3) acidity: asp (D), glu (E); and (4) alkaline: lys (K), arg (R), his (H).

Alternatively, naturally occurring residues can be divided into several groups based on common side chain characteristics: (1) hydrophobicity: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions require the exchange of members of one of these classes for another class. Such substituted residues may also be introduced into conserved substitution sites or into the remaining (non-conserved) sites.

Variations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., carter,1986,Biochem J.237:1-7; and Zoller et al, 1982,Nucl.Acids Res.10:6487-500), cassette mutagenesis (see, e.g., wells et al, 1985, gene 34:315-23), or other known techniques may be performed on cloned DNA to generate antibody variant DNA.

Any cysteine residue that does not participate in maintaining the correct conformation of the antibodies provided herein may also be substituted, for example, with another amino acid, such as alanine or serine, to increase the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability (e.g., when the antibody is an antibody fragment such as an Fv fragment).

In some embodiments of each or any of the above or below embodiments, the antibody molecule of the present disclosure is a "deimmunized" antibody. A "deimmunized" antibody is an antibody derived from a humanized or chimeric antibody that has one or more changes in its amino acid sequence that result in a decrease in the immunogenicity of the antibody as compared to the corresponding original non-deimmunized antibody. One method of producing such antibody mutants includes identifying and removing T cell epitopes of the antibody molecule. In a first step, the immunogenicity of an antibody molecule can be determined by several methods, for example, by in vitro determination of T cell epitopes or in silico prediction of such epitopes as known in the art. Once the critical residues for T cell epitope function are identified, mutations can be made to remove immunogenicity and preserve antibody activity. For reviews, see, for example, jones et al, 2009,Methods in Molecular Biology 525:405-23.

5.2.8 in vitro affinity maturation

In some embodiments, antibody variants having improved properties (such as affinity, stability, or expression level) compared to the parent antibody may be prepared by in vitro affinity maturation. In vitro affinity maturation is based on the principle of mutation and selection, as in the natural prototype. The antibody library is displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cells) or associated (e.g., covalent or non-covalent) with its encoding mRNA or DNA. Affinity selection for the displayed antibodies allows for isolation of organisms or complexes carrying genetic information encoding the antibodies. Two or three rounds of mutation and selection using a display method such as phage display typically produce antibody fragments with affinities in the low nanomolar range. Affinity matured antibodies may have nanomolar or even picomolar affinity for the target antigen.

Phage display is a popular method for displaying and selecting antibodies. The antibodies were displayed on the surface of Fd or M13 phage as fusions with phage coat proteins. Selection involves exposure to an antigen to allow phage-displayed antibodies to bind to their target, a process known as "panning". Phage that bind to the antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For reviews, see, e.g., hoogenboom,2002, methods. Mol. Biol.178:1-37; and Bradbury and Marks,2004,J.Immunol.Methods 290:29-49.

In yeast display systems (see, e.g., boder et al, 1997, nat. Biotech.15:553-57; and Chao et al, 2006, nat. Protocols 1:755-68), antibodies can be fused to the adhesion subunit of the yeast lectin protein Aga2p (which is attached to the yeast cell wall by disulfide bonds that bind to Aga1 p). Displaying the protein via Aga2p allows the protein to protrude from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Using magnetic separation and flow cytometry sievesLibraries are selected to select antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling the yeast with a biotinylated antigen and a second reagent such as streptavidin conjugated to a fluorophore. Variants expressed on the surface of the antibody can be measured by immunofluorescent labelling of the hemagglutinin or c-Myc epitope tag flanking the Fab. Expression has been shown to correlate with the stability of the displayed protein and antibodies with improved stability and affinity can therefore be selected (see, e.g., shusta et al 1999, J.mol. Biol. 292:949-56). Another advantage of yeast display is that the displayed protein is folded in the endoplasmic reticulum of eukaryotic yeast cells using an endoplasmic reticulum partner and a quality control mechanism. Once maturation is complete, the antibody affinity can be conveniently "titrated" while displayed on the yeast surface, eliminating the need for expression and purification of each clone. The theoretical limit of yeast surface display is that the size of the functional library may be smaller than other display methods; however, the most recent method uses a mating system of yeast cells to produce a size estimate of 10 ¹⁴ (see, e.g., U.S. patent publication 2003/0186374; and Blaise et al, 2004,Gene 342:211-18).

In ribosome display, antibody-ribosome-mRNA (ARM) complexes were generated for selection in a cell-free system. The DNA library encoding a particular antibody library is fused to a spacer gene lacking a stop codon. When translated, the spacer sequence remains attached to the peptidyl tRNA and occupies the ribosomal channel, and thus allows the protein of interest to protrude from the ribosome and fold. The resulting complex of mRNA, ribosome and protein can bind to surface-bound ligands, allowing simultaneous isolation of antibodies and their encoding mRNA by affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back into cDNA, which can then be subjected to mutagenesis and used for the next round of selection (see, e.g., fukuda et al, 2006,Nucleic Acids Res.34:e127). In mRNA display, puromycin was used as an adapter molecule to establish covalent bonds between the antibody and the mRNA (Wilson et al, 2001,Proc.Natl.Acad.Sci.USA 98:3750-55).

Since these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of the bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be easily introduced after each selection round, e.g., by non-proofreading polymerase, since the library does not have to be transformed after any diversification step.

In some embodiments of each or any of the above or below embodiments, a mammalian display system may be used.

Diversity can also be introduced into the CDRs of an antibody library in a targeted manner or by random introduction. The former approach involves targeting all CDRs of an antibody by high or low levels of mutagenesis sequences or targeting isolated hot spots of somatic hypermutation (see, e.g., ho et al 2005, j. Biol. Chem. 280:607-17) or residues suspected to affect affinity based on experimental basis or structural reasons. Diversity can also be introduced by replacing naturally diverse regions, via DNA shuffling or similar techniques (see, e.g., lu et al, 2003, J. Biol. Chem.278:43496-507; U.S. Pat. Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending into framework region residues (see, e.g., bond et al, 2005, mol. Biol. 348:699-709), employ loop deletions and insertions in CDRs or use hybridization-based diversification (see, e.g., U.S. patent publication 2004/0005709). Additional methods of generating CDR diversity are disclosed, for example, in U.S. Pat. No. 7,985,840. Other methods that may be used to generate antibody libraries and/or antibody affinity maturation are disclosed, for example, in U.S. patent nos. 8,685,897 and 8,603,930 and U.S. publications 2014/0170705, 2014/0094392, 2012/0028301, 2011/0183855 and 2009/007538, each of which is incorporated herein by reference.

Screening of the library may be accomplished by a variety of techniques known in the art. For example, the antibodies may be immobilized onto a solid support, column, needle or cellulose/poly (vinylidene fluoride) membrane/other filter, expressed on host cells attached to an adsorption plate or for cell sorting, or conjugated to biotin for capture with streptavidin-coated beads or any other method for panning a display library.

For a review of in vitro affinity maturation methods, see, e.g., hoogenboom,2005, nature Biotechnology 23:23:1105-16; quiroz and Sinclair,2010, revista IngeneriaBiomedia4:39-51; and references therein.

5.2.9 antibody modification

Covalent modifications of antibodies that bind to specific antigens are included within the scope of the present disclosure. Covalent modification includes reacting the targeted amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N-or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, hydroxy phosphorylation of seryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine and histidine side chains (see, e.g., creghton, Proteins:Structure and Molecular Properties79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Other types of covalent modifications of the antibodies provided herein, including within the scope of the present disclosure, include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., beck et al, 2008, curr.pharm.biotechnol.9:482-501; and Walsh,2010,Drug Discov.Today 15:773-80), and attaching the antibody to one of a variety of non-protein polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene) in a manner such as described in U.S. Pat. nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

The antibodies of the present disclosure may also be modified to form chimeric molecules comprising an antibody fused to another heterologous polypeptide or amino acid sequence, such as an epitope tag (see, e.g., terpe,2003, appl. Microbiol. Biotechnol. 60:523-33) or the Fc region of an IgG molecule (see, e.g., aruffo,Antibody Fusion Proteins221-42 (Chamow and Ashkenazi editions, 1999)).

Also provided herein are antibody panels that bind to specific antigens. In particular embodiments, the antibody panels have different association rates, different dissociation rates, different affinities for specific antigens, and/or different specificities for specific antigens. In some embodiments of each or any of the above or below embodiments, the panel comprises or consists of: about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 antibodies or more. In some embodiments of each or any of the above or below embodiments, the panel comprises about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 2000, or about 3000 antibodies, or any number of antibodies within a range defined by any two of the foregoing values. Antibody panels can be used, for example, in 96-well or 384-well plates for assays such as ELISA.

5.2.10 immunoconjugates

The present disclosure also provides conjugates comprising any one of the antibodies of the disclosure covalently bound to one or more non-antibody reagents through a synthetic linker.

In some embodiments of each or any of the embodiments above or below, an antibody provided herein is conjugated or recombinantly fused, for example, to a therapeutic agent (e.g., a cytotoxic agent) or a diagnostic or detectable molecule. Conjugated or recombinant fusion antibodies can be used, for example, to treat or prevent a disease or disorder. Conjugated or recombinant fusion antibodies can be used, for example, to monitor or prognose the onset, progression, progress, and/or severity of a disease or disorder.

Such diagnosis and detection may be accomplished, for example, by coupling antibodies to detectable substances including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin/biotin or avidin/biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansyl chloride or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin or aequorin; chemiluminescent materials such as, but not limited to, acridinium ester-based compounds or HALOTAG; radioactive materials such as, but not limited to, iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115 In, 113In, 112In, and 111 In), technetium (99 Tc), thallium (201 Ti), gallium (68 Ga and 67 Ga), palladium (103 Pd), molybdenum (99 Mo), xenon (133 Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, or 117Sn; positron emitting metals using various positron emission tomography; and non-radioactive paramagnetic metal ions.

Also provided herein are antibodies that are recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) to a heterologous protein or polypeptide (or fragment thereof, e.g., to about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acid polypeptides) to generate fusion proteins, and uses thereof. In particular, provided herein are fusion proteins comprising an antigen binding fragment (e.g., CDR1, CDR2, and/or CDR 3) of an antibody provided herein and a heterologous protein, polypeptide, or peptide. In one embodiment, a heterologous protein, polypeptide, or peptide fused to an antibody can be used to target the antibody to a particular cell type.

In addition, the antibodies provided herein may be fused to a tag or "tag" sequence (such as a peptide) to facilitate purification. In particular embodiments, the tag or tag amino acid sequence is a hexahistidine peptide, such as a tag provided in a pQE vector (see, e.g., QIAGEN, inc.), and the like, many of which are commercially available. For example, hexahistidine provides for convenient purification of fusion proteins as described in Gentz et al, 1989,Proc.Natl.Acad.Sci.USA 86:821-24. Other peptide tags for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin protein (Wilson et al, 1984, cell 37:767-78) and "FLAG" tags.

Methods for fusing or conjugating portions (including polypeptides) to antibodies are known (see, e.g., arnon et al, monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al, 1985), hellstrom et al, antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al, 2 nd edition 1987), thorpe, antibody Carriers ofCytotoxic Agents in Cancer Therapy: A Review, monoclonal Antibodies: biological and Clinical Applications 475-506 (Picthera et al, 1985), analysis, results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, monoclonal Antibodies for Cancer Detection and Therapy-16 (Baldwin et al, 1985), thorpe et al, 1982, immunol. Rev.62:119-58, U.S. Pat. No. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,851; 5,125; 5,783,181, 908,626;5,844,5; and 5,112,946;EP 307,434;EP 367,166;EP 394,827;PCT, WO 91/0696/WO 91/0696, WO 331/1985; WO 97/723/1988, and WO 331/1988, and WO 93/or Prinser.82, and WO 82. Light, and WO 1/or Prinser.A, 1988; light, and WO 1/or Prinser.e.82, 1981).

Fusion proteins may be generated, for example, by techniques of gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively, "DNA shuffling"). DNA shuffling can be used to alter the activity of antibodies as provided herein, including, for example, antibodies with higher affinity and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793;5,811,238;5,830,721;5,834,252; and 5,837,458; patten et al, 1997,Curr.Opinion Biotechnol.8:724-33;Harayama,1998,Trends Biotechnol.16 (2): 76-82; hansson et al, 1999, J.mol. Biol.287:265-76; and Lorenzo and Blasco,1998,Biotechniques 24 (2): 308-13). The antibodies or encoded antibodies may be altered by random mutagenesis prior to recombination by error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding antibodies provided herein may be recombined with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

The antibodies provided herein can also be conjugated with a second antibody to form an antibody heteroconjugate, as described, for example, in U.S. Pat. No. 4,676,980.

Antibodies as provided herein may also be attached to a solid support, which is particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

The linker may be a "cleavable linker" that facilitates release of the conjugation agent in the cell, although non-cleavable linkers are also contemplated herein. Linkers for conjugates of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids such as valine and/or citrulline such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., chari et al, 1992,Cancer Res.52:127-31; and U.S. Pat. No. 5,208,020), thioether linkers, or hydrophilic linkers (see, e.g., kovtun et al, 2010,Cancer Res.70:2528-37).

Conjugates of antibodies and reagents can be prepared using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl- (4-vinyl sulfone) benzoate). The present disclosure also contemplates that conjugates of antibodies and reagents may be prepared using any suitable method as disclosed in the art (see, e.g., bioconjugate Techniques (Hermanson edit, 2 nd edition, 2008)).

Conventional conjugation strategies for antibodies and reagents have been based on random conjugation chemistry involving either epsilon-amino groups of Lys residues or thiol groups of Cys residues, which lead to heteroconjugates. Recently developed techniques allow site-specific conjugation to antibodies, resulting in uniform loading and avoiding subpopulations of conjugates with altered antigen binding or pharmacokinetics. Included in the "thiomab" engineering, which includes cysteine substitutions at positions on the heavy and light chains, provides reactive thiol groups and does not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., junutula et al, 2008, J.Immunol. Meth.332:41-52; and Junutula et al, 2008,Nature Biotechnol.26:925-32). In another approach, selenocysteine co-translation is inserted into antibody sequences by re-encoding the stop codon UGA from termination to selenocysteine insertion, allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of other natural amino acids (see, e.g., hofer et al, 2008,Proc.Natl.Acad.Sci.USA 105:12451-56; and Hofer et al, 2009,Biochemistry 48 (50): 12047-57).

5.3 Polynucleotide

In certain embodiments, the disclosure encompasses polynucleotides encoding antibodies described herein. The term "polynucleotide encoding a polypeptide" encompasses polynucleotides comprising only the coding sequence of the polypeptide and polynucleotides comprising additional coding and/or non-coding sequences. The polynucleotides of the present disclosure may be in RNA form or in DNA form. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand.

In certain embodiments, the polynucleotide comprises a coding sequence for a polypeptide fused in frame to a polynucleotide that facilitates, for example, expression and secretion of the polypeptide from a host cell (e.g., a leader sequence that serves as a secretion sequence for controlling transport of the polypeptide). The polypeptide may have a leader sequence that is cleaved by the host cell to form a "mature" form of the polypeptide.

In certain embodiments, the polynucleotide comprises a coding sequence for a polypeptide fused in frame with a tag or label sequence. For example, in some embodiments, the tag sequence is a hexahistidine tag provided by a vector that allows for efficient purification of the polypeptide fused to the tag in the case of a bacterial host. In some embodiments of each or any of the above or below embodiments, the marker is used in combination with other affinity tags.

The disclosure also relates to variants of the polynucleotides described herein, wherein the variants encode, for example, fragments, analogs, and/or derivatives of the polypeptides. In certain embodiments, the disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising an antibody or antigen binding fragment described herein.

As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, e.g., 95% 'identical' to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 'or 3' end positions of the reference nucleotide sequence or anywhere between those end positions, either interspersed with nucleotides of the reference sequence alone, or in one or more contiguous groups within the reference sequence.

The polynucleotide variants may contain alterations in the coding region, the non-coding region, or both. In some embodiments of each or any of the above or below embodiments, the polynucleotide variant comprises an alteration that produces a silent substitution, addition, or deletion without altering the property or activity of the encoded polypeptide. In some embodiments of each or any of the above or below embodiments, the polynucleotide variant comprises a silent substitution that does not result in a change in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants may be produced for a variety of reasons, for example, in order to optimize codon expression in a particular host (i.e., to alter codons in human mRNA to preferred codons in a bacterial host such as e.coli). In some embodiments of each or any of the above or below embodiments, the polynucleotide variant comprises at least one silent mutation in a non-coding region or coding region of the sequence.

In some embodiments of each or any of the above or below embodiments, the polynucleotide variant is produced to modulate or alter expression (or expression level) of the encoded polypeptide. In some embodiments of each or any of the above or below embodiments, the polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments of each or any of the above or below embodiments, the polynucleotide variant is produced to reduce expression of the encoded polypeptide. In some embodiments of each or any of the above or below embodiments, the polynucleotide variant has increased expression of the encoded polypeptide as compared to the parent polynucleotide sequence. In some embodiments of each or any of the above or below embodiments, the polynucleotide variant has reduced expression of the encoded polypeptide as compared to the parent polynucleotide sequence.

In certain embodiments, the present disclosure provides a polynucleotide comprising a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide listed in the sequence listings provided herein.

In certain embodiments, the present disclosure provides a polynucleotide comprising a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide selected from the group consisting of the polynucleotides provided herein.

In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure.

Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments of each or any of the above or below embodiments, the expression vector comprises a polynucleotide molecule. In some embodiments of each or any of the above or below embodiments, the host cell comprises an expression vector comprising a polynucleotide molecule. In some embodiments of each or any of the above or below embodiments, the host cell comprises one or more expression vectors comprising a polynucleotide molecule. In some embodiments of each or any of the above or below embodiments, the host cell comprises a polynucleotide molecule. In some embodiments of each or any of the above or below embodiments, the host cell comprises one or more polynucleotide molecules.

5.4 constant region library

In another aspect, provided herein is an antibody constant region library comprising a plurality of molecules, each comprising at least one engineered antibody constant region variant (e.g., a CH1 region variant and/or a CL region variant), and constant region variants in the plurality of molecules in the library comprise a plurality of loop regions. In some embodiments of each or any of the above or below embodiments, the library diversity may be in the range of about 10 ⁷ -10 ¹⁶ (for single rings) to about 10 ¹⁸ -10 ³³ (for bicyclic rings). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ⁶ –10 ⁷ Within a range of (2). In some embodiments, for single loops, library diversity may be at about 10 ⁷ –10 ⁸ Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ⁸ –10 ⁹ Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ⁹ –10 ¹⁰ Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ¹⁰ –10 ¹¹ Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ¹¹ –10 ¹² Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ¹² –10 ¹³ Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ¹³ –10 ¹⁴ Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ¹⁴ –10 ¹⁵ Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ¹⁵ –10 ¹⁶ Within a range of (2). In some embodiments of each or any of the above or below embodiments, the library diversity may be about 10 for a single loop ¹⁶ –10 ¹⁷ Within a range of (2).

Libraries of the invention can be constructed by introducing various amino acid sequences into at least one loop region (e.g., the CH1 region or the CL region) in an antibody constant region, and optionally replacing one or more amino acid residues within the loop region in the antibody constant region. For example, in some embodiments of each or any of the embodiments described above or below, the library provided herein comprises a plurality of molecules having different amino acid sequences that are introduced into and/or replace a region within the AB loop of the antibody constant region (e.g., the CH1 region or the CL region). In some embodiments of each or any of the embodiments above or below, the library provided herein comprises a plurality of molecules having different amino acid sequences that are introduced into and/or replace a region within the BC loop of the antibody constant region (e.g., the CH1 region or the CL region). In some embodiments of each or any of the embodiments above or below, the library provided herein comprises a plurality of molecules having different amino acid sequences that are introduced into and/or replace a region within the CD loop of the antibody constant region (e.g., the CH1 region or the CL region). In some embodiments of each or any of the embodiments above or below, the library provided herein comprises a plurality of molecules having different amino acid sequences that are introduced into and/or replace a region within the DE loop of the antibody constant region (e.g., the CH1 region or the CL region). In some embodiments of each or any of the embodiments above or below, the library provided herein comprises a plurality of molecules having different amino acid sequences that are introduced into and/or replace a region within the EF loop of the antibody constant region (e.g., the CH1 region or the CL region). In some embodiments of each or any of the embodiments above or below, the library provided herein comprises a plurality of molecules having different amino acid sequences that are introduced into and/or replace a region within the FG loop of the antibody constant region (e.g., the CH1 region or the CL region).

In some embodiments of each or any of the embodiments above or below, various amino acid sequences are introduced into and/or replace the AB, BC, CD, DE, EF and/or FG loop region amino acid fragments of the CH1 region. In some embodiments of each or any of the embodiments above or below, various amino acid sequences are introduced into and/or replace the AB, BC, CD, DE, EF and/or FG loop region of the CL region. In some embodiments of each or any of the embodiments above or below, various amino acid sequences are introduced into and/or replace the AB, BC, CD, DE, EF and/or FG loop region outside of the CH1 region. In some embodiments of each or any of the embodiments above or below, various amino acid sequences are introduced into and/or replace the AB, BC, CD, DE, EF and/or FG loop region outside of the CL region. In some embodiments of each or any of the above or below embodiments, various amino acid sequences are introduced into and/or replace the A, B, C, D, E and/or fβ chain region of the CH1 region. In some embodiments of each or any of the above or below embodiments, various amino acid sequences are introduced into and/or replace the A, B, C, D, E and/or fβ chain region of the CL region.

In some embodiments of each or any of the above or below embodiments, various amino acid sequences may be inserted into the loop region. Various amino acid sequences may be inserted into the AB loop region of the CH1 or CL region. Various amino acid sequences may be inserted into the BC loop region of the CH1 or CL region. Various amino acid sequences may be inserted into the CD loop region of the CH1 or CL region. Various amino acid sequences may be inserted into the DE loop region of the CH1 or CL region. Various amino acid sequences may be inserted into the EF loop region of the CH1 or CL region. Various amino acid sequences may be inserted into the FG loop region of the CH1 or CL region.

In other embodiments, various amino acid sequences may replace regions within the loop region. Various amino acid sequences may be substituted for the region within the AB loop region of the CH1 or CL region. Various amino acid sequences may be substituted for the regions within the BC loop region of the CH1 or CL region. Various amino acid sequences may be substituted for regions within the CD loop region of the CH1 or CL region. Various amino acid sequences may be substituted for the region within the DE loop region of the CH1 or CL region. Various amino acid sequences may be substituted for regions within the EF loop region of the CH1 or CL region. Various amino acid sequences may be substituted for the regions within the FG loop region of the CH1 or CL region.

In some embodiments of each or any of the embodiments above or below, the molecules in the libraries provided herein comprise different sequences in one loop region. In other embodiments, the molecules in the libraries provided herein comprise different sequences in two or more loop regions within one or more constant regions.

Thus, in some embodiments of each or any of the embodiments above or below, provided herein is a Fab Constant Region Library (CRL) comprising a population of molecules each comprising a region derived from a CH1 region of an antibody and/or a region derived from a CL region of an antibody, wherein the population of molecules comprises different amino acid sequences in the region derived from the CH1 region and/or the region derived from the CL region. In some embodiments of each or any of the embodiments above or below, the different amino acid sequences in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region. In some embodiments of each or any of the embodiments above or below, the different amino acid sequences in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CL region. In some embodiments of each or any of the embodiments above or below, the different amino acid sequences in the region derived from the CH1 region are located outside the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region. In some embodiments of each or any of the embodiments above or below, the different amino acid sequences in the region derived from the CL region are located outside the AB, BC, CD, DE, EF and/or FG loop region of the CL region. In some embodiments of each or any of the embodiments above or below, the different amino acid sequences in the region derived from the CH1 region are located in the A, B, C, D, E and/or fβ chain region of the CH1 region. In some embodiments of each or any of the embodiments above or below, the different amino acid sequences in the region derived from the CL region are located in the A, B, C, D, E and/or fβ chain region of the CL region.

In some embodiments of each or any of the embodiments above or below, the population of molecules comprises different amino acid sequences in one or both loop regions in the region derived from the CH1 region. In some embodiments of each or any of the embodiments above or below, the population of molecules comprises different amino acid sequences in one or both loop regions in the region derived from the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region. In some embodiments of each or any of the embodiments above or below, the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region. In some embodiments of each or any of the embodiments above or below, the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CL region. In some embodiments of each or any of the embodiments above or below, the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region. In some embodiments of each or any of the embodiments above or below, the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region and the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region. In some embodiments of each or any of the above or below embodiments, the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

In some specific embodiments, the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID NO. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO. 1. In some specific embodiments, the region derived from the CL region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the region derived from the CL region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID No. 2. In some specific embodiments, the region derived from the CL region is a region derived from a human CL lambda region comprising the amino acid sequence of SEQ ID No. 3, and wherein the region derived from the CL region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 3.

In some more specific embodiments, the amino acid residues TSG (EU numbering 164-166) of the CD loop of the human IgG1 CH1 region are replaced by a different amino acid sequence in a molecule in the Fab CRL. In some more specific embodiments, amino acid residue S (EU numbering 165) of the CD loop of the human IgG1 CH1 region is replaced with a different amino acid sequence in a molecule in the Fab CRL. In some more specific embodiments, the amino acid residues QSS (EU numbering 175-177) of the DE loop of the human IgG1 CH1 region are replaced by a different amino acid sequence in the molecule in the Fab CRL. In some more specific embodiments, amino acid residues SGNS (EU numbering 156-159) of the CD loop of the human clk region are replaced with a different amino acid sequence in the molecule in the Fab CRL. In some more specific embodiments, amino acid residues SKD (EU numbering 168-170) of the DE loop of the human clk region are replaced with a different amino acid sequence in the molecule in the Fab CRL. In some more specific embodiments, amino acid residue K of the DE loop of the human clk region (EU numbering 169) is replaced by a different amino acid sequence in the molecule in the Fab CRL.

The different amino acid sequences within a loop region in a population of molecules introduced into a library of the invention may be of different lengths or may be of the same length but of different sequences. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 7 to 15 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 7 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 8 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 9 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 10 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 11 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 12 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 13 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 14 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise 15 amino acid residues. In some embodiments of each or any of the above or below embodiments, the different amino acid sequences in one loop comprise more than 15 amino acid residues, such as 16, 17, 18, 19, 20 or more amino acid residues.

In addition to the various constant region variants, the molecules in the libraries of the invention may also comprise additional domains, such as VH and/or VL regions. In some embodiments of each or any of the above or below embodiments, each of the molecules in the library further comprises a VH region and a VL region. Thus, in some embodiments of each or any of the above or below embodiments, provided herein is a Fab Constant Region Library (CRL) comprising a population of binding molecules, wherein each binding molecule in the binding molecule comprises: (i) A first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain; and (ii) a second polypeptide comprising a light chain variable region (VL) and a region derived from a CL region of an antibody light chain, wherein the population of binding molecules comprises different amino acid sequences in the region derived from the CH1 region and/or the region derived from the CL region. In some specific embodiments, the molecules in the library of the invention are Fab fragments. In some embodiments of each or any of the above or below embodiments, the library provided herein can be constructed as described in section 7 below.

Furthermore, any antibody-form molecule can be used to construct the Fab CRLs of the present invention. In some embodiments of each or any of the embodiments above or below, different constant regions (e.g., different CH1 regions and/or CL regions) are introduced into the intact antibodies to generate the libraries of the invention. Antibodies can be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass. In a specific embodiment, the antibodies provided herein are IgG antibodies, such as IgG1 antibodies, igG2 antibodies, or IgG4 antibodies (e.g., igG4 null and variants of IgG4 antibodies). In a specific embodiment, the IgG antibody is an IgG1 antibody. In other embodiments, different constant regions (e.g., different CH1 and/or CL regions) are introduced into the intact antibody fragments to generate the libraries of the invention. Exemplary fragments include, but are not limited to, fab ', F (ab') 2, bispecific Fab, single chain Fab. Antibodies may be derived or derived from any animal source, including birds and mammals (e.g., humans, monkeys, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens). In certain embodiments, the antibodies provided herein are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of human immunoglobulins, and include antibodies isolated from a human immunoglobulin library or from mice expressing antibodies from human genes. In certain embodiments, the antibody is a whole mouse antibody. In certain embodiments, the antibody is a mouse-human chimeric antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a fully human antibody. In other embodiments, the antibodies provided herein are humanized antibodies (e.g., comprise human constant regions and framework regions). In some embodiments of each or any of the above or below embodiments, the antibody is a bispecific antibody. In some embodiments of each or any of the above or below embodiments, the antibody is a trispecific antibody. In some embodiments of each or any of the above or below embodiments, the antibody is a bispecific antibody. In some embodiments of each or any of the above or below embodiments, the antibody is a bivalent antibody. In some embodiments of each or any of the above or below embodiments, the antibody is a trivalent antibody. In some embodiments of each or any of the above or below embodiments, the antibody is a tetravalent antibody.

In yet another aspect, provided herein is a method of producing a binding molecule, the method comprising: a first step for performing a function of identifying antibody constant region variants capable of binding to an antigen; and a second step of constructing a binding molecule comprising the antibody constant region variant. In some embodiments of each or any of the above or below embodiments, the first step comprises screening the Fab CRLs provided herein. In some embodiments of each or any of the embodiments above or below, provided herein is a method for identifying a binding molecule comprising a first binding domain that binds a first antigen and a second binding domain that binds a second antigen, the method comprising screening the Fab CRLs provided herein to identify a binding molecule that binds a second antigen with an affinity that is higher than a reference level, wherein the first binding domain comprises a VH region and a VL region of an antibody, and wherein the second binding domain comprises an antibody constant region variant. Screening of the library may be accomplished by a variety of techniques known in the art. For example, a specific antigen (e.g., a polypeptide, fragment, or epitope of an antigen) can be used to coat the wells of an adsorption plate, expressed on host cells attached to an adsorption plate or used for cell sorting, conjugated to biotin for streptavidin-coated bead capture, or used in any other method to panning a display library. Antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by using long washes and monovalent phage display as described in Bass et al, 1990,Proteins 8:309-14 and WO 92/09690, and by using low antigen coating densities as described in Marks et al, 1992, biotechnol.10:779-83.

In yet another aspect, provided herein is a binding molecule produced according to the methods provided herein using Fab CRL.

5.5 methods for preparing antibodies ("methods" or "processes")

In yet another aspect, provided herein are methods ("methods" or "processes") for preparing the various molecules provided herein. In some embodiments, provided herein is a method for preparing a molecule that binds to more than one target molecule, the method comprising: a step for performing a function of obtaining a binding domain capable of binding to the first antigen; a step for performing a function of obtaining a binding domain capable of binding to the second antigen; and a step for performing a function of providing a molecule capable of binding to the first antigen and the second antigen.

Recombinant expression of an antibody provided herein requires construction of an expression vector containing a polynucleotide encoding the antibody or antigen-binding fragment thereof. Once a polynucleotide encoding an antibody molecule, heavy or light chain of an antibody, or fragment thereof (such as but not necessarily containing heavy and/or light chain variable domains) provided herein has been obtained, vectors for producing the antibody molecule can be produced by recombinant DNA techniques using techniques well known in the art. Thus, described herein are methods for producing proteins by expressing polynucleotides comprising antibody encoding nucleotide sequences. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, recombinant DNA techniques in vitro, synthetic techniques, and genetic recombination in vivo. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody molecule provided herein or a fragment thereof, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors may include nucleotide sequences encoding the constant regions of antibody molecules (see, e.g., international publications WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464), and the variable domains of antibodies may be cloned into such vectors to express the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibodies provided herein. Thus, also provided herein is a host cell comprising a polynucleotide encoding an antibody or fragment thereof, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody provided herein operably linked to a heterologous promoter. In certain embodiments for expressing diabodies, vectors encoding both heavy and light chains may be co-expressed in host cells to express the entire immunoglobulin molecule, as described in detail below.

A variety of host expression vector systems may be utilized to express the antibody molecules provided herein (see, e.g., U.S. patent 5,807,715). Such host expression systems represent vehicles in which the coding sequences of interest can be produced and subsequently purified, and also represent cells in which the antibody molecules provided herein can be expressed in situ upon transformation or transfection with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., E.coli (E. Coli) and B.subtilis)) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; a method for producing antibodies by recombinant expression of recombinant vectors comprising antibody coding sequences in a yeast cell system (e.g., pichia pastoris (Saccharomyces Pichia)), a system of insect cells infected with recombinant viral expression vectors comprising antibody coding sequences (e.g., baculovirus), a plant cell system infected with recombinant viral expression vectors comprising antibody coding sequences (e.g., cauliflower mosaic virus, caMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., ti plasmid), or a mammalian cell system (e.g., COS, CHO, BHK, 293, NS0 and 3T3 cells) comprising a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., adenovirus late promoter; vaccinia 7.5K promoter), bacterial cells such as E.coli (Escherichia coli) or eukaryotic cells, especially for expression of the entire recombinant antibody molecule, e.g., mammalian cells such as Chinese hamster cells (CHO major intermediate gene expression elements from Chinese hamster cells) are provided in the early expression system of Focke, in any of the specific embodiments described herein in FIG. 6, in the early Cocke's, in the specific embodiments of FIG. 45, in the specific embodiments of FIG. 1986, in the antibodies are provided herein, in the specific embodiments of FIG. 45, in the human, and in the human, etc. (1986, expression of a nucleotide sequence encoding an antibody provided herein that immunospecifically binds to a specific antigen is under the control of a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use for expressing the antibody molecule. For example, when large amounts of such antibodies are to be produced, it may be desirable to direct vectors expressing high levels of fusion protein products that are easy to purify in order to generate pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to, E.coli (E.coli) expression vector pUR278 (Ruther et al, 1983, EMBO, 12:1791), in which antibody coding sequences may be ligated into the vector separately in frame with the lac Z coding region, such that fusion proteins are produced; pIN vectors (Inouye and Inouye,1985,Nucleic Acids Res.13:3101-3109; van Heeke and Schuster,1989, J.biol. Chem. 24:5503-5509); etc. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione sepharose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In insect systems, california noctuid (Autographa californica) nuclear polyhedrosis virus (AcNPV) was used as a vector for expression of foreign genes. The virus grew in autumn armyworm (Spodoptera frugiperda) cells. Antibody coding sequences can be cloned separately into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, many viral-based expression systems are available. In the case of adenoviruses used as expression vectors, the antibody coding sequence of interest may be linked to an adenovirus transcription/translation control complex, such as a late promoter and a tripartite leader sequence. The chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., the El or E3 region) will result in a recombinant virus that is viable and capable of expressing antibody molecules in an infected host (see, e.g., logan and Shenk,1984,Proc.Natl.Acad.Sci.USA 81:355-359). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the start codon must be identical to the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. Expression efficiency can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., bittner et al 1987,Methods in Enzymol.153:51-544).

In addition, host cell lines may be selected that regulate expression of the inserted sequences or modify and process the gene product in a particular manner as desired. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system may be selected to ensure proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells with cellular mechanisms for the proper processing, glycosylation and phosphorylation of primary transcripts of gene products can be used. Such mammalian host cells include, but are not limited to CHO, VERY, BHK, hela, COS, MDCK, 293, 3T3, W138, BT483, hs578T, HTB2, BT2O and T47D, NS0 (murine myeloma cell lines that do not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells. In some embodiments of each or any of the embodiments above or below, the fully human monoclonal antibodies provided herein are produced in mammalian cells, such as CHO cells.

For long-term, high-yield production of recombinant proteins, stable expression can be utilized. For example, cell lines stably expressing the antibody molecules may be engineered. Instead of using an expression vector containing a viral origin of replication, the host cell is transformed with DNA and a selectable marker controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). Following the introduction of the foreign DNA, the engineered cells may be allowed to grow in the enrichment medium for 1-2 days, and then switched to the selection medium. Selectable markers in recombinant plasmids confer selective resistance and allow cells to stably integrate the plasmid into their chromosomes and grow into colonies, which can then be cloned and expanded into cell lines. The method can be advantageously used to engineer cell lines expressing antibody molecules. Such engineered cell lines may be particularly useful in screening and evaluating compositions that interact directly or indirectly with antibody molecules.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, 1977, cell 11:223), hypoxanthine guanine phosphoribosyl transferase (Szybalska and Szybalski,1992,Proc.Natl.Acad.Sci.USA 48:202), and adenine phosphoribosyl transferase (Lowy et al, 1980, cell 22:8-17) genes, which may be used for tk-, hgprt-or aprt-cells, respectively. Furthermore, antimetabolite resistance can be used as a selection basis for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, 1980,Natl.Acad.Sci.USA 77:357;O'Hare et al, 1981,Proc.Natl.Acad.Sci.USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg,1981,Proc.Natl.Acad.Sci.USA 78:2072); neo, which confers resistance to aminoglycoside G-418 (Wu and Wu,1991,Biotherapy 3:87-95; tolstoshav, 1993, ann. Rev. Pharmacol. Toxicol.32:573-596;Mulligan,1993,Science 260:926-932; and Morgan and Anderson,1993, ann. Rev. Biochem.62:191-217;1993,TIB TECH 11 (5): l 55-215); and hygro, which confers resistance to hygromycin (Santerre et al, 1984, gene 30:147). Methods generally known in the art of recombinant DNA technology can be routinely applied to select desired recombinant clones, and such methods are described, for example, in Ausubel et al (editing), Current Protocols in Molecular Biology，John Wiley&Sons，NY(1993)；Kriegler，Gene Transfer and ExpressionA Laboratory Manual, stockton Press, NY (1990); and chapter 12 and chapter 13, dragopoli et al (editions),Current Protocols in Human Genetics，John Wiley&sons, NY (1994); colberre-Garapin et al, 1981, J.mol. Biol.150:1, which are incorporated herein by reference in their entirety.

The expression level of the antibody molecules can be increased by vector amplification (for reviews see Bebbington and Hentschel,The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloningvolume 3 (Academic Press, new York, 1987)). When the marker in the antibody-expressing vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. As the amplified region associates with the antibody gene, the antibody produced will also increase (Crouse et al, 1983, mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two or more expression vectors provided herein. Two or more vectors may contain the same selectable marker, which enables equal expression of, for example, heavy and light chain polypeptides. Alternatively, a single vector encoding and capable of expressing different component polypeptides of the antibodies of the disclosure, e.g., both heavy and light chain polypeptides, may be used. The coding sequence may comprise cDNA or genomic DNA.

Once the antibody molecules provided herein are produced by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity (particularly by affinity to a particular antigen after protein a) and size column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. Furthermore, the antibodies provided herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

5.6 pharmaceutical compositions

In one aspect, the present disclosure also provides a pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments of each or any of the embodiments above or below, the pharmaceutical composition comprises a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and a pharmaceutically acceptable excipient.

A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof is prepared by mixing a protein of desired purity with an optional physiologically acceptable excipient for storage in aqueous solution or lyophilized or otherwise dried form (see, e.g., remington,Remington's Pharmaceutical Sciences(18 th edition, 1980)).

The antibodies of the present disclosure, or antigen binding fragments thereof, may be formulated in any suitable form for delivery to target cells/tissues, e.g., as microcapsules or macroemulsions (Remington, supra; park et al 2005,Molecules 10:146-61; malik et al 2007, curr. Drug. Deliv. 4:141-51), as sustained release formulations (Putney and Burke,1998,Nature Biotechnol.16:153-57), or in liposomes (Maclean et al 1997, int. J. Oncol.11:325-32; kontermann,2006, curr. Opin. Mol. Ther. 8:39-45).

The antibodies or antigen-binding fragments thereof provided herein can also be embedded in microcapsules, e.g., prepared by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are disclosed, for example, in Remington, supra.

Various compositions and delivery systems are known and can be used with antibodies or antigen-binding fragments thereof as described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or antigen-binding fragments/receptor-mediated endocytosis thereof (see, e.g., wu and Wu,1987, j. Biol. Chem. 262:4429-32), construction of nucleic acids as part of a retrovirus or other vector, and the like. In another embodimentIn embodiments, the compositions may be provided as a controlled or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see, e.g., langer, supra; sefton,1987, crit. Ref. Biomed. Eng.14:201-40; buchwald et al 1980,Surgery 88:507-16; and Saudek et al 1989, N.Engl. J. Med. 321:569-74). In another embodiment, polymeric materials may be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof as described herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release(Langer and Wise editions, 1974);Controlled Drug Bioavailability,Drug Product Design and Performance(Smolen and Ball editions, 1984); ranger and Peppas,1983, J.macromol. Sci. Rev. Macromol. Chem.23:61-126; levy et al, 1985,Science 228:190-92; during et al, 1989, ann. Neurol.25:351-56; howard et al, 1989, J.Neurosurg.71:105-12; us patent 5,679,377;5,916,597;5,912,015;5,989,463; and 5,128,326; PCT publications WO 99/15154 and WO 99/20253). Examples of polymers for sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid), polyglycolide (PLG), polyanhydrides, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly (lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.

In another embodiment, the controlled or sustained release system may be placed in proximity to a specific target tissue (e.g., nasal passages or lungs), thus requiring only a portion of the systemic dose (see, e.g., goodson, Medical Applications of Controlled ReleaseVolume 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer,1990,Science 249:1527-33. Any technique known to those of skill in the art may be used to produce a sustained release formulation comprising one or more antibodies or antigen-binding fragments thereof as described herein(see, e.g., U.S. Pat. No. 4,526,938, PCT publications WO 91/05548 and WO 96/20698, ning et al, 1996, radio&Oncology 39:179-89; song et al, 1995,PDA J.of Pharma.Sci.&Tech.50:372-97; cleek et al, 1997, pro.int' l.Symp.control. Rel.Bioact.Mater.24:853-54; and Lam et al 1997,Proc.Int'l.Symp.Control Rel.Bioact.Mater.24:759-60).

5.7 methods of use

In yet another aspect, provided herein is a method of enriching, isolating, purifying, sorting, selecting, capturing, detecting or depleting cells expressing a specific antigen, the method comprising: providing a sample comprising cells expressing a specific antigen; contacting the sample with a multispecific antibody; and enriching, isolating, purifying, sorting, selecting, capturing, detecting, or depleting cells expressing a specific antigen that binds to the multispecific antibody, wherein the multispecific antibody comprises a first binding domain capable of binding to a first antigen and a second binding domain capable of binding to a second antigen. In some embodiments of each or any of the above or below embodiments, the sample is a blood sample. In some embodiments of each or any of the above or below embodiments, the sample is a tissue sample.

In yet another aspect, provided herein is a method of treating cancer in a subject, the method comprising administering to the subject one or more antibodies described in any one of the above or below embodiments provided herein. In some embodiments of each or any of the above or below embodiments, the cancer is a solid tumor cancer. In some embodiments of each or any of the above or below embodiments, the cancer is a leukemia.

In another aspect, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof provided herein.

Also provided herein is a method of treating a disease or disorder, wherein one or more therapeutic agents are administered to a subject in combination with an antibody or antigen-binding fragment thereof provided herein.

In another aspect, provided herein is the use of an antibody or antigen binding fragment thereof provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

In another aspect, provided herein is the use of a pharmaceutical composition provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

In a specific embodiment, provided herein is a composition comprising an antibody or antigen-binding fragment thereof provided herein for use in the prevention and/or treatment of a disease or disorder. In one embodiment, provided herein is a composition for preventing a disease or disorder, wherein the composition comprises an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for treating a disease or disorder, wherein the composition comprises an antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the subject is a subject in need thereof. In some embodiments of each or any of the above or below embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of suffering from a disease or disorder. In some embodiments of each or any of the above or below embodiments, the administration results in the prevention, control, treatment, or amelioration of a disease or condition.

In one embodiment, provided herein is a composition for preventing and/or treating a symptom of a disease or disorder, wherein the composition comprises an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for preventing a symptom of a disease or disorder, wherein the composition comprises an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for treating a symptom of a disease or disorder, wherein the composition comprises an antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the subject is a subject in need thereof. In some embodiments of each or any of the above or below embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of suffering from a disease or disorder. In some embodiments of each or any of the above or below embodiments, the administration results in the prevention or treatment of a symptom of the disease or condition.

In another embodiment, provided herein is a method of preventing and/or treating a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of preventing a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the subject is a subject in need thereof. In some embodiments of each or any of the above or below embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of suffering from a disease or disorder. In some embodiments, administration results in the prevention or treatment of a disease or disorder.

In another embodiment, provided herein is a method of preventing and/or treating a symptom of a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of preventing symptoms of a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of treating a symptom of a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the subject is a subject in need thereof. In some embodiments of each or any of the above or below embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of suffering from a disease or disorder. In some embodiments of each or any of the above or below embodiments, the administration results in the prevention or treatment of a symptom of the disease or condition.

Also provided herein are methods of preventing and/or treating a disease or disorder by administering to a subject an effective amount of an antibody or antigen-binding fragment thereof provided herein or a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein. In some embodiments, also provided herein is the use of an antibody or antigen binding fragment provided herein for the prevention and/or treatment of a disease or condition. In some embodiments, provided herein are also antibodies or antigen binding fragments provided herein for use in the prevention and/or treatment of a disease or condition. In some embodiments, provided herein is also the use of an antibody or antigen binding fragment provided herein in the manufacture of a medicament for the prevention and/or treatment of a disease or condition. In one aspect, the antibody or antigen-binding fragment thereof is substantially purified (i.e., substantially free of substances that limit its effectiveness or produce undesirable side effects). The subject to which the treatment is administered can be a mammal, such as a non-primate or primate (e.g., a human). In one embodiment, the subject is a human. In another embodiment, the subject is a human suffering from a disease or disorder.

In some embodiments of each or any of the above or below embodiments, the binding molecules of the invention are used to treat solid tumor cancer. In other embodiments, the binding molecules of the invention are used to treat hematological cancers. In other embodiments, the disease or disorder is an autoimmune disease and an inflammatory disease. In other embodiments, the disease or disorder is an infectious disease.

In some embodiments of each or any of the above or below embodiments, the disease or disorder is a disease in which cell growth is abnormal and/or apoptosis is deregulated. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, bone cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, gastrointestinal cancer (gastric, colorectal and/or duodenal cancer), chronic lymphocytic leukemia, acute lymphoblastic leukemia, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, testicular cancer, hepatocellular carcinoma (liver cancer and/or cholangiocarcinoma), primary or secondary central nervous system tumors, cancer primary or secondary brain tumor, hodgkin's disease, chronic or acute leukemia, chronic myelogenous leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, T-cell or B-cell derived lymphoid malignancy, melanoma, multiple myeloma, oral cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, renal cancer and/or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, non-hodgkin's lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, adrenal cortex cancer, gall bladder cancer, spleen cancer, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or combinations thereof.

In some embodiments of each or any of the above or following embodiments, the disease or disorder is selected from the group consisting of: bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphoblastic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer and spleen cancer.

In some embodiments of each or any of the above or below embodiments, the disorder or condition is a hematological cancer, such as leukemia, lymphoma, or myeloma. In some embodiments of each or any of the above or following embodiments, the cancer is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle Cell Lymphoma (MCL), follicular central lymphoma, transformed lymphoma, medium-differentiated lymphocytic lymphoma, medium-grade lymphocytic lymphoma (ILL), diffuse low-differentiated lymphocytic lymphoma (PDL), central cell lymphoma, diffuse small-cell lymphoma (DSCCL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma, mantle cell lymphoma, low-grade follicular lymphoma, multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T-cell leukemia, acute Myelogenous Leukemia (AML), acute promyelocytic leukemia, acute megakaryoblastic leukemia, precursor B-type acute lymphoblastic leukemia, precursor T-type acute lymphoblastic leukemia, burt-type leukemia (myelogenous leukemia), dual-basal lymphoma, chronic leukemia (CML), chronic leukemia, and chronic leukemia. In a specific embodiment, the disease or disorder is myelodysplastic syndrome (MDS). In another specific embodiment, the disease or disorder is Acute Myelogenous Leukemia (AML). In another specific embodiment, the disease or disorder is Chronic Lymphocytic Leukemia (CLL). In another specific embodiment, the disease or disorder is Multiple Myeloma (MM).

In other embodiments, the disease or disorder is a solid tumor cancer. In some embodiments of each or any of the above or following embodiments, the solid tumor cancer is selected from the group consisting of: cancer, adenocarcinoma, adrenocortical carcinoma, colon adenocarcinoma, colorectal cancer, ductal cell carcinoma, lung cancer, thyroid cancer, nasopharyngeal carcinoma, melanoma, non-melanoma skin cancer, liver cancer, and lung cancer.

In some embodiments of each or any of the above or below embodiments, the cancer is an adrenal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is anal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is appendiceal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is cholangiocarcinoma. In some embodiments of each or any of the above or below embodiments, the cancer is bladder cancer. In some embodiments of each or any of the above or below embodiments, the cancer is bone cancer. In some embodiments of each or any of the above or below embodiments, the cancer is brain cancer. In some embodiments of each or any of the above or below embodiments, the cancer is breast cancer. In some embodiments of each or any of the above or below embodiments, the cancer is cervical cancer. In some embodiments of each or any of the above or below embodiments, the cancer is colorectal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is esophageal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is a cancer of the liver. In some embodiments of each or any of the above or below embodiments, the cancer is a gestational trophoblastic cancer. In some embodiments of each or any of the above or below embodiments, the cancer is a head and neck cancer. In some embodiments of each or any of the above or below embodiments, the cancer is hodgkin's lymphoma. In some embodiments of each or any of the above or below embodiments, the cancer is a bowel cancer. In some embodiments of each or any of the above or below embodiments, the cancer is a renal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is leukemia. In some embodiments of each or any of the above or below embodiments, the cancer is liver cancer. In some embodiments of each or any of the above or below embodiments, the cancer is lung cancer. In some embodiments of each or any of the above or below embodiments, the cancer is melanoma. In some embodiments of each or any of the above or below embodiments, the cancer is mesothelioma. In some embodiments of each or any of the above or below embodiments, the cancer is Multiple Myeloma (MM). In some embodiments of each or any of the above or below embodiments, the cancer is a neuroendocrine tumor. In some embodiments of each or any of the above or below embodiments, the cancer is non-hodgkin's lymphoma. In some embodiments of each or any of the above or below embodiments, the cancer is oral cancer. In some embodiments of each or any of the above or below embodiments, the cancer is ovarian cancer. In some embodiments of each or any of the above or below embodiments, the cancer is pancreatic cancer. In some embodiments of each or any of the above or below embodiments, the cancer is prostate cancer. In some embodiments of each or any of the above or below embodiments, the cancer is sinus cancer. In some embodiments of each or any of the above or below embodiments, the cancer is skin cancer. In some embodiments of each or any of the above or below embodiments, the cancer is soft tissue sarcoma spinal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is gastric cancer. In some embodiments of each or any of the above or below embodiments, the cancer is testicular cancer. In some embodiments of each or any of the above or below embodiments, the cancer is laryngeal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is thyroid cancer. In some embodiments of each or any of the above or below embodiments, the cancer is endometrial cancer. In some embodiments of each or any of the above or below embodiments, the cancer is a vaginal cancer. In some embodiments of each or any of the above or below embodiments, the cancer is vulvar cancer.

In some embodiments of each or any of the above or below embodiments, the adrenal cancer is an Adrenal Cortical Cancer (ACC), a pararenal cortical cancer, a pheochromocytoma, or a neuroblastoma. In some embodiments of each or any of the above or below embodiments, the anal cancer is squamous cell carcinoma, a hole anorectal carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma. In some embodiments of each or any of the above or below embodiments, the appendiceal cancer is a neuroendocrine tumor (NET), mucous adenocarcinoma, goblet cell carcinoid, intestinal adenocarcinoma, or print-stop cell adenocarcinoma. In some embodiments of each or any of the above or below embodiments, the bile duct cancer is extrahepatic bile duct cancer, adenocarcinoma, portal bile duct cancer, perihepatic bile duct cancer, distal bile duct cancer, or intrahepatic bile duct cancer. In some embodiments of each or any of the above or below embodiments, the bladder cancer is Transitional Cell Carcinoma (TCC), papillary carcinoma, squamous cell carcinoma, adenocarcinoma, small cell carcinoma or sarcoma. In some embodiments of each or any of the above or below embodiments, the bone cancer is a primary bone cancer, a sarcoma, an osteosarcoma, a chondrosarcoma, a sarcoma, a fibrosarcoma, a malignant fibrous histiocytoma, a bone giant cell tumor, a chordoma, or a metastatic bone cancer. In some embodiments of each or any of the embodiments above or below, the brain cancer is an astrocytoma, a brain stem glioma, a glioblastoma, a meningioma, a ependymoma, an oligodendroglioma, a mixed glioma, a pituitary adenocarcinoma, a pituitary adenoma, a craniopharyngeal tube tumor, a germ cell tumor, a pineal zone tumor, a medulloblastoma, or a primary CNS lymphoma. In some embodiments of each or any of the above or below embodiments, the breast cancer is breast adenocarcinoma, invasive breast cancer, non-invasive breast cancer, breast sarcoma, metastatic cancer, adenoid cystic cancer, phyllotor, angiosarcoma, HER2 positive breast cancer, triple negative breast cancer, or inflammatory breast cancer. In some embodiments of each or any of the above or below embodiments, the cervical cancer is squamous cell carcinoma or adenocarcinoma. In some embodiments of each or any of the above or below embodiments, the colorectal cancer is colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucous adenocarcinoma, print-guard cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma. In some embodiments of each or any of the above or below embodiments, the esophageal cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments of each or any of the above or below embodiments, the gallbladder carcinoma is adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma or sarcoma. In some embodiments of each or any of the above or below embodiments, the Gestational Trophoblastic Disease (GTD) is grape embryo, gestational trophoblastic tumor (GTN), choriocarcinoma, placental Site Trophoblastic Tumor (PSTT), or epithelial-like trophoblastic tumor (ETT). In some embodiments of each or any of the above or below embodiments, the head and neck cancer is laryngeal, nasopharyngeal, hypopharynx, nasal cavity, sinus, salivary gland, oral cavity, oropharyngeal or tonsil cancer. In some embodiments of each or any of the above or below embodiments, the hodgkin's lymphoma is classical hodgkin's lymphoma, nodular sclerosis, mixed cell type, lymphocyte-rich type, lymphocyte depletion type, or nodular lymphocyte-based hodgkin's lymphoma (NLPHL). In some embodiments of each or any of the above or below embodiments, the bowel cancer is a small bowel cancer (small intestine cancer), small bowel cancer (small bowel cancer), adenocarcinoma, sarcoma, gastrointestinal stromal tumor, carcinoid tumor, or lymphoma. In some embodiments of each or any of the above or below embodiments, the renal cancer is Renal Cell Carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, manifold RCC, unclassified RCC, transitional cell carcinoma, urothelial carcinoma, renal pelvis carcinoma, or renal sarcoma. In some embodiments of each or any of the above or below embodiments, the leukemia is Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), hairy Cell Leukemia (HCL), or myelodysplastic syndrome (MDS). In a specific embodiment, the leukemia is AML. In some embodiments of each or any of the above or below embodiments, the liver cancer is hepatocellular carcinoma (HCC), fiberboard layer-like HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis. In some embodiments of each or any of the above or below embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large cell undifferentiated carcinoma, lung nodules, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland type lung cancer, lung carcinoid, mesothelioma, lung sarcoidosis, or malignant granulosa cell lung tumor. In some embodiments of each or any of the embodiments above or below, the melanoma is superficial diffuse melanoma, nodular melanoma, acro-lentigo, malignant lentigo, non-pigmented melanoma, pro-fibrotic melanoma, eyeball melanoma, or metastatic melanoma. In some embodiments of each or any of the above or below embodiments, the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In some embodiments of each or any of the above or below embodiments, the multiple myeloma is active myeloma or smoky myeloma. In some embodiments of each or any of the above or below embodiments, the neuroendocrine tumor is a gastrointestinal neuroendocrine tumor, a pancreatic neuroendocrine tumor, or a lung neuroendocrine tumor. In some embodiments of each or any of the above or below embodiments, non-hodgkin's lymphoma is anaplastic large cell lymphoma, lymphoblastic lymphoma, peripheral T cell lymphoma, follicular lymphoma, cutaneous T cell lymphoma, lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, MALT lymphoma, small cell lymphocytic lymphoma, burkitt's lymphoma, chronic Lymphocytic Leukemia (CLL), small Lymphocytic Lymphoma (SLL), precursor T lymphoblastic leukemia/lymphoma, acute Lymphoblastic Leukemia (ALL), adult T cell lymphoma/leukemia (ATLL), hairy cell leukemia, B cell lymphoma, diffuse Large B Cell Lymphoma (DLBCL), primary mediastinal B cell lymphoma, primary Central Nervous System (CNS) lymphoma Mantle Cell Lymphoma (MCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, lymph node marginal zone B cell lymphoma, spleen marginal zone B cell lymphoma, lymphoplasmacytic lymphoma, B-cell non-hodgkin's lymphoma, T-cell non-hodgkin's lymphoma, natural killer cell lymphoma, cutaneous T-cell lymphoma, albert syndrome, sezary syndrome, primary cutaneous anaplastic large cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), anaplastic Large Cell Lymphoma (ALCL), systemic ALCL, enteropathic T-cell lymphoma (EATL), or hepatosplenic gamma/delta T-cell lymphoma. In some embodiments of each or any of the above or below embodiments, the oral cancer is squamous cell carcinoma, warty carcinoma, small salivary gland carcinoma, lymphoma, benign oral tumor, eosinophilic granuloma, fibroma, granuloma, keratoacanthoma, smooth myoma, osteochondrioma, lipoma, schwannoma, neurofibroma, papilloma, condyloma acuminatum, warty xanthoma, suppurative granuloma, rhabdomyoma, odontogenic tumor, white spot, erythema, squamous cell lip carcinoma, basal cell lip carcinoma, oral cancer, gum carcinoma, or tongue carcinoma. In some embodiments of each or any of the above or below embodiments, the ovarian cancer is ovarian epithelial cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, clear cell epithelial ovarian cancer, undifferentiated epithelial ovarian cancer, ovarian low malignant potential tumor, primary peritoneal cancer, fallopian tube cancer, germ cell tumor, teratoma, asexual cell tumor, ovarian germ cell cancer, endoplasmic sinus tumor, sex cord-stromal tumor, sex cord-gonadal stromal tumor, ovarian stromal tumor, granulosa cell tumor, granulosa-follicular membrane tumor, seltoli-Leydig cell tumor, ovarian sarcoma, ovarian carcinoma sarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, kukenbo tumor, or ovarian cyst. In some embodiments of each or any of the embodiments above or below, the pancreatic cancer is pancreatic exocrine adenocarcinoma, pancreatic endocrine adenocarcinoma or pancreatic adenocarcinoma, islet cell tumor or neuroendocrine tumor. In some embodiments of each or any of the above or below embodiments, the prostate cancer is a prostate cancer, a prostate sarcoma, a transitional cell carcinoma, a small cell carcinoma, or a neuroendocrine tumor. In some embodiments of each or any of the above or below embodiments, the sinus cancer is squamous cell carcinoma, mucosal cell carcinoma, adenoid cystic cell carcinoma, acinar cell carcinoma, paranasal undifferentiated carcinoma, nasal cancer, paranasal carcinoma, maxillary sinus carcinoma, ethmoid sinus carcinoma, or nasopharyngeal carcinoma. In some embodiments of each or any of the above or below embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, melanoma, merkel cell carcinoma, kaposi's Sarcoma (KS), actinic keratosis, cutaneous lymphoma, or keratoacanthoma. In some embodiments of each or any of the above or below embodiments, the soft tissue carcinoma is angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), kaposi's sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated Liposarcoma (DL), myxoid/round cell liposarcoma (MRCL), hyper-differentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma. In some embodiments of each or any of the above or below embodiments, the spinal cancer is spinal metastasis. In some embodiments of each or any of the embodiments above or below, the gastric cancer is gastric adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumor, carcinoid tumor, gastric carcinoid tumor, ECL cell carcinoid type I, ECL cell carcinoid type II, or ECL cell carcinoid type III. In some embodiments of each or any of the above or below embodiments, the testicular cancer is a seminoma, a non-seminoma, an embryonal carcinoma, a yolk sac carcinoma, a choriocarcinoma, a teratoma, a gonadal stromal tumor, a testicular stromal tumor, or a testicular supportive tumor. In some embodiments of each or any of the above or below embodiments, the laryngeal carcinoma is squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal carcinoma, pharyngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, lymphoepithelioma, spindle cell carcinoma, wart carcinoma, undifferentiated carcinoma, or lymph node carcinoma. In some embodiments of each or any of the above or below embodiments, the thyroid cancer is papillary carcinoma, follicular carcinoma, hurthle cell carcinoma, medullary thyroid carcinoma, or undifferentiated carcinoma. In some embodiments of each or any of the embodiments above or below, the uterine cancer is endometrial cancer, endometrial adenocarcinoma, endometrioid carcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinoma sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, or undifferentiated sarcoma. In some embodiments of each or any of the above or below embodiments, the vaginal cancer is squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In some embodiments of each or any of the above or below embodiments, the vulvar cancer is squamous cell carcinoma or adenocarcinoma.

In some embodiments of each or any of the above or below embodiments, the disease or disorder is caused by a pathogen. In some embodiments of each or any of the above or following embodiments, the pathogen causes an infectious disease selected from the group consisting of: acute myelitis (AFM), anabrosis, anthrax, babesiosis, botulism, brucellosis, campylobacter, carbapenem-resistant infections, chancre, chikungunya virus infections, chlamydia, fish botulinum, clostridium difficile infections, clostridium perfringens, coccidioidomycosis, coronavirus infections, covid-19 (SARS-CoV-2), creutzfeldt-Jakob disease/infectious spongiform encephalopathy, cryptosporidiosis (Crypto), cyclosporin, dengue 1, 2, 3 or 4, diphtheria, escherichia coli infection/shiga toxin production (STEC), eastern equine encephalitis, hemorrhagic fever (Ebola), epstein-Barr disease, encephalitis, arbovirus or paraminosis, non-polio enterovirus, D68 enterovirus (EV-D68), giardiasis, meldonia disease, gonococcal infection, inguinal granuloma, haemophilus influenzae type B (Hib or H-influenza), hantavirus Pulmonary Syndrome (HPS), hemolytic Uremic Syndrome (HUS), hepatitis A (hepA), hepatitis B (hepB), hepatitis C (hepC), hepatitis D (hepD), hepatitis E (hepE), herpes zoster, histoplasmosis infection, human immunodeficiency virus/AIDS (HIV/AIDS), influenza (Legioma), human influenza (Legioma), influenza (HPV (Legioma), leprosy (hansen's disease), leptospirosis, listeriosis (listeria), lyme disease, venereal lymphogranulomatosis infection (LGV), malaria, measles, meliosis, meningitis (viral), meningococcal disease (meningitis (bacterial)), middle east respiratory syndrome coronavirus (MERS-CoV), mumps, norovirus, pediculosis, pelvic Inflammatory Disease (PID), pertussis (wheezing), plague (inguinal adenitis, septicaemia, pneumonitis), pneumococosis (pneumonia), poliomyelitis (poliomyelitis), bovaccina, psittacosis, pubic lice, impetigo (smallpox, monkey pox, vaccinia), Q fever, rabies, pneumonitis rickettsia (falling mountain zebra fever), rubella (german measles), salmonella gastritis (salmonella), scabies, mackerel toxin, septicemia, severe Acute Respiratory Syndrome (SARS), shigella gastroenteritis (shigella), smallpox, methicillin-resistant staphylococcal infection (MRSA), staphylococcal food poisoning enterotoxin B poisoning (staphylococcal food poisoning), staphylococcal infection Vancomycin Intermediate (VISA), staphylococcal infection Vancomycin Resistant (VRSA), streptococcal disease group a (invasive) (group a streptococcus (invasive)), streptococcal disease group B (group B streptococcus), streptococcal midbody STSS toxic shock, syphilis (primary, secondary, early latent, late latent, congenital), tetanus infection, trichomonas vaginalis, trichomonas infection, tuberculosis (TB), latent Tuberculosis (LTBI), rabbit fever, typhoid group D, vaginosis, varicella virus (varicella), vibrio cholerae, vibriosis (vibrio), ebola hemorrhagic fever, lassa hemorrhagic fever, marburg hemorrhagic fever, west nile virus, yellow fever, yersinia, and zika virus infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is acute myelitis weakness (AFM). In some embodiments of each or any of the above or below embodiments, the infectious disease is an intangible disease. In some embodiments of each or any of the above or below embodiments, the infectious disease is anthrax. In some embodiments of each or any of the above or below embodiments, the infectious disease is babesia. In some embodiments of each or any of the above or below embodiments, the infectious disease is botulism. In some embodiments of each or any of the above or below embodiments, the infectious disease is brucellosis. In some embodiments of each or any of the above or below embodiments, the infectious disease is campylobacter. In some embodiments of each or any of the above or below embodiments, the infectious disease is resistant to carbapenem infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is chancre. In some embodiments of each or any of the above or below embodiments, the infectious disease is a chikungunya virus infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is chlamydia. In some embodiments of each or any of the above or below embodiments, the infectious disease is fish toxicity. In some embodiments of each or any of the above or below embodiments, the infectious disease is clostridium difficile infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is clostridium perfringens. In some embodiments of each or any of the above or below embodiments, the infectious disease is a coccidioidomycosis fungal infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is a coronavirus. In some embodiments of each or any of the above or below embodiments, the infectious disease is Covid-19 (SARS-CoV-2). In some embodiments of each or any of the above or below embodiments, the infectious disease is creutzfeldt-jakob disease/infectious spongiform encephalopathy. In some embodiments of each or any of the above or below embodiments, the infectious disease is cryptosporidiosis (Crypto). In some embodiments of each or any of the above or below embodiments, the infectious disease is cyclosporin. In some embodiments of each or any of the above or below embodiments, the infectious disease is dengue 1, 2, 3, or 4. In some embodiments of each or any of the above or below embodiments, the infectious disease is diphtheria. In some embodiments of each or any of the above or below embodiments, the infectious disease is escherichia coli infection/shiga toxin production (STEC). In some embodiments of each or any of the above or below embodiments, the infectious disease is eastern equine encephalitis. In some embodiments of each or any of the above or below embodiments, the infectious disease is hemorrhagic fever (ebola). In some embodiments of each or any of the above or below embodiments, the infectious disease is irinotecan disease. In some embodiments of each or any of the above or below embodiments, the infectious disease is encephalitis. In some embodiments of each or any of the above or below embodiments, the infectious disease is an arbovirus or a parainfection. In some embodiments of each or any of the above or below embodiments, the infectious disease is a non-polio enterovirus. In some embodiments of each or any of the above or below embodiments, the infectious disease is D68 enterovirus (EV-D68). In some embodiments of each or any of the above or below embodiments, the infectious disease is giardiasis. In some embodiments of each or any of the above or below embodiments, the infectious disease is a melioidosis. In some embodiments of each or any of the above or below embodiments, the infectious disease is gonococcal infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is inguinal granuloma. In some embodiments of each or any of the above or below embodiments, the infectious disease is haemophilus influenzae type B (Hib or H-influenza). In some embodiments of each or any of the above or below embodiments, the infectious disease is hantavirus lung syndrome (HPS). In some embodiments of each or any of the above or below embodiments, the infectious disease is Hemolytic Uremic Syndrome (HUS). In some embodiments of each or any of the above or below embodiments, the infectious disease is hepatitis a (Hep a). In some embodiments of each or any of the above or below embodiments, the infectious disease is hepatitis B (Hep B). In some embodiments of each or any of the above or below embodiments, the infectious disease is hepatitis C (Hep C). In some embodiments of each or any of the above or below embodiments, the infectious disease is hepatitis D (Hep D). In some embodiments of each or any of the above or below embodiments, the infectious disease is hepatitis E (Hep E). In some embodiments of each or any of the above or below embodiments, the infectious disease is herpes. In some embodiments of each or any of the above or below embodiments, the infectious disease is shingles (shingles). In some embodiments of each or any of the above or below embodiments, the infectious disease is histoplasmosis infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is human immunodeficiency virus/AIDS (HIV/AIDS). In some embodiments of each or any of the above or below embodiments, the infectious disease is Human Papillomavirus (HPV). In some embodiments of each or any of the above or below embodiments, the infectious disease is influenza (Flu). In some embodiments of each or any of the above or below embodiments, the infectious disease is legionellosis (legionnaires disease). In some embodiments of each or any of the above or below embodiments, the infectious disease is leprosy (hansen's disease). In some embodiments of each or any of the above or below embodiments, the infectious disease is leptospirosis. In some embodiments of each or any of the above or below embodiments, the infectious disease is listeriosis (listeria). In some embodiments of each or any of the above or below embodiments, the infectious disease is lyme disease. In some embodiments of each or any of the above or below embodiments, the infectious disease is a venereal lymphogranulomatous infection (LGV). In some embodiments of each or any of the above or below embodiments, the infectious disease is malaria. In some embodiments of each or any of the above or below embodiments, the infectious disease is measles. In some embodiments of each or any of the above or below embodiments, the infectious disease is melioidosis. In some embodiments of each or any of the above or below embodiments, the infectious disease is meningitis (viral). In some embodiments of each or any of the embodiments above or below, the infectious disease is meningococcal disease (meningitis (bacterial)). In some embodiments of each or any of the embodiments mentioned below, the infectious disease is middle east respiratory syndrome coronavirus (MERS-CoV). In some embodiments of each or any of the above or below embodiments, the infectious disease is mumps. In some embodiments of each or any of the above or below embodiments, the infectious disease is norovirus. In some embodiments of each or any of the above or below embodiments, the infectious disease is pediculosis. In some embodiments of each or any of the above or below embodiments, the infectious disease is Pelvic Inflammatory Disease (PID). In some embodiments of each or any of the above or below embodiments, the infectious disease is pertussis (asthma). In some embodiments of each or any of the above or below embodiments, the infectious disease is plague (inguinal adenitis). In some embodiments of each or any of the above or below embodiments, the infectious disease is sepsis. In some embodiments of each or any of the above or below embodiments, the infectious disease is pneumonic. In some embodiments of each or any of the above or below embodiments, the infectious disease is pneumococcal disease (pneumonia). In some embodiments of each or any of the above or below embodiments, the infectious disease is poliomyelitis (poliomyelitis). In some embodiments of each or any of the above or below embodiments, the infectious disease is polifeprosan. In some embodiments of each or any of the above or below embodiments, the infectious disease is psittacosis. In some embodiments of each or any of the above or below embodiments, the infectious disease is pubic lice. In some embodiments of each or any of the above or below embodiments, the infectious disease is impetigo (smallpox). In some embodiments of each or any of the above or below embodiments, the infectious disease is monkey pox. In some embodiments of each or any of the above or below embodiments, the infectious disease is vaccinia. In some embodiments of each or any of the above or below embodiments, the infectious disease is Q fever. In some embodiments of each or any of the above or below embodiments, the infectious disease is rabies. In some embodiments of each or any of the above or below embodiments, the infectious disease is rickettsia (fever of the mountain spot). In some embodiments of each or any of the above or below embodiments, the infectious disease is a rubella (german measles). In some embodiments of each or any of the above or below embodiments, the infectious disease is salmonella gastroenteritis (salmonella). In some embodiments of each or any of the above or below embodiments, the infectious disease is scabies. In some embodiments of each or any of the above or below embodiments, the infectious disease is mackerel toxin. In some embodiments of each or any of the above or below embodiments, the infectious disease is sepsis. In some embodiments of each or any of the above or below embodiments, the infectious disease is Severe Acute Respiratory Syndrome (SARS). In some embodiments of each or any of the above or below embodiments, the infectious disease is shigella gastroenteritis (shigella). In some embodiments of each or any of the above or below embodiments, the infectious disease is smallpox. In some embodiments of each or any of the above or below embodiments, the infectious disease is methicillin-resistant staphylococcus infection (MRSA). In some embodiments of each or any of the above or below embodiments, the infectious disease is staphylococcal food poisoning enterotoxin B poisoning (staphylococcal food poisoning). In some embodiments of each or any of the above or below embodiments, the infectious disease is staphylococcal Vancomycin Intermediate (VISA). In some embodiments of each or any of the above or below embodiments, the infectious disease is staphylococcal Vancomycin Resistance (VRSA). In some embodiments of each or any of the above or below embodiments, the infectious disease is streptococcal group a (invasive) (group a streptococci (invasive)). In some embodiments of each or any of the above or below embodiments, the infectious disease is streptococcal disease. In some embodiments of each or any of the above or below embodiments, the infectious disease is group B (group B streptococcus). In some embodiments of each or any of the above or below embodiments, the infectious disease is streptococcal toxic shock syndrome STSS toxic shock. In some embodiments of each or any of the above or below embodiments, the infectious disease is primary. In some embodiments of each or any of the above or below embodiments, the infectious disease is secondary. In some embodiments of each or any of the above or below embodiments, the infectious disease is early latent. In some embodiments of each or any of the above or below embodiments, the infectious disease is late-stage latent. In some embodiments, the infectious disease is congenital. In some embodiments of each or any of the above or below embodiments, the infectious disease is tetanus infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is trichomonas vaginalis. In some embodiments of each or any of the above or below embodiments, the infectious disease is a trichomonas infection. In some embodiments of each or any of the above or below embodiments, the infectious disease is Tuberculosis (TB). In some embodiments of each or any of the above or below embodiments, the infectious disease is Latent Tuberculosis (LTBI). In some embodiments of each or any of the above or below embodiments, the infectious disease is rabbit fever. In some embodiments of each or any of the above or below embodiments, the infectious disease is typhoid D. In some embodiments of each or any of the above or below embodiments, the infectious disease is a vaginal disease. In some embodiments of each or any of the above or below embodiments, the infectious disease is varicella virus (varicella), vibrio cholerae (cholera). In some embodiments of each or any of the above or below embodiments, the infectious disease is vibriosis (vibrios). In some embodiments of each or any of the above or below embodiments, the infectious disease is ebola virus hemorrhagic fever. In some embodiments of each or any of the above or below embodiments, the infectious disease is lassa virus hemorrhagic fever. In some embodiments of each or any of the above or below embodiments, the infectious disease is marburg hemorrhagic fever. In some embodiments of each or any of the above or below embodiments, the infectious disease is west nile virus. In some embodiments of each or any of the above or below embodiments, the infectious disease is yellow fever. In some embodiments of each or any of the above or below embodiments, the infectious disease is yersinia. In some embodiments of each or any of the above or below embodiments, the infectious disease is a zika virus infection.

In some embodiments of each or any of the above or below embodiments, the pathogen is a bacterium. In some embodiments of each or any of the embodiments above or below, the bacterium is a bacterium of the genus bacillus, bartonella, bordetella, borrelia, brucella, campylobacter, chlamydia, chlamydophila, clostridium, corynebacterium, enterococcus, escherichia, franciscensis, haemophilus, helicobacter, legionella, leptospira, listeria, mycobacterium, mycoplasma, neisseria, pseudomonas, rickettsia, salmonella, shigella, staphylococcus, streptococcus, treponema, urinary, vibrio or yersinia. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacillus bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus bartonella. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus bordetella. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus borrelia. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus brucella. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus campylobacter. In some embodiments of each or any of the above or below embodiments, the bacterium is a chlamydia bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a chlamydophila bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a clostridium bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a coryneform bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus enterococcus. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus escherichia. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus francissia. In some embodiments of each or any of the above or below embodiments, the bacterium is a haemophilus bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a helicobacter bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus legionella. In some embodiments of each or any of the above or below embodiments, the bacterium is a leptospira bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a listeria bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a mycobacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a mycoplasma bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a neisseria species bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a pseudomonas bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus rickettsia. In some embodiments of each or any of the above or below embodiments, the bacterium is a salmonella bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a shigella bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a staphylococcus bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a streptococcus bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a treponema bacterium. In some embodiments of each or any of the above or below embodiments, the bacterium is a bacterium of the genus uroplasma. In some embodiments of each or any of the above or below embodiments, the bacteria are of the genus vibrio. In some embodiments of each or any of the above or below embodiments, the bacterium is a yersinia bacterium.

In some embodiments of each or any of the above or below embodiments, the pathogen is a parasite. In some embodiments of each or any of the above or below embodiments, the parasite is a protozoa, helminth, or ectoparasite. In some embodiments of each or any of the above or below embodiments, the protozoa is amoeba, giardia, leishmania, intestinal bag worm, plasmodium or cryptosporidium. In some embodiments of each or any of the embodiments above or below, the worm is a trematode, cestode, acanthocellatus or roundworm. In some embodiments of each or any of the above or below embodiments, the ectoparasite is an arthropod.

In some embodiments of each or any of the above or below embodiments, the pathogen is a virus. In some embodiments of each or any of the embodiments above or below, the virus is a virus of the family adenoviridae, arenaviridae, astroviridae, bunaeae, caliciviridae, coronaviridae, filoviridae, flaviviridae, hepadnaviridae, hepaciviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae, poxviridae, reoviridae, retrovirus, rhabdoviridae, or togaviridae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family adenoviridae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the arenaviridae family. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the astroviridae family. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family euphorbiaceae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family caliciviridae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family coronaviridae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family filoviridae. In some embodiments of each or any of the above or below embodiments, the virus is a flaviviridae virus. In some embodiments of each or any of the above or below embodiments, the virus is a hepadnaviridae virus. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the hepaciviridae family. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the orthomyxoviridae family. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the papillomaviridae family. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family paramyxoviridae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the parvoviridae family. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the picornaviridae family. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family polyomaviridae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family poxviridae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the reoviridae family. In some embodiments of each or any of the above or below embodiments, the virus is a retrovirus. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the family Rhabdoviridae. In some embodiments of each or any of the above or below embodiments, the virus is a virus of the togaviridae family.

In some embodiments of each or any of the above or below embodiments, the virus is adenovirus, coronavirus, coxsackie virus, epstein-barr virus, hepatitis a virus, hepatitis b virus, hepatitis c virus, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human papilloma virus, parainfluenza virus, polio virus, rabies virus, respiratory syncytial virus, rubella virus, or varicella-zoster virus. In some embodiments of each or any of the above or below embodiments, the virus is an adenovirus. In some embodiments of each or any of the above or below embodiments, the virus is a coronavirus. In some embodiments of each or any of the above or below embodiments, the coronavirus virus is Covid-19 (SARS-CoV-2). In some embodiments of each or any of the above or below embodiments, the virus is a coxsackievirus. In some embodiments of each or any of the above or below embodiments, the virus is epstein-barr virus. In some embodiments of each or any of the above or below embodiments, the virus is hepatitis a virus. In some embodiments of each or any of the above or below embodiments, the virus is hepatitis b virus. In some embodiments of each or any of the above or below embodiments, the virus is hepatitis c virus. In some embodiments of each or any of the above or below embodiments, the virus is herpes simplex virus type 2. In some embodiments of each or any of the above or below embodiments, the virus is a cytomegalovirus. In some embodiments of each or any of the above or below embodiments, the virus is human herpesvirus 8. In some embodiments of each or any of the above or below embodiments, the virus is a human immunodeficiency virus. In some embodiments of each or any of the above or below embodiments, the virus is an influenza virus. In some embodiments of each or any of the above or below embodiments, the virus is measles virus. In some embodiments of each or any of the above or below embodiments, the virus is mumps virus. In some embodiments of each or any of the above or below embodiments, the virus is a human papilloma virus. In some embodiments of each or any of the above or below embodiments, the virus is parainfluenza virus. In some embodiments of each or any of the above or below embodiments, the virus is poliovirus. In some embodiments of each or any of the above or below embodiments, the virus is rabies virus. In some embodiments of each or any of the above or below embodiments, the virus is a respiratory syncytial virus. In some embodiments of each or any of the above or below embodiments, the virus is a rubella virus. In some embodiments of each or any of the above or below embodiments, the virus is varicella-zoster virus.

In some embodiments of each or any of the above or below embodiments, the disease or disorder is an immune or autoimmune disorder. Such conditions include autoimmune bullous diseases, non-betalipoproteinemia, acquired immunodeficiency-related diseases, acute immune diseases associated with organ transplantation, acquired acrocyanosis, acute and chronic parasitic or infectious processes, acute pancreatitis, acute renal failure, acute rheumatic fever, acute transverse myelitis, adenocarcinoma, high altitude ectopic beats, adult (acute) respiratory distress syndrome, AIDS dementia complex, alcoholic cirrhosis, alcohol-induced liver injury, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allergies and asthma, allograft rejection, alpha-l-antitrypsin deficiency, alzheimer's disease, amyotrophic lateral sclerosis, anemia, angina, ankylosing spondylitis-related lung disease, anterior angular cell degeneration antibody-mediated cytotoxicity, antiphospholipid syndrome, anti-receptor hypersensitivity, aortic and peripheral aneurysms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, arthrosis, asthma, ataxia, specific allergy, atrial fibrillation (persistent or paroxysmal), atrial flutter, atrioventricular block, atrophic autoimmune hypothyroidism, autoimmune hemolytic anemia, autoimmune hepatitis type 1 (typical autoimmune or lupus hepatitis), autoimmune hypoglycemia, autoimmune neutropenia, autoimmune thrombocytopenia, autoimmune thyroid diseases, B-cell lymphomas, bone graft rejection, bone Marrow Transplantation (BMT) rejection, obstructive bronchitis, bundle branch block, burn, cachexia, arrhythmia, sudden cardiac arrest syndrome, heart tumor, cardiomyopathy, cardiopulmonary bypass inflammatory response, cartilage graft rejection, cerebellar degeneration, cerebellar dysfunction, chaotic or multifocal atrial tachycardia, chemotherapy-related diseases, chlamydia infection, cholecystitis, chronic alcoholism, chronic active hepatitis, chronic fatigue syndrome, chronic immune diseases associated with organ transplantation, chronic eosinophilic pneumonia, chronic inflammatory lesions, chronic skin mucosal candidiasis, chronic Obstructive Pulmonary Disease (COPD), chronic salicylic acidosis, common variability immunodeficiency of colorectal (common variability hypopigmentation), conjunctivitis, connective tissue disease-related interstitial lung disease, contact dermatitis, kums positive hemolytic anemia pulmonary heart disease, creutzfeldt-Jakob disease, cryptogenic autoimmune hepatitis, cryptogenic fibrosing alveolitis, culture negative sepsis, cystic fibrosis, cytokine therapy-related diseases, crohn's disease, dementia pugilistica, demyelinating disease, dengue hemorrhagic fever, dermatitis, scleroderma dermatitis, skin diseases, dermatomyositis/multiple myositis-related lung diseases, diabetes, diabetic arteriosclerotic diseases, diabetes, diffuse lewy body disease, dilated cardiomyopathy, dilated congestive cardiomyopathy, discoid lupus erythematosus, basal ganglia disorders, diffuse intravascular coagulation, down syndrome in middle-aged years, drug-induced interstitial lung disease, drug hepatitis, drug-induced dyskinesia that blocks CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, enteropathic synovitis, epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial lymphocytopenia, fetal thymus implantation rejection, friedrich ataxia, functional peripheral arterial disease, female infertility, fibrosis, pulmonary fibrosis disease, mycotic sepsis, gas gangrene, gastric ulcers, giant cell arteritis, glomerulonephritis, glomerulonephropathy, goodpasture syndrome, thyroid autoimmune hypothyroidism (hashimoto's disease), gouty arthritis, graft rejection of any organ or tissue, graft versus host disease, gram negative sepsis, gram positive sepsis, granuloma caused by intracellular organisms Group B Streptococcus (GBS) infection, graves' disease, blood calcium-related lung disease, hairy cell leukemia, hashimoto's disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hematopoietic malignancies (leukemia and lymphoma), hemolytic anemia, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, hemorrhage, sjog-Hensch purpura (Henoch-Schoenlein purpura), hepatitis A, hepatitis B, hepatitis C, HIV infection/HIV neuropathy, hodgkin's disease, hypoparathyroidism, huntington's chorea, hyperkinesia, hypersensitivity pneumonitis, hyperthyroidism, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis assessment, idiopathic addison's disease, idiopathic leukopenia, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia, idiopathic liver disease, infantile spinal muscular atrophy, infectious diseases, aortic inflammation, inflammatory bowel disease, insulin dependent diabetes mellitus, interstitial pneumonia, iridocyclitis/uveitis/optic neuritis, ischemia reperfusion injury, ischemic stroke, juvenile pernicious anemia, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, kaposi's sarcoma, kawasaki's disease, renal transplant rejection, leishmania, leprosy, corticospinal lesions, linear IgA disease, lipid disorders, liver transplant rejection, lyme disease, lymphogranuloma, lymphocytic infiltration lung disease, malaria, idiopathic or NOS male infertility, leishmaniasis malignant tissue cell hyperplasia, malignant melanoma, meningitis, meningococcemia, renal microvascular inflammation, migraine, mitochondrial multisystem disease, mixed connective tissue disease-related lung disease, monoclonal gammaglobular disease, multiple myeloma, multisystem degeneration (Mencel, dejerine-Thomas, shy-Drager and Machado-Joseph), myalgia encephalitis/Royal free disease (Royal Free Disease), myasthenia gravis, renal microvascular inflammation, intracellular mycobacteria, mycobacterium tuberculosis, myelodysplastic syndrome, myocardial infarction, myocardial ischemic disease, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephropathy, nephrotic syndrome, neurodegenerative diseases, neurogenic type I muscle atrophy, neutropenia fever, nonalcoholic steatohepatitis, abdominal aorta and branch occlusion thereof, occlusive arterial disease, organ graft rejection, orchitis/hydrocele, orchitis/vasectomy reversal, organ hypertrophy, osteoarthropathy, osteoporosis, ovarian failure, pancreatic graft rejection, parasitic disease, parathyroid graft rejection, parkinson's disease, pelvic inflammatory disease, pemphigus vulgaris, pemphigoid, perennial rhinitis, pericardial disease, peripheral arteriosclerotic disease, peripheral vascular disease, peritonitis, pernicious anemia, uveitis pigmentosa, pneumosporopneumonia, pneumonia, ms syndrome (multiple neuropathy, organ hypertrophy, endocrinopathy, monoclonal gammaglobosis and skin change syndrome), post-perfusion syndrome, post-pump syndrome, post MI cardiotomy syndrome, post-infection interstitial lung disease, premature ovarian failure primary biliary cirrhosis, primary sclerotic hepatitis, primary myoedema, primary pulmonary hypertension, primary sclerosing cholangitis, primary vasculitis, progressive supranuclear palsy, psoriasis, type 1 psoriasis, type 2 psoriasis, psoriatic arthrosis, pulmonary hypertension secondary to connective tissue disease, pulmonary manifestations of polyarteritis nodosa, post-inflammatory interstitial lung disease, radiofibrosis, radiation therapy, raynaud's phenomenon and disease, raynaud's disease, lei Fusa m disease (Refsum's disease), regular narrow QRS tachycardia, rate's disease, nephrotic NOS, neovascular hypertension, reperfusion injury, restrictive cardiomyopathy, rheumatoid arthritis-related interstitial lung disease, rheumatoid spondylitis, sarcoidosis, compact syndrome, rheumatoid arthritis-related diseases, (Schmidt's syndrome), scleroderma, senile chorea, lewy body type senile dementia, septicemia syndrome, septic shock, seronegative arthropathy, shock, sickle cell anemia, T cell or FAB ALL, gaoan's disease/arteritis, (Takayasu's disease/arteris), telangiectasia, th2 and Thl mediated diseases, thromboangiitis obliterans, thrombocytopenia, thyroiditis, poisoning, toxic shock syndrome, transplantation, trauma/massive hemorrhage, autoimmune hepatitis type 2 (anti-LKM antibody hepatitis), insulin resistance type B with black acanthosis, type III hypersensitivity, type IV hypersensitivity, ulcerative colitis, unstable angina, uremia, diabetes insipidus, urticaria, uveitis, valvular heart disease, measles varicose veins, vasculitis, vascular diffuse lung disease, venous diseases, venous thrombosis, ventricular fibrillation, vitiligo acute liver disease, viral and fungal infections, viral encephalitis/suppurative meningitis, viral hemocyte syndrome, wegener's granulomatosis (Wegener's disease), wernicke-kosakov syndrome (wernike-Korsakoff syndrome), wilson's disease, xenograft rejection of any organ or tissue, yersinia and salmonella-associated arthropathy, acquired immunodeficiency syndrome (AIDS), autoimmune lymphoproliferative syndrome, hemolytic anemia, inflammatory diseases, thrombocytopenia, acute and chronic immune diseases associated with organ transplantation, addison's disease, allergic diseases, alopecia areata, alopecia, and other diseases, atherosclerosis/arteriosclerosis, atherosclerosis, arthritis (including osteoarthritis, juvenile chronic arthritis, suppurative arthritis, lyme arthritis, psoriatic arthritis and reactive arthritis), sjogren's disease-related lung disease, sjogren's syndrome, skin allograft rejection, skin modification syndrome, small intestine transplant rejection, sperm autoimmunity, multiple sclerosis (all subtypes), spinocerebellar degeneration, spinal arthropathy, sporadic polyadenying type I, sporadic polyadenying type II, stell's disease, streptococcal myositis, stroke, cerebellar structural lesions, subacute sclerotic panencephalitis, sympathetic ophthalmitis, syncope, cardiovascular system syphilis, systemic anaphylactic shock, systemic inflammatory response syndrome, systemic juvenile rheumatoid arthritis, systemic lupus erythematosus-related lung disease, lupus nephritis, systemic sclerosis and systemic sclerosis-related interstitial lung disease.

In some embodiments of each or any of the above or below embodiments, the disease or disorder is an inflammatory disease. Inflammation plays a fundamental role in host defense and immune-mediated disease progression. Inflammatory responses are triggered by complex cascade events including chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., leukocytes) in response to injury (e.g., trauma, ischemia, and foreign particles) and infection (e.g., bacterial or viral infection). Inflammatory responses are characterized by increased blood flow, increased capillary permeability, and phagocytic influx. These events lead to swelling, redness, heating (altered thermal pattern) and pus formation at the site of injury or infection.

Cytokines and prostaglandins control the inflammatory response and are released into the blood or affected tissues in an orderly and self-limiting cascade. This release of cytokines and prostaglandins increases blood flow to the injured or infected area and may lead to redness and fever. Some of these chemicals cause fluid leakage into the tissue, resulting in swelling. This protective process may stimulate nerves and cause pain. These changes are beneficial to the body when they occur in the relevant area for a limited time.

The subtle well-balanced interaction between humoral and cellular immune elements in the inflammatory response enables the elimination of harmful agents and the initiation of repair of damaged tissues. When this delicate equilibrium interaction is disrupted, the inflammatory response may cause considerable damage to normal tissue and its hazard may exceed the original injury that triggered the response. In the case of these uncontrolled inflammatory reactions, clinical intervention is required to prevent tissue damage and organ dysfunction. Psoriasis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, crohn's disease, asthma, allergies, or inflammatory bowel disease are characterized by chronic inflammation. Inflammatory diseases such as arthritis, related arthritic conditions (e.g., osteoarthritis, rheumatoid arthritis, and psoriatic arthritis), inflammatory bowel disease (e.g., crohn's disease and ulcerative colitis), sepsis, psoriasis, atopic dermatitis, contact dermatitis, and chronic obstructive pulmonary disease, chronic inflammatory pulmonary disease, and the like are also common and problematic diseases.

Various delivery systems are known and can be used to administer prophylactic or therapeutic agents (e.g., antibodies or antigen binding fragments thereof provided herein), including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or antigen binding fragments/receptor-mediated endocytosis thereof (see, e.g., wu and Wu, j. Biol. Chem.262:4429-4432 (1987)), construction of nucleic acids as part of a retrovirus or other vector, and the like. Methods of administering a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein) or pharmaceutical composition include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, and mucosal administration (e.g., intranasal and oral routes). In a specific embodiment, a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein) or pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agent or composition can be administered by any convenient route, such as by infusion or bolus injection, by absorption through the epithelial layer or mucosal layer (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.), and can be administered with other bioactive agents. Administration may be systemic or local. In addition, pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer and formulation with an aerosol. See, for example, U.S. patent nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540 and 4,880,078; and PCT publications WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, each of which is incorporated herein by reference in its entirety.

In a particular embodiment, it may be desirable to administer a prophylactic or therapeutic agent or pharmaceutical composition provided herein topically to an area in need of treatment. This may be accomplished, for example, but not limited to, by local infusion, by local administration (e.g., by intranasal spray), by injection, or by means of an implant having a porous, non-porous, or gel-like material, including a membrane (such as a siallastic membrane) or fibers. In some embodiments of each or any of the embodiments described above or below, care must be taken that the material that is not absorbed by the antibody or antigen-binding fragment thereof provided herein is used when administering the antibody or antigen-binding fragment thereof.

In another embodiment, the prophylactic or therapeutic agent or composition provided herein may be delivered in vesicles, particularly liposomes (see Langer,1990,Science 249:1527-1533; treat et al in Liposomes in the Therapy of Infectious Disease and Cancer, lopez-Berestein and Fidler (eds.), lists, new York, pages 353-365 (1989); lopez-Berestein, ibid., pages 317-327; see ibid.).

In another embodiment, the prophylactic or therapeutic agent or composition provided herein may be delivered in a controlled or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; sefton,1987,CRC Crit.Ref.Biomed.Eng.14:20;Buchwald et al, 1980,Surgery 88:507;Saudek et al, 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials may be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., an antibody provided herein) or a composition provided herein (see, e.g., medical Applications of Controlled Release, langer and Wise (editions), CRC pres., boca Raton, florida (1974); controlled Drug Bioavailability, drug Product Design and Performance, smolen and Ball (editions), wiley, new York (1984); range and Peppas,1983, j., macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al, 1985, science228:190; during et al, 1989, ann. Neurol.25:351; howard et al, 1989,J.Neurosurg.7 1:105); us patent 5,679,377; us patent 5,916,597; U.S. Pat. nos. 5,912,015; us patent 5,989,463; us patent 5,128,326; PCT publication WO 99/15154; and PCT publication WO 99/20253. Examples of polymers for sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid), polyglycolide (PLG), polyanhydrides, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly (lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable. In another embodiment, a controlled or sustained release system may be placed in proximity to the therapeutic target (i.e., nasal passage or lung) so that only a portion of the systemic dose is required (see, e.g., goodson, medical Applications of Controlled Release, supra, volume 2, pages 115-138 (1984)). Controlled release systems are discussed in the overview by Langer (1990,Science 249:1527-1533). Any technique known to those of skill in the art may be used to produce a sustained release formulation comprising one or more antibodies or antigen-binding fragments thereof provided herein. See, for example, U.S. Pat. nos. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; ning et al, 1996, "Intratumoral Radioimmunotherapy of aHuman Colon Cancer Xenograft Using a Sustained-Release Gel", radius & Oncology 39:179-189; song et al, 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA Journal of Pharmaceutical Science & Technology 50:372-397; cleek et al, 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application", pro.int' l.Symp.control. Rel.Bioact.Mater.24:853-854; and Lam et al, 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery", proc.int' l.symp.control rel.bio.mater.24:759-760, each of which is incorporated herein by reference in its entirety.

In a particular embodiment, where the compositions provided herein are nucleic acids encoding a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein), the nucleic acids can be administered in vivo to facilitate expression of the prophylactic or therapeutic agent encoded thereby by: for example, nucleic acids are constructed as part of an appropriate nucleic acid expression vector and administered to render it intracellular, either by use of retroviral vectors (see U.S. Pat. No. 4,980,286) or by direct injection or by use of microprojectile bombardment (e.g., gene gun; biolistic, dupont), or by coating with lipids or cell surface receptors or transfection agents, or by ligation of nucleic acids with homologous frame-like peptides known to enter the nucleus (see, e.g., joliot et al, 1991,Proc.Natl.Acad.Sci.USA 88:1864-1868), and the like. Alternatively, the nucleic acid may be introduced into the cell by homologous recombination and incorporated into the host cell DNA for expression.

In a specific embodiment, the compositions provided herein comprise one, two, or more antibodies or antigen-binding fragments thereof provided herein. In another embodiment, a composition provided herein comprises one, two, or more antibodies or antigen-binding fragments thereof provided herein and a prophylactic or therapeutic agent other than an antibody or antigen-binding fragment thereof provided herein. In one embodiment, a known prophylactic or therapeutic agent may be used or has been used or is currently used to prevent, manage, treat and/or ameliorate a disease or disorder. In addition to prophylactic or therapeutic agents, the compositions provided herein may also include excipients.

The compositions provided herein include pharmaceutical compositions of raw materials useful for making pharmaceutical compositions (e.g., compositions suitable for administration to a subject or patient) useful for preparing unit dosage forms. In some embodiments of each or any of the embodiments above or below, the composition provided herein is a pharmaceutical composition. Such compositions comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., an antibody or antigen-binding fragment thereof provided herein or other prophylactic or therapeutic agent) and a pharmaceutically acceptable excipient. The pharmaceutical composition may be formulated into a route suitable for administration to a subject.

In a particular embodiment, the term "excipient" may also refer to a diluent, adjuvant (e.g., freund's adjuvant (complete or incomplete)), or vehicle. The pharmaceutical excipients may be sterile liquids, such as water and oils, including those derived from petroleum, animal, vegetable or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is an exemplary excipient. Saline and dextrose in water and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing co., easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, such as in purified form, and a suitable amount of excipient, so as to provide a form for appropriate administration to a patient. The formulation should be compatible with the mode of administration.

In one embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a sterile isotonic buffered aqueous solution. The composition may also contain a solubilizing agent and a local anesthetic such as lidocaine, as necessary, to reduce pain at the injection site. However, such compositions may be administered by a route other than intravenous administration.

Typically, the components of the compositions provided herein are provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or anhydrous concentrate, in a hermetically sealed container such as an ampoule or pouch that indicates the amount of active agent. Where the composition is administered by infusion, the composition may be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of compositions for administration by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

The antibodies or antigen-binding fragments thereof provided herein can be packaged in hermetically sealed containers such as ampules or sachets that indicate the amount of antibody. In some embodiments of each or any of the embodiments above or below, the antibody or antigen-binding fragment thereof is provided as a dry sterilized lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be reconstituted to an appropriate concentration, for example, with water or saline for administration to a subject. The lyophilized antibody or antigen binding fragment thereof may be stored in its original container at between 2 ℃ and 8 ℃ and the antibody or antigen binding fragment thereof may be administered within 12 hours after reconstitution, such as within 6 hours, within 5 hours, within 3 hours, or within 1 hour. In some embodiments of each or any of the embodiments above or below, the antibodies or antigen binding fragments thereof provided herein are provided in liquid form in a hermetically sealed container that indicates the amount and concentration of the antibodies.

The compositions provided herein may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed from anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like; and those formed from cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The amount of a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein) or composition provided herein effective to prevent and/or treat a disease or disorder can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help determine optimal dosage ranges. The precise dosage to be employed in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be determined according to the judgment of the practitioner and each patient's circumstances.

The effective dose can be deduced from dose response curves derived from in vitro or animal model test systems.

In some embodiments of each or any of the embodiments above or below, the route of administering a dose of an antibody or antigen binding fragment thereof provided herein to a patient is intranasal, intramuscular, intravenous, subcutaneous, or a combination thereof, although other routes described herein are also acceptable. Each dose may or may not be administered by the same route of administration. In some embodiments of each or any of the embodiments described above or below, the antibodies or antigen-binding fragments thereof provided herein can be administered to other doses of the same or different antibodies or antigen-binding fragments thereof provided herein via multiple routes of administration simultaneously or subsequently.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein is administered to a subject prophylactically or therapeutically. The antibodies or antigen-binding fragments thereof provided herein can be administered to a subject prophylactically or therapeutically in order to prevent, reduce, or ameliorate a disease or symptom thereof.

5.8 Gene therapy

In a particular embodiment, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof is administered to a subject for use in the methods provided herein, e.g., to prevent, manage, treat, and/or ameliorate a disease, disorder, or condition by gene therapy. Such therapies include therapies by administering an expressed or expressible nucleic acid to a subject. In some embodiments of each or any of the embodiments above or below, the nucleic acid produces an antibody that it encodes, and the antibody mediates a prophylactic or therapeutic effect. Any method available in the art for recombinant gene expression (or gene therapy) may be used.

In some embodiments, the nucleic acid for gene therapy encodes a binding molecule or fragment thereof as disclosed herein. In some embodiments, a nucleic acid for gene therapy encodes a binding molecule comprising a region derived from the CH1 region of an antibody heavy chain and/or a region derived from the CL region of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops. In some embodiments, a nucleic acid for gene therapy encodes a first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain, wherein the region derived from the CH1 region comprises one or more antigen binding loops. In some embodiments, the nucleic acid for gene therapy encodes a second polypeptide comprising a light chain variable region (VL) and a region derived from a CL region of an antibody light chain, wherein the region derived from the CL region comprises one or more antigen binding loops. In some embodiments, one or more nucleic acid molecules for gene therapy encode: (i) A first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain; and (ii) a second polypeptide comprising a light chain variable region (VL) and a region derived from the CL region of the antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops.

For a general review of gene therapy methods, see Goldspiel et al, 1993,Clinical Pharmacy 12:488-505; wu and Wu,1991,Biotherapy 3:87-95; tolstophav, 1993, ann. Rev. Pharmacol. Toxicol.32:573-596; mulligan,1993,Science 260:926-932; and Morgan and Anderson,1993, ann. Rev. Biochem.62:191-217; 5 months 1993, TIBTECH 11 (5): 155-215. A commonly known method for use in the field of recombinant DNA technology is described in Ausubel et al (eds.), current Protocols in Molecular Biology, john Wiley & Sons, NY (1993); and Kriegler, gene Transfer and Expression, A Laboratory Manual, stock Press, NY (1990).

In a specific embodiment, the composition comprises a nucleic acid encoding an antibody provided herein, which nucleic acid is part of an expression vector for expressing the antibody or chimeric protein or heavy or light chain thereof in a suitable host. In particular, such nucleic acids have a promoter, such as a heterologous promoter, operably linked to the antibody coding region, which is inducible or constitutive, and optionally tissue specific. In another particular embodiment, a nucleic acid molecule is used in which the antibody coding sequence and any other desired sequences are flanked by regions that promote homologous recombination at desired sites in the genome, thereby providing for the in-chromosomal expression of the antibody coding nucleic acid (Koller and Smithies,1989,Proc.Natl.Acad.Sci.USA 86:8932-8935; zijlstra et al, 1989,Nature 342:435-438). In some embodiments of each or any of the above or below embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequence includes sequences encoding both the heavy and light chains of an antibody or fragments thereof.

The delivery of the nucleic acid into the subject may be direct, in which case the subject is directly exposed to the nucleic acid or a vector carrying the nucleic acid, or indirect, in which case the cell is first transformed with the nucleic acid in vitro and then transplanted into the subject. These two methods are known as in vivo gene therapy or ex vivo gene therapy, respectively.

In a specific embodiment, the nucleic acid sequence is administered directly in vivo, wherein the sequence is expressed to produce the encoded product. This can be accomplished by any of a variety of methods known in the art, for example, by constructing them as part of a suitable nucleic acid expression vector and administering the vector such that the sequences become intracellular, for example, by infection with defective or attenuated retroviruses or other viral vectors (see U.S. patent 4,980,286), or by direct injection of naked DNA, or by bombardment with microparticles (e.g., gene gun; biolistic, dupont), or coating with lipid or cell surface receptors or transfection agents, encapsulation in liposomes, microparticles or microcapsules, or by administering them in conjunction with peptides known to enter the nucleus, by administering them in conjunction with ligands that undergo receptor-mediated endocytosis (see, e.g., wu and Wu,1987, j. Biol. Chem. 262:4429-4432), which can be used to target cell types that specifically express receptors, and the like. In another embodiment, a nucleic acid-ligand complex may be formed in which the ligand comprises a fusion viral peptide to disrupt endosomes, thereby allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can target cell-specific uptake and expression in vivo by targeting specific receptors (see, e.g., PCT publication WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188, WO 93/20221). Alternatively, the nucleic acid may be introduced into the cell by homologous recombination and incorporated into the host cell DNA for expression (Koller and Smithies,1989,Proc.Natl.Acad.Sci.USA 86:8932-8935; and Zijlstra et al, 1989,Nature 342:435-438).

In a specific embodiment, a viral vector containing a nucleic acid sequence encoding an antibody is used. For example, retroviral vectors can be used (see Miller et al, 1993, meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into the host cell DNA. Nucleic acid sequences encoding antibodies for gene therapy may be cloned into one or more vectors, which facilitate the delivery of genes into a subject. More details on retroviral vectors can be found in Boesen et al, 1994,Biotherapy 6:291-302, which describe the use of retroviral vectors to deliver MDR1 genes to hematopoietic stem cells to render the stem cells more resistant to chemotherapy. Other references describing the use of retroviral vectors in gene therapy are: clowes et al, 1994, J.Clin.Invest.93:644-651; klein et al, 1994,Blood 83:1467-1473; salmons and Gunzberg,1993,Human Gene Therapy 4:129-141; and Grossman and Wilson,1993,Curr.Opin.in Genetics and level.3:110-114.

Adenoviruses are other viral vectors that can be used for recombinant production of antibodies. Adenoviruses are particularly attractive vectors for delivering genes to airway epithelial cells. Adenovirus naturally infects respiratory epithelial cells and causes mild disease at the epithelial cells. Other targets of adenovirus-based delivery systems are liver, central nervous system, endothelial cells and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson,1993,Current Opinion in Genetics and Development 3:499-503 provide an overview of adenovirus-based gene therapy. Bout et al 1994,Human Gene Therapy 5:3-10 demonstrate that gene transfer to rhesus respiratory epithelial cells can be accomplished using adenovirus vectors. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al, 1991,Science 252:431-434; rosenfeld et al, 1992, cell 68:143-155; mastrangeli et al, 1993, J.Clin. Invest.91:225-234; PCT publication WO94/12649; and Wang et al, 1995,Gene Therapy 2:775-783. In a specific embodiment, an adenovirus vector is used.

Adeno-associated virus (AAV) may also be used (Walsh et al, 1993, proc.Soc.exp.biol. Med.204:289-300; and U.S. Pat. No. 5,436,146). In a specific embodiment, an AAV vector is used to express an antibody provided herein. In certain embodiments, the AAV comprises a nucleic acid encoding a VH domain. In other embodiments, the AAV comprises a nucleic acid encoding a VL domain. In certain embodiments, the AAV comprises nucleic acids encoding VH and VL domains. In some embodiments of the methods provided herein, an AAV comprising a nucleic acid encoding a VH domain and an AAV comprising a nucleic acid encoding a VL domain are administered to a subject. In other embodiments, an AAV comprising nucleic acids encoding VH and VL domains is administered to a subject. In certain embodiments, the VH domain and VL domain are overexpressed.

Another method of gene therapy involves the transfer of genes into cells of tissue culture by methods such as electroporation, liposome infection, calcium phosphate mediated transfection or viral infection. Typically, the transfer method involves transferring the selectable marker to the cell. The cells are then placed under selection to isolate those cells that have taken up and expressed the transgene. These cells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into the cells prior to in vivo administration of the resulting recombinant cells. Such introduction may be performed by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or phage vector containing a nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, minicell-mediated gene transfer, spheroplast fusion, and the like. A variety of techniques for introducing exogenous genes into cells are known in the art (see, e.g., loeffler and Behr,1993, meth. Enzymol.217:599-618; cohen et al, 1993, meth. Enzymol.217:618-644; clin. Pharma. Ther.29:69-92 (1985)) and can be used according to the methods provided herein, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide stable transfer of the nucleic acid to the cell so that the nucleic acid can be expressed by the cell, such as heritable and reachable by its post-cellular representation.

The resulting recombinant cells can be delivered to a subject by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) can be administered intravenously. The amount of cells contemplated for use depends on the desired effect, patient status, etc., and can be determined by one of skill in the art.

For gene therapy purposes, cells into which nucleic acid may be introduced include any desired, available cell type, and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, myocytes, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem cells or progenitor cells, particularly hematopoietic stem cells or progenitor cells, such as hematopoietic stem cells or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and the like.

In a specific embodiment, the cells used for gene therapy are autologous to the subject.

In embodiments where recombinant cells are used in gene therapy, nucleic acid sequences encoding antibodies are introduced into the cells so that they can be reached by the cells or their post-representation, and then the recombinant cells are administered in vivo to obtain a therapeutic effect. In a specific embodiment, stem cells or progenitor cells are used. According to embodiments of the methods provided herein, any stem and/or progenitor cell that can be isolated and maintained in vitro can potentially be used (see, e.g., PCT publication WO 94/08598; stemple and Anderson,1992,Cell 7 1:973-985;Rheinwald,1980,Meth.Cell Bio.21A:229; and Pittelkow and Scott,1986,Mayo Clinic Proc.61:771).

In a specific embodiment, the nucleic acid introduced for gene therapy purposes comprises an inducible promoter operably linked to a coding region such that expression of the nucleic acid can be controlled by controlling the presence or absence of a suitable transcriptional inducer.

5.9 diagnostic assays and methods

Labeled antibodies and derivatives and analogs thereof that immunospecifically bind to an antigen provided herein can be used for diagnostic purposes to detect, diagnose, or monitor a disease or disorder.

The antibodies provided herein can be used to determine antigen levels in biological samples using classical immunohistological methods described herein or known to those of skill in the art (see, e.g., jalkanen et al, 1985, J. Cell. Biol.101:976-985; and Jalkanen et al, 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods that can be used to detect protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISA) and Radioimmunoassays (RIA). Suitable antibody assay markers are known in the art and include enzyme markers such as glucose oxidase; radioisotopes such as iodine (125 i,121 i), carbon (14C), sulfur (35S), tritium (3H), indium (121 In), and technetium (99 Tc); luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin. One aspect provided herein is the detection and diagnosis of a disease or disorder in a human.

It will be appreciated in the art that the size of the subject and the imaging system used will determine the amount of imaging portion required to produce the diagnostic image. In the case of radioisotope moieties, the amount of radioactivity injected is typically in the range of about 5 millicuries to 20 millicuries of 99Tc for a human subject. The labeled antibodies will then accumulate at the location of the cells containing the particular protein. In vivo Tumor Imaging is described in S.W. Burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Tumor Imaging: the Radiochemical Detection of Cancer chapter 13, S.W. Burchiel and B.A. Rhodes editions, masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following administration to allow concentration of the labeled antibody at the site in the subject and to allow unbound labeled antibody to be cleared to background levels is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval after administration is from 5 days to 20 days or from 5 days to 10 days.

In one embodiment, the monitoring of the disease or condition is performed by repeating the method for diagnosing the disease or condition, e.g., one month after the initial diagnosis, six months after the initial diagnosis, one year after the initial diagnosis, etc.

The presence of the marker molecule can be detected in the subject using methods known in the art for in vivo scanning. These methods depend on the type of label used. The skilled person will be able to determine the appropriate method for detecting a particular label. Methods and apparatus useful in the diagnostic methods provided herein include, but are not limited to, computed Tomography (CT), whole-body scanning such as Positron Emission Tomography (PET), magnetic Resonance Imaging (MRI), and ultrasonography.

In a specific embodiment, the molecules are labeled with a radioisotope and detected in the patient using a radiation-responsive surgical instrument (Thurston et al, U.S. Pat. No. 5,441,050). In another embodiment, the molecules are labeled with a fluorescent compound and detected in the patient using a fluorescent response scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In another embodiment, the molecules are labeled with a paramagnetic marker and detected in the patient using Magnetic Resonance Imaging (MRI).

5.10 kit

Also provided herein are kits comprising an antibody provided herein or a composition provided herein (e.g., a pharmaceutical composition) packaged into a suitable packaging material. The kit optionally includes a label or package insert that includes a description of the component or instructions for in vitro, in vivo, or ex vivo use of the component therein.

The term "packaging material" refers to the physical structure that contains the components of the kit. The packaging material may maintain the components aseptically and may be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoule, vial, tube, etc.).

Kits provided herein can include a label or instructions for use. The label or instructions for use include "printed matter", such as paper or cardboard, alone or attached to a component, kit or packaging material (e.g., a cartridge), or attached to, for example, an ampoule, tube or vial containing the kit components. The label or instructions may additionally include a computer-readable medium, such as a disk (e.g., hard disk, card, storage disk), optical disk (such as CD or DVD-ROM/RAM, DVD, MP3, magnetic tape), or electronic storage medium (such as RAM and ROM), or a mix of these (such as magnetic/optical storage medium, FLASH media, or memory cards). The label or instructions may include information identifying the manufacturer, lot number, manufacturer location, and date.

Kits provided herein can additionally include other components. Each component of the kit may be enclosed in a separate container, and all of the various containers may be in a single package. The kit may also be designed for refrigeration. The kit may also be designed to contain an antibody provided herein, or a cell containing a nucleic acid encoding an antibody provided herein. The cells in the kit may be maintained under suitable storage conditions until ready for use.

Also provided herein are antibody panels that immunospecifically bind to a specific antigen. In particular embodiments, provided herein are antibody panels having different association rate constants, different dissociation rate constants, different affinities for an antigen, and/or different specificities for an antigen. In certain embodiments, provided herein are panels of about 10, preferably about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 antibodies or more. Antibody panels can be used, for example, in 96-well or 384-well plates, such as for assays such as ELISA.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

As used herein, numerical values are generally given throughout this document in a range format. The use of range format is merely for convenience and brevity and should not be construed as an absolute limitation on the scope of the invention, unless the context clearly indicates otherwise. Thus, a range used is intended to include all possible sub-ranges, all individual values within the range, and all values or ranges of values (including integers within such range and fractions or integers of values within the range) unless the context clearly dictates otherwise. This configuration applies in all contexts throughout this patent document regardless of the breadth of the range. Thus, for example, references to a range of 90% to 100% include 91% to 99%, 92% to 98%, 93% to 95%, 91% to 98%, 91% to 97%, 91% to 96%, 91% to 95%, 91% to 94%, 91% to 93%, and the like. References to a range of 90% to 100% also include 91%, 92%, 93%, 94%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., and 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc.

In addition, the ranges mentioned 1 to 3, 3 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, 190 to 200, 200 to 225, 225 to 250 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and the like. In another example, references to a range of 25 to 250, 250 to 500, 500 to 1,000, 1,000 to 2,500, 2,500 to 5,000, 5,000 to 25,000, 25,000 to 50,000 include any numerical value or range within or encompassing such values, e.g., 25, 26, 27, 28, 29 … 250, 251, 252, 253, 254 … 500, 501, 502, 503, 504 …, etc.

A range of ranges also used herein is disclosed throughout the literature. The use of a range includes combinations of upper and lower limits to provide another range. This configuration applies in all contexts throughout this patent document regardless of the breadth of the range. Thus, for example, reference to a range of ranges such as 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100, 100 to 150 includes ranges such as 5 to 20, 5 to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150 and 10 to 30, 10 to 40, 10 to 50, 10 to 75, 10 to 100, 10 to 150 and 20 to 40, 20 to 50, 20 to 75, 20 to 100, 20 to 150, and the like.

For brevity, certain abbreviations are used herein. One example is a single letter abbreviation that refers to an amino acid residue. Amino acids and their corresponding three-letter and one-letter abbreviations are as follows:

alanine Ala (A)

Arginine Arg (R)

Asparagine Asn (N)

Asp (D)

Cysteine Cys (C)

Glutamic acid Glu (E)

Glutamine Gln (Q)

Glycine Gly (G)

Histidine His (H)

Isoleucine Ile (I)

Leucine Leu (L)

Lysine Lys (K)

Met methionine (M)

Phe (F)

Proline Pro (P)

Serine Ser (S)

Threonine Thr (T)

Trp tryptophan (W)

Tyrosine Tyr (Y)

Valine Val (V)

The present invention is generally disclosed herein using a positive language to describe various embodiments. The invention also specifically includes embodiments in which specific subject matter such as materials or substances, method steps and conditions, protocols, procedures, assays or analyses are all or partially excluded. Thus, even though the invention is not generally expressed in terms of what the invention does not include, aspects not expressly included in the invention are disclosed herein.

Various embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate and not limit the scope of the invention as described in the claims.

6. Description of the embodiments

The present invention provides the following non-limiting embodiments.

In one set of embodiments (set a embodiments), there is provided:

A1. (embodiment 1) a binding molecule comprising:

(i) A first polypeptide comprising a heavy chain variable region (VH) and a region derived from a CH1 region of an antibody heavy chain; and

(ii) A second polypeptide comprising a light chain variable region (VL) and a region derived from the CL region of an antibody light chain,

wherein the CH1 domain derived region and/or the CL domain derived region comprises one or more antigen binding loops.

A2. (embodiment 2) the binding molecule according to embodiment A1, wherein the one or more antigen binding loops in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region.

A3. (embodiment 3) the binding molecule according to embodiment A1 or A2, wherein the one or more antigen binding loops in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CL region.

A4. (embodiment 4) the binding molecule according to any one of embodiments A1-A3, wherein the region derived from the CH1 region comprises one or two antigen binding loops.

A5. (embodiment 5) the binding molecule according to any one of embodiments A1-A4, wherein the region derived from the CL region comprises one or two antigen binding loops.

A6. (embodiment 6) the binding molecule according to any one of embodiments A1-A5, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region.

A7. (embodiment 7) the binding molecule according to any one of embodiments A1-A6, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region.

A8. (embodiment 8) the binding molecule according to any one of embodiments A1-A7, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region.

A9. (embodiment 9) the binding molecule according to any one of embodiments A1-A8, wherein the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region.

A10. (embodiment 10) the binding molecule according to any one of embodiments A1-A9, wherein the region derived from the CL region comprises one antigen binding loop at the DE loop region of the CL region.

A11. (embodiment 11) the binding molecule according to any one of embodiments A1-a10, wherein the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region.

A12. (embodiment 12) the binding molecule according to any one of embodiments A1-A6 or A9, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

A13. (embodiment 13) the binding molecule according to any one of embodiments A1-A6 or a10, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

A14. (embodiment 14) the binding molecule according to any one of embodiments A1-A6 or A9-a13, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

A15. (embodiment 15) the binding molecule according to any one of embodiments A1-A5, A7 or A9, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

A16. (embodiment 16) the binding molecule of any one of embodiments A1-A5, A7 or a10, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

A17. (embodiment 17) the binding molecule according to any one of embodiments A1-A5, A7, A9-a11, a15 or a16, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

A18. (embodiment 18) the binding molecule according to any one of embodiments A1-A9, a12 or a15, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

A19. (embodiment 19) the binding molecule of any one of embodiments A1-A8, a10, a13, or a16, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

A20. (embodiment 20) the binding molecule according to any one of embodiments A1-a19, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

A21. (embodiment 21) the binding molecule according to any one of embodiments A1-a20, wherein the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1.

A22. (embodiment 22) the binding molecule according to any one of embodiments A1-a21, wherein the region derived from the CL region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the region derived from the CL region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID No. 2.

A23. (embodiment 23) the binding molecule according to embodiment a21 or a22, wherein the antigen binding loop at the CD loop region of the CH1 region replaces the amino acid residue TSG of the CD loop of the human IgG1 CH1 region.

A24. (embodiment 24) the binding molecule according to embodiment a21 or a22, wherein the antigen binding loop at the DE loop region of the CH1 region replaces the amino acid residue QSS of the DE loop of the human IgG1 CH1 region.

A25. (embodiment 25) the binding molecule according to embodiment a21 or a22, wherein the antigen binding loop at the CD loop region of the CL region replaces the amino acid residue SGNS of the CD loop of the human clk region.

A26. (embodiment 26) the binding molecule of embodiment a21 or a22, wherein the antigen binding loop at the DE loop region of the CL region replaces the amino acid residue SKD of the DE loop of the human clk region.

A27. (embodiment 27) the binding molecule of any one of embodiments A1-a26, wherein each of the one or more antigen binding loops comprises 7 to 15 amino acid residues.

A28. (embodiment 28) the binding molecule of any one of embodiments A1-a27, wherein the VH region and the VL region bind to a first antigen; and the region derived from the CH1 region and/or the region derived from the CL region binds to a second antigen.

A29. (embodiment 29) the binding molecule of embodiment a28, wherein the first antigen and the second antigen are the same antigen.

A30. (embodiment 30) the binding molecule of embodiment a28, wherein the first antigen and the second antigen are two different antigens.

A31. (embodiment 31) a nucleic acid encoding a binding molecule according to any one of embodiments A1-a 30.

A32. (embodiment 32) a vector comprising the nucleic acid according to embodiment a31.

In another set of embodiments (set B embodiments), there is provided:

B1. (embodiment 1) a Constant Region Library (CRL) comprising a population of binding molecules wherein each of the binding molecules comprises:

(ii) A second polypeptide comprising a light chain variable region (VL) and a region derived from a CL region of an antibody light chain, wherein the population of binding molecules comprises different amino acid sequences in the region derived from a CH1 region and/or the region derived from a CL region.

B2. (embodiment 2) the CRL according to embodiment B1, wherein the different amino acid sequences in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region.

B3. (embodiment 3) the CRL according to embodiment B1 or B2, wherein the different amino acid sequences in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop regions of the CL region.

B4. (embodiment 4) the CRL according to any one of embodiments B1-B3, wherein the population of binding molecules comprises different amino acid sequences in one or both loop regions in the region derived from the CH1 region.

B5. (embodiment 5) the CRL according to any one of embodiments B1-B4, wherein the population of binding molecules comprises different amino acid sequences in one or both loop regions in the CL-derived region.

B6. (embodiment 6) the CRL according to any one of embodiments B1-B5, wherein the population of binding molecules comprises different amino acid sequences at the CD loop region of the CH1 region.

B7. (embodiment 7) the CRL of any one of embodiments B1-B6 wherein the population of binding molecules comprises different amino acid sequences at the DE loop region of the CH1 region.

B8. (embodiment 8) the CRL of any one of embodiments B1-B7 wherein the population of binding molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region.

B9. (embodiment 9) the CRL according to any one of embodiments B1-B8, wherein the population of binding molecules comprises different amino acid sequences at the CD loop region of the CL region.

B10. (embodiment 10) the CRL of any one of embodiments B1-B9 wherein the population of binding molecules comprises different amino acid sequences at the DE loop region of the CL region.

B11. (embodiment 11) the CRL according to any one of embodiments B1-B10, wherein the population of binding molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

B12. (embodiment 12) the CRL of any one of embodiments B1-B6 or B9 wherein the population of binding molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the CD loop region of the CL region.

B13. (embodiment 13) the CRL of any one of embodiments B1-B6 or B10 wherein the population of binding molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the DE loop region of the CL region.

B14. (embodiment 14) the CRL according to any one of embodiments B1-B6 or B9-B13, wherein the population of binding molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

B15. (embodiment 15) the CRL of any one of embodiments B1-B5, B7 or B9 wherein the population of binding molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the CD loop region of the CL region.

B16. (embodiment 16) the CRL of any one of embodiments B1-B5, B7 or B10 wherein the population of binding molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the DE loop region of the CL region.

B17. (embodiment 17) the CRL of any one of embodiments B1-B5, B7, B9-B11, B15, or B16 wherein the population of binding molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

B18. (embodiment 18) the CRL of any one of embodiments B1-B9, B12 or B15 wherein the population of binding molecules comprises different amino acid sequences at the CD loop region of the CH1 region and the DE loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the CD loop region of the CL region.

B19. (embodiment 19) the CRL of any one of embodiments B1-B8, B10, B13, or B16 wherein the population of binding molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the DE loop region of the CL region.

B20. (embodiment 20) the CRL of any one of embodiments B1-B19 wherein the population of binding molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and the population of binding molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

B21. (embodiment 21) the CRL according to any one of embodiments B1-B20, wherein the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1.

B22. (embodiment 22) the CRL according to any one of embodiments B1-B21, wherein the CL-region derived region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the CL-region derived region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 2.

B23. (embodiment 23) the CRL according to embodiment B21 or B22, wherein the amino acid residue TSG of the CD loop of the human IgG1 CH1 region is replaced by a different amino acid sequence in the binding molecule in the CRL.

B24. (embodiment 24) the CRL according to embodiment B21 or B22, wherein the amino acid residue QSS of the DE loop of the human IgG1 CH1 region is replaced by a different amino acid sequence in the binding molecule in the CRL.

B25. (embodiment 25) the CRL according to embodiment B21 or B22, wherein the amino acid residue SGNS of the CD loop of the human clk region is replaced by a different amino acid sequence in the binding molecule in the CRL.

B26. (embodiment 26) the CRL according to embodiment B21 or B22, wherein the amino acid residue SKD of the DE loop of the human clk region is replaced by a different amino acid sequence in the binding molecule in the CRL.

B27. (embodiment 27) the CRL according to any one of embodiments B1-B26, wherein the different amino acid sequences comprise 7 to 15 amino acid residues.

B28. (embodiment 28) a Constant Region Library (CRL) comprising a population of molecules each comprising a region derived from a CH1 region of an antibody and/or a region derived from a CL region of an antibody, wherein the population of molecules comprises different amino acid sequences in the region derived from a CH1 region and/or the region derived from a CL region.

B29. (embodiment 29) the CRL of embodiment B28 wherein the different amino acid sequences in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region.

B30. (embodiment 30) the CRL of embodiment B28 wherein the different amino acid sequences in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CL region.

B31. (embodiment 31) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences in one or both loop regions in the region derived from the CH1 region.

B32. (embodiment 32) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences in one or both loop regions in the CL-derived region.

B33. (embodiment 33) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region.

B34. (embodiment 34) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region.

B35. (embodiment 35) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region.

B36. (embodiment 36) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region of the CL region.

B37. (embodiment 37) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the DE loop region of the CL region.

B38. (embodiment 38) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CL region.

B39. (embodiment 39) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region of said CL region.

B40. (embodiment 40) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region.

B41. (embodiment 41) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region and said DE loop region of said CL region.

B42. (embodiment 42) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region of said CL region.

B43. (embodiment 43) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region.

B44. (embodiment 44) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region and said DE loop region of said CL region.

B45. (embodiment 45) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region of the CH1 region and the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region of said CL region.

B46. (embodiment 46) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and the population of molecules comprises different amino acid sequences at the DE loop region of the CL region.

B47. (embodiment 47) the CRL of embodiment B28 wherein the population of molecules comprises different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region and said DE loop region of said CL region.

B48. (embodiment 48) the CRL according to any one of embodiments B28-B47, wherein the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1.

B49. (embodiment 49) the CRL according to any one of embodiments B28-B47, wherein the CL-region derived region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the CL-region derived region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 2.

B50. (embodiment 50) the CRL according to embodiment B48 or B49, wherein the amino acid residue TSG of the CD loop of the human IgG1 CH1 region is replaced by a different amino acid sequence in the molecule in the CRL.

B51. (embodiment 51) the CRL according to embodiment B48 or B49, wherein the amino acid residue QSS of the DE loop of the human IgG1 CH1 region is replaced by a different amino acid sequence in the molecule in the CRL.

B52. (embodiment 52) the CRL according to embodiment B48 or B49, wherein the amino acid residue SGNS of the CD loop of the human clk region is replaced by a different amino acid sequence in the molecule in the CRL.

B53. (embodiment 53) the CRL of embodiment B48 or B49 wherein the amino acid residue SKD of the DE loop of the human clk region is replaced by a different amino acid sequence in the molecule in the CRL.

B54. (embodiment 54) the CRL according to any one of embodiments B28-B53, wherein the different amino acid sequences comprise 7 to 15 amino acid residues.

B55. (embodiment 55) the CRL according to any one of embodiments B28-B54, wherein each of the molecules further comprises a VH region and a VL region.

B56. (embodiment 56) the CRL according to any one of embodiments B1-B55, wherein the binding molecule or the molecule is a Fab fragment.

B57. (embodiment 57) the CRL of any one of embodiments B1-B56, wherein the CRL having one loop region has a diversity ranging from 10 ⁷ To 10 ¹⁶ 。

B58. (embodiment 58) the CRL of any one of embodiments B1-B56, wherein the CRL having two loop regions has a diversity ranging from 10 ¹⁸ To 10 ³³ 。

B59. (embodiment 59) a method for identifying a binding molecule comprising a first binding domain that binds a first antigen and a second binding domain that binds a second antigen, the method comprising screening the CRL according to any one of embodiments B1-B58 to identify a binding molecule that binds the second antigen with an affinity that is higher than a reference level, wherein the first binding domain comprises a VH region and a VL region of an antibody, and wherein the second binding domain comprises an antibody constant region variant.

B60. (embodiment 60) a method of producing a binding molecule, the method comprising: a first step for performing a function of identifying antibody constant region variants capable of binding to an antigen; and a second step of constructing the binding molecule comprising the antibody constant region variant.

B61. (embodiment 61) the method of embodiment B60, wherein the first step comprises screening the CRL according to any one of embodiments B1-B58.

B62. (embodiment 62) a binding molecule produced according to the method of any one of embodiments B59-B61.

In another set of embodiments (set C embodiments), there is provided:

C1. (embodiment 1) a method of making a binding molecule, the method comprising expressing in a host cell a polynucleotide encoding a binding molecule, wherein the binding molecule comprises:

C2. (embodiment 2) the method of embodiment C1, wherein the one or more antigen binding loops in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region.

C3. (embodiment 3) the method of embodiment C1 or C2, wherein the one or more antigen binding loops in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop regions of the CL region.

C4. (embodiment 4) the method of any one of embodiments C1-C3, wherein the region derived from the CH1 region comprises one or two antigen binding loops.

C5. (embodiment 5) the method of any one of embodiments C1-C4, wherein the region derived from the CL region comprises one or two antigen binding loops.

C6. (embodiment 6) the method of any one of embodiments C1-C5, wherein the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region.

C7. (embodiment 7) the method of any one of embodiments C1-C6, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region.

C8. (embodiment 8) the method of any one of embodiments C1-C7, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region.

C9. (embodiment 9) the method of any one of embodiments C1-C8, wherein the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region.

C10. (embodiment 10) the method of any one of embodiments C1-C9, wherein the region derived from the CL region comprises one antigen binding loop at the DE loop region of the CL region.

C11. (embodiment 11) the method of any one of embodiments C1-C10, wherein the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region.

C12. (embodiment 12) the method of any one of embodiments C1-C6 or C9, wherein the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

C13. (embodiment 13) the method of any one of embodiments C1-C6 or C10, wherein the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

C14. (embodiment 14) the method of any one of embodiments C1-C6 or C9-C13, wherein said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

C15. (embodiment 15) the method of any one of embodiments C1-C5, C7, or C9, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

C16. (embodiment 16) the method of any one of embodiments C1-C5, C7, or C10, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

C17. (embodiment 17) the method of any one of embodiments C1-C5, C7, C9-C11, C15, or C16, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

C18. (embodiment 18) the method of any one of embodiments C1-C9, C12, or C15, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

C19. (embodiment 19) the method of any one of embodiments C1-C8, C10, C13, or C16, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

C20. (embodiment 20) the method of any one of embodiments C1-C19, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

C21. (embodiment 21) the method of any one of embodiments C1-C20, wherein the region derived from the CH1 region is a region derived from a human IgG1CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1.

C22. (embodiment 22) the method of any one of embodiments C1-C21, wherein the CL-region derived region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the CL-region derived region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID No. 2.

C23. (embodiment 23) the method of embodiment C21 or C22, wherein the antigen binding loop at the CD loop region of the CH1 region replaces the amino acid residue TSG of the CD loop of the human IgG1CH1 region.

C24. (embodiment 24) the method of embodiment C21 or C22, wherein the antigen binding loop at the DE loop region of the CH1 region replaces the amino acid residue QSS of the DE loop of the human IgG1CH1 region.

C25. (embodiment 25) the method of embodiment C21 or C22, wherein the antigen binding loop at the CD loop region of the CL region replaces the amino acid residue SGNS of the CD loop of the human clk region.

C26. (embodiment 26) the method of embodiment C21 or C22, wherein the antigen binding loop at the DE loop region of the CL region replaces the amino acid residue SKD of the DE loop of the human clk region.

C27. (embodiment 27) the method of any one of embodiments C1-C26, wherein each of the one or more antigen binding loops comprises 7 to 15 amino acid residues.

C28. (embodiment 28) the method of any one of embodiments C1-C27, wherein the VH region and the VL region bind to a first antigen; and the region derived from the CH1 region and/or the region derived from the CL region binds to a second antigen.

C29. (embodiment 29) the method of embodiment C28, wherein the first antigen and the second antigen are the same antigen.

C30. (embodiment 30) the method of embodiment C28, wherein the first antigen and the second antigen are two different antigens.

In yet another set of embodiments (set D embodiments), there is provided:

D1. (embodiment 1) a pharmaceutical composition comprising:

(a) A binding molecule, the binding molecule comprising:

wherein said CH1 domain-derived region and/or said CL domain-derived region comprises

Containing one or more antigen binding loops; and

(b) Pharmaceutically acceptable excipients.

D2. (embodiment 2) the pharmaceutical composition according to embodiment D1, wherein the one or more antigen binding loops in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region.

D3. (embodiment 3) the pharmaceutical composition according to embodiment D1 or D2, wherein the region derived from the CL region of the pharmaceutical composition has one or more antigen binding loops located in the AB, BC, CD, DE, EF and/or FG loop regions of the CL region.

D4. (embodiment 4) the pharmaceutical composition according to any one of embodiments D1-D3, wherein the region derived from the CH1 region comprises one or two antigen binding loops.

D5. (embodiment 5) the pharmaceutical composition according to any one of embodiments D1-D4, wherein the region derived from the CL region comprises one or two antigen binding loops.

D6. (embodiment 6) the pharmaceutical composition according to any one of embodiments D1-D5, wherein said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region.

D7. (embodiment 7) the pharmaceutical composition according to any one of embodiments D1-D6, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region.

D8. (embodiment 8) the pharmaceutical composition according to any one of embodiments D1-D7, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region.

D9. (embodiment 9) the pharmaceutical composition according to any one of embodiments D1-D8, wherein the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region.

D10. (embodiment 10) the pharmaceutical composition according to any one of embodiments D1-D9, wherein the region derived from the CL region comprises one antigen binding loop at the DE loop region of the CL region.

D11. (embodiment 11) the pharmaceutical composition according to any one of embodiments D1-D10, wherein the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region.

D12. (embodiment 12) the pharmaceutical composition according to any one of embodiments D1-D6 or D9, wherein said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

D13. (embodiment 13) the pharmaceutical composition according to any one of embodiments D1-D6 or D10, wherein said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

D14. (embodiment 14) the pharmaceutical composition according to any one of embodiments D1-D6 or D9-D13, wherein said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

D15. (embodiment 15) the pharmaceutical composition according to any one of embodiments D1-D5, D7 or D9, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

D16. (embodiment 16) the pharmaceutical composition of any one of embodiments D1-D5, D7 or D10, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

D17. (embodiment 17) the pharmaceutical composition according to any one of embodiments D1-D5, D7, D9-D11, D15 or D16, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

D18. (embodiment 18) the pharmaceutical composition according to any one of embodiments D1-D9, D12 or D15, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

D19. (embodiment 19) the pharmaceutical composition of any one of embodiments D1-D8, D10, D13, or D16, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

D20. (embodiment 20) the pharmaceutical composition of any one of embodiments D1-D19, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

D21. (embodiment 21) the pharmaceutical composition according to any one of embodiments D1-D20, wherein the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1.

D22. (embodiment 22) the pharmaceutical composition according to any one of embodiments D1-D21, wherein the CL-region derived region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the CL-region derived region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID No. 2.

D23. (embodiment 23) the pharmaceutical composition according to embodiment D21 or D22, wherein the antigen binding loop at the CD loop region of the CH1 region replaces the amino acid residue TSG of the CD loop of the human IgG1 CH1 region.

D24. (embodiment 24) the pharmaceutical composition according to embodiment D21 or D22, wherein the antigen binding loop at the DE loop region of the CH1 region replaces the amino acid residue QSS of the DE loop of the human IgG1 CH1 region.

D25. (embodiment 25) the pharmaceutical composition according to embodiment D21 or D22, wherein the antigen binding loop at the CD loop region of the CL region replaces the amino acid residue SGNS of the CD loop of the human clk region.

D26. (embodiment 26) the pharmaceutical composition of embodiment D21 or D22, wherein the antigen binding loop at the DE loop region of the CL region replaces the amino acid residue SKD of the DE loop of the human clk region.

D27. (embodiment 27) the pharmaceutical composition of any one of embodiments D1-D26, wherein each of the one or more antigen binding loops comprises 7 to 15 amino acid residues.

D28. (embodiment 28) the pharmaceutical composition of any one of embodiments D1-D27, wherein the VH region and the VL region bind to a first antigen; and the region derived from the CH1 region and/or the region derived from the CL region binds to a second antigen.

D29. (embodiment 29) the pharmaceutical composition of embodiment D28, wherein the first antigen and the second antigen are the same antigen.

D30. (embodiment 30) the pharmaceutical composition of embodiment D28, wherein the first antigen and the second antigen are two different antigens.

In yet another set of embodiments (set E embodiments), there is provided:

E1. (embodiment 1) a method of treating a disease or disorder in a subject, the method comprising administering to the subject a binding molecule, wherein the binding molecule comprises:

E2. (embodiment 2) the method of embodiment E1, wherein the one or more antigen binding loops in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region.

E3. (embodiment 3) the method according to embodiment E1 or E2, wherein the one or more antigen binding loops in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop regions of the CL region.

E4. (embodiment 4) the method of any one of embodiments E1-E3, wherein the region derived from the CH1 region comprises one or two antigen binding loops.

E5. (embodiment 5) the method of any one of embodiments E1-E4, wherein the region derived from the CL region comprises one or two antigen binding loops.

E6. (embodiment 6) the method of any one of embodiments E1-E5, wherein the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region.

E7. (embodiment 7) the method of any one of embodiments E1-E6, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region.

E8. (embodiment 8) the method of any one of embodiments E1-E7, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region.

E9. (embodiment 9) the method of any one of embodiments E1-E8, wherein the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region.

E10. (embodiment 10) the method of any one of embodiments E1-E9, wherein the region derived from the CL region comprises one antigen binding loop at the DE loop region of the CL region.

E11. (embodiment 11) the method of any one of embodiments E1-E10, wherein the region derived from the CL region comprises one antigen binding loop at the CD loop region of the CL region and one antigen binding loop at the DE loop region of the CL region.

E12. (embodiment 12) the method of any one of embodiments E1-E6 or E9, wherein the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

E13. (embodiment 13) the method of any one of embodiments E1-E6 or E10, wherein the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

E14. (embodiment 14) the method of any one of embodiments E1-E6 or E9-E13, wherein the region derived from the CH1 region comprises an antigen binding loop at the CD loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

E15. (embodiment 15) the method of any one of embodiments E1-E5, E7, or E9, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

E16. (embodiment 16) the method of any one of embodiments E1-E5, E7, or E10, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

E17. (embodiment 17) the method of any one of embodiments E1-E5, E7, E9-E11, E15, or E16, wherein the region derived from the CH1 region comprises one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

E18. (embodiment 18) the method of any one of embodiments E1-E9, E12, or E15, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region.

E19. (embodiment 19) the method of any one of embodiments E1-E8, E10, E13, or E16, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from the CL region comprises an antigen binding loop at said DE loop region of said CL region.

E20. (embodiment 20) the method of any one of embodiments E1-E19, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

E21. (embodiment 21) the method of any one of embodiments E1-E20, wherein the region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1.

E22. (embodiment 22) the method of any one of embodiments E1-E21, wherein the CL-region-derived region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the CL-region-derived region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID No. 2.

E23. (embodiment 23) the method of embodiment E21 or E22, wherein the antigen binding loop at the CD loop region of the CH1 region replaces the amino acid residue TSG of the CD loop of the human IgG1CH1 region.

E24. (embodiment 24) the method of embodiment E21 or E22, wherein the antigen binding loop at the DE loop region of the CH1 region replaces the amino acid residue QSS of the DE loop of the human IgG1CH1 region.

E25. (embodiment 25) the method of embodiment E21 or E22, wherein the antigen binding loop at the CD loop region of the CL region replaces the amino acid residue SGNS of the CD loop of the human clk region.

E26. (embodiment 26) the method of embodiment E21 or E22, wherein the antigen binding loop at the DE loop region of the CL region replaces the amino acid residue SKD of the DE loop of the human clk region.

E27. (embodiment 27) the method of any one of embodiments E1-E26, wherein each of the one or more antigen binding loops comprises 7 to 15 amino acid residues.

E28. (embodiment 28) the method of any one of embodiments E1-E27, wherein the VH region and the VL region bind to a first antigen; and the region derived from the CH1 region and/or the region derived from the CL region binds to a second antigen.

E29. (embodiment 29) the method of embodiment E28, wherein the first antigen and the second antigen are the same antigen.

E30. (embodiment 30) the method of embodiment E28, wherein the first antigen and the second antigen are two different antigens.

E31. (embodiment 31) the method of any one of embodiments E1-E30, wherein the disease or disorder is associated with the first antigen and/or the second antigen.

7. Examples

The following is a description of various methods and materials used in the study. They are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the following experiments are performed and all experiments that can be performed. It should be understood that the exemplary descriptions written in the present tense are not necessarily performed, but may be performed to generate data or the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.

EXAMPLE 1 construction of Fab constant region library

Construction of a Fab Constant Region Library (CRL) was based on anti-XO 1B1 Fab fused to the M13 filamentous phage coat protein pIX. The variable region of the anti-XO 1B1 parent Fab binds to XO1B1 (a monoclonal antibody directed against human thrombin). A library of designs (shown in figure 1B) was constructed in the solvent accessible region of the CD and DE loops of the CH1 and/or CL constant regions of the anti-XO 1B1 parent Fab to enable binding to the second antigen. CRL was generated using synthetic DNA fragments (obtained from DNA manufacturers) containing the library variants. These DNA library fragments were subcloned into a double gene e.coli phage display vector using standard molecular biology techniques, wherein the Fab heavy chain was in frame with the amino terminus of the pIX phage coat protein and the light chain was expressed under the control of a second promoter.

Fab CRLs were constructed using different loop combinations and lengths to replace portions of the CD and DE loops of the CH1 and/or CL constant regions of the parent Fab. For example, three amino acids (TSG) within the (A) CH1CD loop are replaced with a diversified 7 or 9 amino acid binding loop _164-166 EU numbering), three amino acids within the (B) clk DE loop (SKD) _168-170 EU numbering), or (C) two loops to construct a first generation (G1) Fab CRL; by replacing three amino acids (QSS) within the CH1CD loop of (A) with identical 7 and 9 amino acid binding loops _175-177 EU numbering), (B) four amino acids within the clk DE loop (SGNS) _156-159 EU numbering), or (C) two loops to construct a second generation (G2) Fab CRL; substitution of S in the CH1 CD ring of (A) by using a diversified 15 amino acid binding ring ₁₆₅ (B) K in the CoppaDE Ring ₁₆₉ Or (C) two loops to construct a fifth generation (G5) Fab CRL.

The different compositions of the Fab CRL binding loop are shown in figure 1C. Amino acid cysteines are excluded from CRLs because they are prone to form unwanted disulfide bonds. Methionine is excluded because of the tendency to oxidize. The steric and hydraulic properties of phenylalanine are largely summarized by the side chains of tyrosine and tryptophan, and thus phenylalanine is excluded from CRL. Similarly, the steric and hydraulic properties of isoleucine are largely summarized by the side chains of valine and leucine, and therefore isoleucine is excluded from CRL. The first and last residues of the diverse sequence are referred to as anchors. For a CRL of 15 amino acids, alanine and glycine were selected as anchors at positions-1, -2, 16 and 17. Glycine provides maximum backbone flexibility in cases where backbone twist is required for presentation of diverse loops to their targets, which is disadvantageous for the other 19 typical amino acids. Alanine provides minimal backbone constraints that stabilize proper presentation of diverse regions if such presentation Allowing for reduced torsional flexibility. For CRLs of 7 and 9 amino acids, the anchoring residues are selected according to their small hydrophilic side chains. For amino acids with repeating side chain functionalities (aspartic acid and glutamic acid, asparagine and glutamine), larger amino acids are excluded from the anchor position. Arginine is included in the anchor position due to its ability of guanidine side-chain functionality to form stable interactions with the backbone and side-chain atoms of adjacent residues. The anchor residue group thus comprises alanine, aspartic acid, glycine, asparagine, arginine, serine and threonine. In the diverse loop of a CRL of 9 amino acids, only the bulky hydrophobic residues tryptophan, valine and leucine are allowed to be located at alternating positions to prevent the appearance of large hydrophobic surfaces that may bind non-specifically to the target. Likewise, these residues are only allowed as clusters at positions 3, 4 and 5 in a CRL of 7 amino acids. CRL of 15 amino acids was limited only by exclusion of cysteine, methionine, phenylalanine and isoleucine. Due to the different compositions of the binding loops of Fab CRL, the diversity range of Fab CRL with single loop is 10 ⁷ To 10 ¹⁶ The method comprises the steps of carrying out a first treatment on the surface of the The diversity range of Fab CRL with bicyclic ring is 10 ¹⁸ To 10 ³³ 。

Example 2 selection of Fab constant region conjugates of anti-polyhistidine monoclonal antibodies

To verify that phage displayed CRLs were used to evolve completely new molecular recognition of targets of interest, a selection was first made for commercial anti-polyhistidine monoclonal antibodies, as the binding motif (polyhistidine) is known and contained in the designed library. A total of three panning pools were used in this selection. All three pools were based on CRL generation 1 (G1) as described in example 1. Specifically, three amino acids within the clk DE loop were replaced by using diversified 7 and 9 amino acid binding loops (SKD _168-170 EU numbering), fab CRLs for use in panning pool 3 (P3) were constructed; replacement of three amino acids within the CH1 CD loop by using a diversity of 7 and 9 amino acid binding loops (TSG _164-166 EU numbering), fab CRLs for use in panning pool 4 (P4) were constructed; by replacing clk with 9 amino acid binding loopsThree amino acids within the DE loop (SKD _168-170 EU numbering) and three amino acids (TSG) within the CH1 CD loop _164-166 EU numbering), fab CRL was constructed for use in panning pool 5 (P5). The anti-polyhistidine monoclonal antibodies used in round 1, round 3, round 4, round 5 and round 6 of panning were at concentrations of 100nM, 50nM, 10nM and 10nM, respectively. Round 2 panning against XO1B1 to remove any CRL Fab that does not maintain CDR-mediated target binding.

After each round of panning (except round 2), aliquots of output phage from each of the three pools were analyzed for anti-polyhistidine monoclonal antibody binding and XO1B1 binding by a polyclonal phage ELISA. As shown in fig. 2A, these pools were gradually enriched with Fab constant region conjugates conjugated to anti-polyhistidine monoclonal antibodies. As shown in fig. 2B, the binding of the enriched pool to XO1B1 was comparable to that of the parent Fab. Furthermore, as demonstrated by the sequencing results shown in fig. 2C, the initially diverse binding loops of Fab CRLs are enriched for histidine, confirming that Fab constant region conjugates can be selected from Fab CRLs for targets of interest.

Example 3 selection of Fab constant region conjugates against mEphA2-Fc

De novo FabBRL panning against mEphA2-Fc

The overall process of selecting Fab constant region conjugates from Fab CRLs for recombinant murine EphA 2-human IgG1 Fc chimeras (referred to herein as mhepha 2-Fc) is shown in fig. 3. G1, G2 and G5 single-and double-loop Fab CRLs underwent multiple rounds of panning against megepha 2-Fc under specific panning conditions. After each round of panning, the pools were analyzed for binding to the mhepha 2-Fc, XO1B1 and negative control Fc fusion proteins by polyclonal phage ELISA. Any pool that shows enrichment for EphA2 binding was further analyzed by monoclonal phage ELISA to identify cloned mhap 2-Fc binders, which were subsequently sequenced. Next Generation Sequencing (NGS) was also applied to selected panning pools to identify additional potential Fab constant region conjugates to the mhepha 2-Fc. Finally, hits identified by two methods (phage ELISA and NGS) were further characterized after purification from mammalian expression.

A total of seventeen panning conditions (referred to as panning pools P1-P17) were explored to identify the most productive conditions in producing hits against the mha 2-Fc antigen. For a general panning method, reference is made to Antibody Phage Display, methods and Protocols by Robert Aitken (ISBN 978-1-60327-302-2). In all cases, selection was performed by binding CRL to non-specific biotinylated mhap a2-Fc captured on neutravidin beads in the presence of the blocking mixture. Human IgG1 Fc competitors were used in all rounds of panning in all pools to prevent enrichment of the conjugate against the Fc region of the mhap 2-Fc antigen. After extensive washing, MC1061F E.coli bacterial cells were infected with the bound phage and expanded for subsequent panning rounds or characterized by phage ELISA. Different panning conditions include (1) which library was used in panning, (2) the amount of antigen used in each round of panning, (3) the length of antigen binding time, and (4) the method of maintaining CDR-mediated binding to XO1B 1. The specific panning conditions for each pool during the first through sixth rounds of panning are listed in table 2 below.

TABLE 2 FabCRL panning conditions for mEphA2-Fc

* Additional two rounds of panning with overnight antigen binding

Each of the seventeen pools underwent six to eight rounds of panning against mhepha 2-Fc. After six to eight rounds of panning, aliquots of output phage from each of seventeen pools were analyzed for binding to mhha 2-Fc relative to anti-XO 1B1 parent Fab by polyclonal phage ELISA.

Twelve of seventeen pools did not show enrichment of binding of mhha 2-Fc as a result of detection of mhha 2-Fc binding by polyclonal phage ELISA; four pools (P5-R8, P9-R8, P15-R8, P17-R8) showed weak enrichment of the binding of mEphA2-Fc (3.4-fold to 34-fold binding signal relative to the anti-XO 1B1 parent Fab); one pool (P8-R6) showed a strong enrichment of the binding of the mha 2-Fc (binding signal relative to the anti-XO 1B1 parental Fab, 8335-fold phage library pool). After four to eight rounds of panning, a summary of the results of the mhha 2-Fc binding polyclonal phage ELISA for five enrichment pools (P5, P8, P9, P15, P17) is shown in fig. 4A, highlighting that the G5 library with the longer 15 amino acid loop was most effectively enriched in the mhha 2-Fc panning.

To assess the binding properties of individual clones within the enriched phage pools, the binding of total 378 clones from the five enriched pools (P5, P8, P9, P15, P17) to the mhap a2-Fc (fig. 4B) and to XO1B1 (fig. 4C) was further analyzed by monoclonal phage ELISA. If the binding signal of the mha 2-Fc is not less than 60 compared to the negative control and the binding signal of XO1B1 is not less than 50 compared to the parent Fab, a single clone is selected for Sanger sequencing analysis. The selection criteria resulted in sixteen clones selected from P8, which P8 was derived from the G5 CH1-CD loop library. The P5 (G2 CH1-DE loop library) clone lost all CDR-mediated binding to XO1B1, indicating potential misfolding or truncation of the clone in the panning pool. XO1B1 binding was also greatly reduced for P9 (G5 bicyclic library) clones and EphA2 binding was only moderately increased, so no clones were selected from the pool for further sequence analysis. Although clones from P15 and P17 (G5 clk-DE loop library) maintained XO1B1 binding similar to the parent anti-XO 1B1 Fab, the mhap 2-Fc binding signal did not meet selection criteria and no clones were selected for further sequence analysis. Sixteen clones selected from P8 after six rounds of panning are shown in fig. 4D, with most clones having similar binding spectra for mha 2-Fc and XO1B 1. Sixteen selected clones were sequenced and all sixteen selected clones had the same anti-mha 2-Fc binding sequence in the CH1 CD loop, which is shown in fig. 4E. Interestingly, the selected loops were 13 amino acids in length, which represents truncated variants derived from a designed 15 amino acid loop library.

To confirm the binding properties of the identified clones, this single Fab constant region conjugate that binds to mha 2-Fc (identified as EPAXB 1) was purified as a mammalian expressed His from HEK Expi293 cells ₆ A labelled fusion. After one purification step by Immobilized Metal Affinity Chromatography (IMAC), a yield of 269mg protein per liter of expression volume was obtained. Proteins were characterized by analytical size exclusion high performance liquid chromatography (SE-HPLC), reducing (R) and non-reducing (NR) sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE and complete Mass Spectrometry (MS). Unimodal (except histidine buffer peak) was observed by SE-HPLC, and high purity was observed by SDS-PAGE. The observed molecular weight of the intact purified protein (49,119 Da) was close to the predicted molecular weight of 49,110Da. Purified protein analysis is shown in fig. 4F.

To confirm the dual specificity of the purified Fab, the binding kinetics and affinity of EPAXB1 to both mhap 2-Fc and XO1B1 were analyzed using Surface Plasmon Resonance (SPR). The binding kinetics and affinity of the anti-XO 1B1 parent Fab to both the mhha 2-Fc and XO1B1 were also analyzed as a reference. As shown in fig. 4G, the parental Fab did not bind to the mhepha 2-Fc, while EPAXB1 bound with an affinity of 2.18nM, confirming that the CRL loop identified during panning confers new binding in the Fab constant region. Meanwhile, EPAXB1 can bind to XO1B1 with an affinity (kd=0.36 nM) comparable to that of the parent Fab (kd=0.35 nM), indicating that CDR-mediated binding is not interfered with by the addition of CRL loops.

Since the number of hits identified by phage ELISA may be limited by assay sensitivity, a subset of the panning pools were also submitted for Next Generation Sequencing (NGS) to identify additional Fab constant region binders by sequence enrichment. Based on results from the polyclonal phage ELISA, the later round of panning (round 4, round 5, round 6, or round 8) from thirteen of the seventeen panning pools was selected for AmpliconEZ NGS analysis in the reservoir region. The enrichment was calculated as the ratio of the number of instances of a particular sequence to the total number of full length sequences observed multiplied by 100%. Anti-mhap 2 hits identified by phage ELISA were also observed by NGS, andthe sequence had an enrichment of 94.2%. Enrichment of more than 0.5% without significant non-specific binding motifs (such as arginine-rich motifs) was used as a guide to select sequences for further binding analysis. Thus, a total of forty-three NGS-based hits (including two bicyclic variants) were taken as His ₆ The labeled Fab is cloned into a mammalian expression vector. These forty three fabs were expressed from HEK Expi293 cells plus a single Fab constant region conjugate (EPAXB 1) and a parent anti-XO 1B1 Fab identified by phage ELISA, and the supernatants were analyzed by SPR. Four out of forty-three Fab showed binding to the mha 2-Fc in the nM range. However, one of the potential hits identified later showed negligible expression levels and was therefore not identified as EphA2 conjugate. As a result, three novel Fab constant region conjugates (identified as EPAXB17, EPAXB27 and EPAXB 28) were identified by NGS coupled to SPR screening and are shown in FIG. 5.

Taken together, starting from seventeen different panning conditions based on loops containing 7, 9 or 15 diversified amino acids in the Fab CH1 and/or CL regions, a total of four Fab constant region conjugates were identified for the mhap 2-Fc. They are all derived from monocyclic 5 th generation (G5) Fab CRLs. The characteristics of the four Fab constant region conjugates are shown in table 3 below. Of the four identified sequences, three present an Ala-Tyr-Pro motif.

TABLE 3 characterization of Fab constant region conjugates against mEphA2-Fc

Thermal stability of Fab with engineered anti-EphA 2 binding loop

Each of the four fabs with engineered anti-EphA 2 binding loops was analyzed by Differential Scanning Fluorometry (DSF) to measure thermostability. Thermal stability was determined by Differential Scanning Fluorometry (DSF) using a Prometheus NanoDSF instrument (Nanotemper technologies). Samples were diluted to 0.5mg/mL in PBS pH 7.4 and loaded from 384 well sample plates into 24 well capillaries. Repeated runs were performed on each sample. The sample was heated from 20 ℃ to 95 ℃ at a rate of 1.0 ℃/min, and intrinsic tryptophan and tyrosine fluorescence was measured using an excitation wavelength of 330nm and an emission wavelength of 350 nm. The onset temperature of aggregation was also monitored using back reflection techniques. Melting temperature and onset of aggregation were determined with Pr.stability Analysis v1.0.2 software.

As shown in table 4 below, each of the four fabs with engineered anti-EphA 2 binding loops had some instability compared to the parent Fab. Two fabs that bind EphA2 through an engineered CD loop in the CH1 domain (i.e., EPAXB1 and EPAXB 17) have only a2 ℃ loss in Tm compared to the parent Fab. However, a very broad transition was observed, which may indicate early unfolding of CH 1. Due to this broad transition, the onset temperature (T-onset) is the best measure for comparison and the bispecific Fab starts 10 ℃ to 12 ℃ earlier than the parent Fab. Two fabs that bind EphA2 through the engineered DE loop in the CLk domain (i.e., EPAXB27 and EPAXB 28) have a 9 ℃ loss in Tm compared to the parent Fab, and an onset temperature 9 ℃ earlier. Notably, bispecific antibodies with engineered loops have Tm and onset temperatures in the range of an unrelated control antibody (CNTO 5825), which is known to be stable and have good biophysical properties.

TABLE 4 thermostability of Fab constant region conjugates

Reformatting Fab constant region conjugates to mEphA2-Fc

Although the mhap 2-Fc binding loop in the CH1 domain of EPAXB1 was found in the context of anti-XO 1B1 Fab, it was paired with a new variable region derived from a different human germline sequence (anti-human IL23R and HER 2) to determine if the mhap 2-Fc binding would be retained. In addition, EPAXB1 was reformatted as a monoclonal antibody (fig. 6A). Each form was expressed in HEK 293Expi cells and purified by IMAC (for Fab) or protein a purification (for monoclonal antibodies). The expression yield of bispecific Fab and monoclonal antibodies ranged from 231mg to 481mg protein expressed per liter, and for each new form, a single peak (except buffer peak) was observed by SE-HPLC. The purification yields and SE-HPLC retention times of bispecific Fab were comparable to their corresponding monospecific Fab parent (fig. 6B).

The binding affinities of purified monospecific and bispecific Fab and purified bispecific monoclonal antibodies to each target (mhepha 2-Fc, XO1B1, human IL23R and HER 2-Fc) were determined by Surface Plasmon Resonance (SPR). In all novel forms, ephA2 binding loops remain bound with an affinity of 2.18nM in anti-XO 1B1 bispecific Fab, an affinity of 8.84nM in anti-IL 23R bispecific Fab, and an affinity of 16.57nM in anti-HER 2 bispecific Fab. In each Fab, variable region affinity to its cognate target is maintained in the anti EphA2 bispecific format. In the anti-XO 1B 1X anti-EphA 2 monoclonal antibody format, the apparent affinity of the variable region for XO1B1 is 0.08nM and the apparent affinity of the anti-EphA 2 loop for mEphA2-Fc is 0.09nM. Due to the avidity, the apparent binding of the monoclonal antibody form is tighter compared to Fab (fig. 6C).

Target binding of additional bispecific mabs

anti-IL 23R x anti-EphA 2 and anti-HER 2 x anti-EphA 2 Fab are reformatted into standard monoclonal antibody forms and evaluated for their ability to bind to their respective targets on the cell.

HER2 x EphA2 and IL23 x EphA2 bispecific antibodies and corresponding HER2 and IL23R monospecific mabs were expressed in HEK 293Expi cells and purified by protein a resin. Protein purity was assessed by SE-HPLC (size exclusion high performance liquid chromatography).

Purification yields ranged from 91mg to 157mg protein/L expression, and a single peak was observed by SE-HPLC (FIG. 7). Bispecific purification yields and SE-HPLC retention times were comparable to their corresponding monospecific mAb parents.

Plasmids encoding full-length human EphA2 and HER2 receptors were transfected into 293F cells to prepare stable cell pools under hygromycin selection. Selected pools were screened by FACS using anti-EphA 2 antibodies conjugated to phycoerythrin fluorophores (R & D Systems catalog number: FAB 3035P) or anti-HER 2 antibodies (abCam catalog number: 11710), and detected using anti-rat secondary antibodies conjugated to phycoerythrin fluorophores (Jackson catalog number: 112-116-143). All selected pools were separated together with their primary antibodies and isotype specific Dynabead. For EphA2, dynabead goat anti-mouse (Thermo catalog number: 11033) was used, and for HER2, dynabead sheep anti-rat (Thermo catalog number: 11035) was used.

For binding studies, cells were isolated from the flask with Accutase, washed and resuspended in BD staining buffer. Cells were seeded into 96-well v-plates at 150,000 cells/well and incubated on ice for 1 hour with primary stained antibodies serially diluted 1:2 starting at 400 nM. Cells were then washed and incubated with goat AF488 anti-hIgG Fcg specific F (ab') 2 secondary detection reagent for 1 hour on ice. Cells were washed, fixed with BD Cytofix, washed again and resuspended in 50 μl staining buffer. The cells were then analyzed on an iQue VBR Plus flow cytometer.

Minimal binding of HER2 x EphA2 and IL23 x EphA2 bispecific antibodies or their corresponding monospecific control antibodies to untransfected HEK cells was observed (fig. 8A). Each of the antibodies containing EphA2 binding loops in the antibody constant regions bound to HEK cells stably expressing human EphA2, while isotype control did not bind significantly (fig. 8C). The binding of anti-HER 2X anti-EphA 2 bispecific to HEK-HER2 cells was similar to that of the parent anti-HER 2 mAb (FIG. 8B), and the binding of anti-IL 23R X anti-EphA 2 bispecific to HEK-IL23R cells was similar to that of the parent anti-IL 23RmAb (FIG. 8D), indicating that the presence of the EphA2 binding loop did not affect variable region mediated cell binding.

To further confirm the specificity of the constant region binding interactions, bispecific Fab and mAb and their corresponding monospecific parent antibodies were also evaluated by the SPR method to determine if they had a non-specific interaction with a subset of unrelated recombinant test proteins with a range of biophysical properties. For most bispecific Fab and mabs containing engineered EphA2 binding loops, no binding to recombinant test proteins was observed. In one case, non-specific interactions were observed, but this was due to the parent antibody, not the engineered constant region loop (data not shown).

Simultaneous target binding of bispecific Fab and mAb

Bispecific Fab and mAb were evaluated for their ability to bind their targets simultaneously by variable region-mediated binding and constant region loop binding.

Using Octet384 systems (ForteBio, sartorius) evaluate dual target engagement by Biological Layer Interferometry (BLI). Biotinylated antigen was loaded onto streptavidin coated biosensors to achieve 1nm Response Unit (RU) shift. Monospecific or bispecific mAb (1. Mu.M) or Fab (400 nM) were associated for 3 min. The secondary antigen was then associated for 3 minutes. All samples were diluted into running buffer of DPBS, 0.1% bsa and 0.02% surfactant P20.

anti-HER 2 x anti-EphA 2 and anti-IL 23R x anti-EphA 2 bispecific Fab bind to their respective targets simultaneously as shown by Biological Layer Interferometry (BLI) (fig. 9A and 9B, respectively). Regardless of which antigen the biosensor first captures, the dual target conjugation was successful. anti-HER 2 x anti-EphA 2 bispecific in mAb format was also able to bind both targets simultaneously (fig. 9C).

<110> Jansen biotechnology Co

<120> molecules with engineered antibody constant region variants

<130> 14620-683-228

<140>

<141>

<150> US 63/176,736

<151> 2021-04-19

<150> US 63/176,731

<151> 2021-04-19

<150> US 63/176,725

<151> 2021-04-19

<150> US 63/176,720

<151> 2021-04-19

<150> US 63/176,718

<151> 2021-04-19

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Ala Gly Trp Ala Ser Pro Arg Leu Ala Tyr Pro Asp Thr Val Pro Val

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Ala Gly Ala

<210> 47

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic peptides

<400> 47

Gly Ala Leu Thr

1

<210> 48

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic peptides

<400> 48

Ser Gly Val His

1

Claims

1. A binding molecule comprising a region derived from the CH1 region of an antibody heavy chain and/or a region derived from the CL region of an antibody light chain, wherein the region derived from the CH1 region and/or the region derived from the CL region comprises one or more antigen binding loops.

2. A binding molecule comprising:

3. The binding molecule of claim 1 or 2, wherein:

(i) The one or more antigen binding loops in the region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CH1 region; and/or

(ii) The one or more antigen binding loops in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop regions of the CL region.

4. A binding molecule according to any one of claims 1 to 3, wherein:

(i) The region derived from the CH1 region comprises one or two antigen binding loops; and/or

(ii) The region derived from the CL region comprises one or two antigen binding loops.

5. The binding molecule of any one of claims 1-4, wherein:

(i) Said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region; and/or

(ii) The region derived from the CH1 region comprises an antigen binding loop at the DE loop region of the CH1 region.

6. The binding molecule of any one of claims 1-5, wherein the region derived from the CH1 region comprises one antigen binding loop at the CD loop region of the CH1 region and one antigen binding loop at the DE loop region of the CH1 region.

7. The binding molecule of any one of claims 1-6, wherein the region derived from the CL region:

(i) An antigen binding loop at the CD loop region of the CL region;

(ii) An antigen binding loop at the DE loop region of the CL region; or alternatively

(iii) An antigen binding loop is included at the CD loop region of the CL region and an antigen binding loop is included at the DE loop region of the CL region.

8. The binding molecule of any one of claims 2-7, wherein:

(i) Said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region;

(ii) Said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said DE loop region of said CL region;

(iii) Said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region and an antigen binding loop at said DE loop region of said CL region;

(iv) Said region derived from the CH1 region comprises an antigen binding loop at said DE loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region;

(v) Said region derived from the CH1 region comprises an antigen binding loop at said DE loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said DE loop region of said CL region;

(vi) Said region derived from the CH1 region comprises an antigen binding loop at said DE loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region and an antigen binding loop at said DE loop region of said CL region;

(vii) Said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region and an antigen binding loop at said DE loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said CD loop region of said CL region;

(viii) Said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region and an antigen binding loop at said DE loop region of said CH1 region; and said region derived from a CL region comprises an antigen binding loop at said DE loop region of said CL region; or alternatively

(ix) Said region derived from the CH1 region comprises an antigen binding loop at said CD loop region of said CH1 region and an antigen binding loop at said DE loop region of said CH1 region; and said region derived from a CL region comprises one antigen binding loop at said CD loop region of said CL region and one antigen binding loop at said DE loop region of said CL region.

9. The binding molecule of any one of claims 1-8, wherein:

(i) The region derived from the CH1 region is a region derived from a human IgG1CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1; and/or

(ii) The CL-derived region is a region derived from a human clk region comprising the amino acid sequence of SEQ ID No. 2, and wherein the CL-derived region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID No. 2.

10. The binding molecule of claim 9, wherein:

(i) The antigen binding loop at the CD loop region of the CH1 region replaces the amino acid residue TSG of the CD loop of the human IgG1CH1 region; and/or

(ii) The antigen binding loop at the DE loop region of the CH1 region replaces the amino acid residue QSS of the DE loop of the human IgG1CH1 region.

11. The binding molecule of claim 9, wherein:

(i) The antigen binding loop at the CD loop region of the CL region replaces the amino acid residue SGNS of the CD loop of the human clk region; and/or

(ii) The antigen binding loop at the DE loop region of the CL region replaces amino acid residue SKD of the DE loop of the human clk region.

12. The binding molecule of any one of claims 1-11, wherein each antigen binding loop of the one or more antigen binding loops comprises 7 to 15 amino acid residues.

13. The binding molecule of any one of claims 2 to 12, wherein the VH region and the VL region bind to a first antigen; and the region derived from the CH1 region and/or the region derived from the CL region binds to a second antigen.

14. The binding molecule of claim 13, wherein:

(i) The first antigen and the second antigen are the same antigen; or alternatively

(ii) The first antigen and the second antigen are two different antigens.

15. A nucleic acid encoding the binding molecule of any one of claims 1-14.

16. A vector comprising the nucleic acid of claim 15.

17. A method of making a binding molecule comprising expressing in a host cell a polynucleotide encoding the binding molecule of any one of claims 1-14.

18. A pharmaceutical composition comprising (a) a binding molecule according to any one of claims 1-14 or a nucleic acid according to claim 15, and (b) a pharmaceutically acceptable excipient.

19. A method of treating a disease or disorder in a subject, comprising administering to the subject a binding molecule according to any one of claims 1-14 and/or a nucleic acid according to claim 15, optionally wherein the disease or disorder is associated with the first antigen and/or the second antigen.

20. Use of a binding molecule according to any one of claims 1-14 and/or a nucleic acid according to claim 15 in the manufacture of a medicament for the treatment of a disease or disorder.

21. A Constant Region Library (CRL) comprising a population of binding molecules, wherein each of the binding molecules is a binding molecule according to any one of claims 1-14, wherein the population of binding molecules comprises different amino acid sequences in the region derived from the CH1 region and/or the region derived from the CL region.

22. A Constant Region Library (CRL) comprising a population of molecules each comprising a region derived from a CH1 region of an antibody and/or a region derived from a CL region of an antibody, wherein the population of molecules comprises different amino acid sequences in the region derived from a CH1 region and/or the region derived from a CL region.

23. The CRL of claim 21, wherein:

(i) Said different amino acid sequences in said region derived from the CH1 region are located in the AB, BC, CD, DE, EF and/or FG loop region of said CH1 region; and/or

(ii) The different amino acid sequences in the region derived from the CL region are located in the AB, BC, CD, DE, EF and/or FG loop region of the CL region.

24. The CRL of claim 21, wherein the population of molecules:

(i) Comprising different amino acid sequences in one or both loop regions in the region derived from the CH1 region;

(ii) Comprising different amino acid sequences in one or both loop regions in the CL-derived region;

(iii) Comprising different amino acid sequences at the CD loop region of the CH1 region;

(iv) Comprising different amino acid sequences at the DE loop region of the CH1 region;

(v) Comprising different amino acid sequences at the CD loop region and the DE loop region of the CH1 region;

(vi) Comprising different amino acid sequences at the CD loop region of the CL region;

(vii) Comprising different amino acid sequences at the DE loop region of the CL region;

(viii) Comprising different amino acid sequences at the CD loop region and the DE loop region of the CL region;

(ix) Comprising different amino acid sequences at the CD loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region of said CL region;

(x) Comprising different amino acid sequences at the CD loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said DE loop region of said CL region;

(xi) Comprising different amino acid sequences at the CD loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region and said DE loop region of said CL region;

(xii) Comprising different amino acid sequences at the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region of said CL region;

(xiii) Comprising different amino acid sequences at the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said DE loop region of said CL region;

(xiv) Comprising different amino acid sequences at the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region and said DE loop region of said CL region;

(xv) Comprising different amino acid sequences at the CD loop region of the CH1 region and the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region of said CL region;

(xvi) Comprising different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said DE loop region of said CL region; or alternatively

(xvii) Comprising different amino acid sequences at the CD loop region and the DE loop region of the CH1 region; and said population of molecules comprises different amino acid sequences at said CD loop region and said DE loop region of said CL region.

25. The CRL of any one of claims 21-24, wherein:

(i) The region derived from the CH1 region is a region derived from a human IgG1 CH1 region comprising the amino acid sequence of SEQ ID No. 1, and wherein the region derived from the CH1 region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID No. 1; and/or

26. The CRL of claim 25, wherein:

(i) Said amino acid residue TSG of the CD loop of said human IgG1 CH1 region is replaced by a different amino acid sequence in said molecule in said CRL; or alternatively

(ii) Said amino acid residue QSS of the DE loop of said human IgG1 CH1 region is replaced by a different amino acid sequence in said molecule in said CRL.

27. The CRL of claim 25, wherein:

(i) Said amino acid residue SGNS of the CD loop of said human clk region is replaced by a different amino acid sequence in said molecule in said CRL; or alternatively

(ii) Said amino acid residue SKD of the DE loop of said human clk region is replaced by a different amino acid sequence in said molecule in said CRL.

28. The CRL of any one of claims 21-27, wherein:

(i) The different amino acid sequences comprise 7 to 15 amino acid residues;

(ii) Each of the molecules further comprises a VH region and a VL region;

(iii) The binding molecule or the molecule is a Fab fragment;

(iv) The CRL having one loop region has a diversity range of 10 ⁷ To 10 ¹⁶ The method comprises the steps of carrying out a first treatment on the surface of the Or alternatively

(v) The CRL having two loop regions has a diversity of 10 ¹⁸ To 10 ³³ 。

29. A method for identifying a binding molecule comprising a first binding domain that binds a first antigen and a second binding domain that binds a second antigen, the method comprising screening the CRL of any one of claims 21-28 to identify a binding molecule that binds the second antigen with an affinity that is higher than a reference level, wherein the first binding domain comprises a VH region and a VL region of an antibody, and wherein the second binding domain comprises an antibody constant region variant.

30. A method of producing a binding molecule comprising: a first step for performing a function of identifying antibody constant region variants capable of binding to an antigen; and a second step of constructing the binding molecule comprising the antibody constant region variant, optionally wherein the first step comprises screening for a CRL according to any one of claims 21-28.

31. A binding molecule identified according to the method of claim 29.

32. A binding molecule produced according to the method of claim 30.

33. A method for treating a disease or disorder in a subject, comprising administering to the subject a binding molecule according to any one of claims 1-14, 31 and 32 and/or a nucleic acid according to claim 15.